Enhancing biological nitrogen fixation in common bean (Phaseolus vulgaris L)

Graduate Theses and Dissertations Graduate College 2013 Enhancing biological nitrogen fixation in common bean (Phaseolus vulgaris L) MERCY KASUZI K...
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Graduate Theses and Dissertations

Graduate College

2013

Enhancing biological nitrogen fixation in common bean (Phaseolus vulgaris L) MERCY KASUZI KABAHUMA Iowa State University

Follow this and additional works at: http://lib.dr.iastate.edu/etd Part of the Agricultural Science Commons, Agriculture Commons, and the Agronomy and Crop Sciences Commons Recommended Citation KABAHUMA, MERCY KASUZI, "Enhancing biological nitrogen fixation in common bean (Phaseolus vulgaris L)" (2013). Graduate Theses and Dissertations. Paper 13162.

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ENHANCING biological nitrogen fixation in Common bean (Phaseolus vulgaris L.) by Mercy Kasuzi Kabahuma

A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE

Major: Crop Production and Physiology Program of Study Committee: Mark E. Westgate, Major Professor Gwyn Beattie Kathleen Delate

Iowa State University Ames, Iowa 2013 Copyright © Mercy Kasuzi Kabahuma, 2013.

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To my family and friends

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TABLE OF CONTENTS LIST OF FIGURES

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LIST OF TABLES

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ABSTRACT CHAPTER 1. GENERAL INTRODUCTION

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Hypotheses

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Expected Outcomes

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References

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CHAPTER 2. GENOTYPIC VARIATION IN UREIDE CONTENT IN PHASEOLUS VULGARIS L.

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Abstract

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Introduction

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Materials and Methods

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Results and Discussion

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Conclusions

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References

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CHAPTER 3. SHOOT AND ROOT CONTROL OF UREIDE ACCUMULATION AND PARTITIONING IN PHASEOLUS VULGARIS L. GENOTYPES 46 Abstract

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Introduction

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Materials and Methods

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Results and Discussion

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Conclusions

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References

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CHAPTER 4. GENERAL DISCUSSION

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ACKNOWLEDGEMENTS

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LIST OF FIGURES Chapter 2 Figure 1. Number of nodules on R32, R99, Puebla, and Eagle roots of greenhouse-grown plants sampled at flowering.

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Figure 2. Biomass of tissues collected from R32, R99, Puebla, and Eagle bean lines during flowering/early pod set.

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Figure 3. Ureide concentration in plant tissues of R32, R99, Puebla, and Eagle bean lines during flowering/early pod set.

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Figure 4. Ureide content of tissues of R32, R99, Puebla, and Eagle bean lines during flowering /early pod set.

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Figure 5. N concentration in tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set.

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Figure 6. Total N accumulation in tissues of R32, R99, Puebla, and Eagle bean lines harvested during flowering/early pod set.

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Chapter 3 Figure 1. Biomass of tissues collected from R32, R99, Puebla and Eagle controls and grafted bean lines during flowering/early pod set.

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Figure 2. Ureide content in tissues of R32, R99, Puebla and Eagle controls and grafts harvested during flowering /early pod set.

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Figure 3. Total N accumulation in tissues of R32, R99, Puebla and Eagle controls and grafts harvested during flowering/early pod set.

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LIST OF TABLES Chapter 2 Table 1. Biomass of tissues from R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set.

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Table 2. Ureide concentration in tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set.

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Table 3. Ureide content of tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set.

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Table 4. N concentration in tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set.

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Table 5. Total N accumulation in tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set.

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Table 6. Percentage plant ureide and percentage N derived from N2 fixation.

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Table 7. Nodule effectiveness.

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Chapter 3 Table 1. Number of nodules on controls, self- and intra-species graft of R32, R99, Puebla, and Eagle roots sampled at flowering.

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Table 2. Biomass of controls, self- and intra-species graft tissues from R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set.

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Table 3. Shoot: Root ratios of controls, self- and intra-species graft of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set.

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Table 4. Ureide concentration in controls, self- and intra-species graft tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set.

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Table 5. Ureide content of controls, self- and intra-species graft tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set.

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Table 6. N concentration in controls, self- and intra-specie graft tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set.

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Table 7. Total N accumulation in controls, self- and intra-specie graft tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set.

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Table 8. Percent plant N derived from N2 fixation.

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ABSTRACT Common bean (Phaseolus vulgaris L.) is a herbaceous annual which, in a symbiotic relationship with specific soil bacteria, ‘fixes’ atmospheric nitrogen (N2) into amino form that can be used for plant growth. Efforts to optimize biological nitrogen fixation (BNF) in common beans are critical because of widespread increase in soil degradation in Africa. Among legumes, common beans derive the least percent N2 from N2 fixation. This has been attributed partly to susceptibility of common beans to physical and chemical environmental stresses, inconsistent response to inoculum, and lack of selection for the BNF trait. Improvement in productivity of this leguminous crop could be achieved through identification of genotypes with greatest capacity for BNF and nitrogen assimilation from BNF. Chapter 2 presents phenotypic traits that could possibly be associated with BNF and N assimilation. Bean lines varying in ability to form nodules and fix nitrogen were analyzed for root, stem, leaf, petiole and pod biomass, ureide concentration, nitrogen concentration, and nodule numbers. There was significant variation in ureide accumulation across plant tissues and genotypes. A combination of phenotypic traits, however, could be used to select for improved BNF. Moderate nodule number, leaf ureide content, and total biomass at flowering were consistent with greater BNF. Nodule effectiveness should be considered for increasing % N derived from N2 fixation. In Chapter 3 a grafting technique was used to determine shoot and/ or root control of ureide accumulation and partitioning among four genotypes noted for variation in phenotypic traits related to nitrogen fixation. The extent of nodulation, as modified by super-nodulating scions or non-nodulating rootstocks, only indirectly affected ureide and N accumulation. Plants with a greater number of nodules did not accumulate more nitrogen, indicating most nodules were not effective in fixing N.

The results indicate shoot regulation of nodulation, ureide metabolism, and nodule

effectiveness would be ideal physiological targets for further investigations aimed at improving BNF and yield.

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CHAPTER 1. GENERAL INTRODUCTION Legumes, such as common beans (Phaseolus vulgaris L.) have the ability to form a symbiotic relationship with soil bacteria capable of trapping nitrogen gas (N2) from the atmosphere and converting it into ammonia, which can be used by the plant for growth, development and seed production. Atmospheric nitrogen is converted to ammonia by the nitrogenase enzyme in a process known as biological nitrogen fixation (BNF) (Postgate, 1998; Bhatia et al., 2001). The capacity of legumes to fix atmospheric nitrogen gives them an advantage over non-leguminous crops when grown on soils low in nitrogen (N). As such, they are an integral part of most small-landholder cropping systems (Bhatia et al., 2001). The need to improve productivity of legumes as a global source of dietary protein, however, has made it vital to understand the factors that influence nitrogen fixation (Schulze, 2004). BNF is a symbiotic relationship between specific nitrogen-fixing bacteria (rhizobia) and legume plants (Caetano-Anolles and Gresshoff, 1991). Upon perception of flavonoids exuded by legume roots, rhizobia activate nod genes which induce production of Nod factors (Burdman et al., 1997). Nod factors are calcium dependent proteins that promote attachment of the rhizobia to root hair surfaces (Smit et al., 1987). Subsequent curling of the root hairs encloses the attached rhizobia (Heidstra et al., 1994), which induce hydrolysis of the root hair cell wall. Invagination of the plasma membrane by the plant forms an infection thread within which rhizobia multiply and penetrate host cells (Ridge et al., 1985). Once inside the parenchyma cells of the root, rhizobia and infected cells proliferate forming a nodule initial. Whether the nodule initial develops into a functional nodule is determined by the plant and a host of environmental factors. Within the successful nodule, rhizobia are surrounded by a peribacteroid membrane where they differentiate into bacteroids and begin fixing atmospheric nitrogen (Hedtke and Newcomb, 1977). In many regions of the world where common beans are grown, nitrogen fixation is limited by unfavorable soil conditions and temperature and water stress. Temperature has been reported to affect nodulation, survival and persistence of rhizobial strains in soil. Depending on their natural habitat, tolerance of rhizobia to temperature varies across various strains

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(Mohammadi et al., 2012). Additionally, temperatures greater than 40 C inhibit infection of root hairs by rhizobia (Hungaria and Franco, 1993) and nitrogen fixation (Long, 2001). Moreover, nitrogenase activity was lower when common beans formed nodules at 35 C (Piha and Munns, 1987). In their study, Michiels et al. (1994) attributed ineffectiveness of rhizobium symbiosis at temperatures greater than 40 C to deletion of the symbiotic plasmids. Low temperatures also limit nitrogen assimilation (Gibson, 1965), photosynthate assimilation, malate production, and nitrogenase activity (Bordeleau and Prevost, 1994).

Moisture stress has greater impact on

nodulation and nodule activity than on plant root and shoot metabolism (Albrecht et al., 1984). Rhizobial strains vary in their adaptability to water stress. Busse and Bottomley (1989) demonstrated growth rate of Rhizobium meliloti slowed and cell morphology became irregular as water potential of the growth media decreased from −0.15 and −1.5 MPa. Low soil water potentials inhibit nodulation and growth of rhizobia. For example, nodulation in subterrainian clover (Trifolium subterraneum) was inhibited at soil water potential below -0.8MPa; growth of Rhizobium leguminosarum bv. Trifolii was impaired at water potentials below -1.0MPa (Leung and Bottomley, 1994). Ramos et al. (1999) reported lower leghemoglobin (Lb) levels in nodules, and loss of nodule nitrogenase and sucrose synthase activities due to water stress. Indirect effects of reduced root growth in dry soils decreases the number of potential infection sites for rhizobia (Bordeleau and Prevost, 1994). Also, accumulation of ureides in leaf and nodule tissues brought about by water stress has been associated with inhibition of nitrogen fixation (Sinclair and Serraj, 1995). Zahran (1999) reported the toxic effect of soil salinity on plant growth especially in arid and semi-arid regions. Saline soils in arid areas are dominated by Na+ and Cl- . Na+ displaces Ca2+ from root membranes of non-halophytes altering their functionality. Legumes and rhizobia can be extremely sensitive to low soil pH (Corea and Barneix, 1997). The optimum pH range for rhizobial growth is between 6.0 and 7.0 below which very few rhizobia are capable of growing (Brockwell et al., 1991; Graham et al., 1994). Very few species survive in soils at pH below 5.0 (Mohammadi et al., 2012).

McKay and Djordjevic (1993) demonstrated a reduction in

production and excretion of nodulation factors in R. leguminosarum bv. Trifolii at pH below 5.0. Furthermore, Zahran (1999) attributed failure of nodulation at pH below 5.0 to failure of rhizobia to survive in such acidic soils. More than 23% of the soils for bean production in East Africa

have pH at or below 5.0 (Beebe et al., 2012).

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Soil nutrient concentrations also affect legume hosts, soil bacteria, and their symbiotic relationships (Mohammadi et al., 2012). Low phosphorus levels in acidic soils, for example, delay nodulation and infection of primary roots (Mullen et al., 1988). Aluminum and manganese toxicity and low levels of calcium inhibit growth of rhizobia and nodulation at soil pH less than 5 (Alva et al., 1990; Bordeleau and Prevost, 1994; Campo, 1995). During the initial stages of symbiosis, rhizobia secrete calcium-dependent proteins to promote attachment onto root hair surfaces (Gage, 2004). The low concentration of calcium ions in acidic soils, however, inhibits attachment of rhizobia onto root surfaces (Cartano-Anolles et al., 1989). Numerous studies have documented the negative impact of high nitrate concentration in soil on nodulation and nodule weight (Malik et al., 1987; Muller and Pereira, 1995; Abaidoo et al., 1990; Streeter, 1984). Loss of nodule permeability and depletion of internal O2 concentration has been implicated as a common factor underlying the loss of N fixation under unfavorable environmental conditions. Oxygen concentration less than 40 µM in infected nodule cells is required for active nitrogenase. Oxygen levels around the bacteroids are kept low by oxygen binding leghemoglobin (Lb) within the nodules, which gives them their distinctive pink color (Dakora et al., 1991). The nodule is made up of few and small intercellular spaces and is surrounded by a diffusion barrier both of which limit entry of oxygen into the infected cells (Witty et al., 1987; Walsh et al., 1989). Nodule cytochrome oxidase, the terminal electron acceptor in oxidase phosphorylation, has a high affinity for oxygen, which also promotes low levels of oxygen in nodules (Preisig et al. 1996). Plant intake of high levels of nitrate reduces permeability of nodules to oxygen diffusion decreasing oxygen available for respiration (ArreseIgor et al., 1990; Minchin et al., 1986) resulting in reduction of ATP and NAD(P)H which are required for nitrogenase activity (Sprent et al., 1987). In saline soils, permeability of nodules to oxygen is reduced resulting in high levels of oxygen accumulating in nodules (Delgado et al., 1994). High oxygen levels in infected cells of nodules lowers nitrogenase enzyme activity. Limited supply of water also increases resistance of nodules to O2 diffusion, limiting bacteroid respiration and inducing nodule senescence (Walsh, 1995; González et al., 1995, 1998 and Galvez et al., 2005).

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Despite the numerous factors that compromise nodulation and nitrogen fixation, legumes generally assimilate 50 to 70% of their nitrogen via symbiotic nitrogen fixation (Philips and Bennett, 1978; Vance, 1997). Relative to other legumes, however, common beans (Phaseolus vulgaris L.) are poor nitrogen fixers (Westermann et al., 1981 and Bliss, 1993; MartinezRomero, 2003). Yet there are reports of some varieties of Phaseolus vulgaris L. and strains of rhizobia exhibiting high rates of nitrogen fixation (Bliss, 1993; Hardarson and Atkins, 2003). And there has been considerable investment in improving the capacity of Phaseolus vulgaris L. for fixing atmospheric nitrogen (Peña-Cabriales and Castellanos, 1993; Buttery and Park, 1993; Hardarson et al., 1993; Kipe-Nolt and Giller, 1993; Devi et al., 2013). This includes development of complex inoculant formulations designed to improve early plant growth, biological nitrogen fixation,

and

overall

plant

productivity (Noel

et

al.,

1996).

Becker

(beckerunderwood.com), for example, manufactures as series of BioStacked

Underwood

inoculants that

contain compounds and biological agents to promote nodulation and improve nitrogen fixation. Because the number of nodules formed could set an upper limit to plant supported BNF, particular attention has been paid to understanding the host plant’s role in regulating nodulation through autoregulation. Autoregulation is a process within legume plants which controls the number of nodules formed on roots that are already supporting nodules (Bhatia et al., 2001). Evidently, once a critical number of nodules are formed, a signal is produced that suppresses formation of new nodules (Renee and Bohlool, 1984; Olsson, 1989 and Caetano-Anollés and Gresshoff, 1991). Numerous studies have been conducted to determine the origin and control of the autoregulatory signal (Lee et al., 1991; Sheng and Harper, 1997; Dasharath and VandenBosch, 2005 and Sayuri et al., 2008). In an attempt to discern the source of the autoregulatory signal, scientists have used Ethyl Methane Sulphonate (EMS) mutagenesis in Phaseolus vulgaris L., N-nitroso-N-methylurea (NMU) mutagenesis in soybeans, and Gamma rays in chickpeas (Cicer arietinum) to develop nodulation mutants, including non-nodulating, ineffective nodulating, and super-nodulating (with autoregulation deficiencies) lines (Park and Buttery, 1988; Gremaud and Harper, 1989; Melki, et al., 2012). These mutants have revealed both root and shoot tissues have major roles in control of nodulation and nitrogen fixation (Buttery and Park, 1990; Hamaguchi et al., 1993; Harper et al., 1997 and Abd-Alla, 2011). Efforts using these lines to improve symbiotic nitrogen fixation have been directed towards

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developing breeding lines capable of nodulation and nitrogen fixation at high soil nitrate or ammonium concentrations (Herridge and Rose, 2000). There is great interest in understanding how nitrogen fixed by BNF is converted to forms that can easily be transported to other plant parts for use in growth and development. Some leguminous plants translocate fixed N2 as amides asparagine and glutamine (known as amidic plants); others such as common bean produce and translocate ureides allantoin and allantoate (known as ureidic plants) (Thomas et al., 1980; Tajima et al., 2004; Todd et al., 2006; Alamillo et al., 2010; Díaz-Leal et al., 2012). Because ureides have a highly condensed N: C ratio of 1:1, it is considered more energetically favorable in terms of g C g -1 fixed N to transport N as ureides from the nodules to the shoot (Atkins, 1991; Todd et al., 2006). Schubert (1986) demonstrated that only half as much energy (ATP) was required to produce ureides than to produce amides. Using less carbon and ATP to transport N has given ureidic legumes an advantage over amidic legumes under drought conditions (Todd et al., 2006).

Whether transporting N as ureides

provides a competitive advantage for common beans is not known. Ureides (allantoin and allantoate) are synthesized primarily in root nodules via purine biosynthesis and oxidation (Smith and Atkins, 2002; Boldt and Zrenner 2003; Zrenner et al. 2006; Todd et al., 2006; Alamillo et al., 2010). Xanthine dehydrogenase (XAN) in infected cells converts the purines to uric acid, which is oxidized to allantoin in adjacent uninfected cells (Smith and Atkins, 2002; Todd et al., 2006). Allantoinase reduces allantoin to allantoate (Webb and Lindell, 1993; Todd et al., 2006; Díaz-Leal et al., 2012); allantoate is transported in the xylem to the leaves and ultimately hydrolyzed to NH4+ and CO2. In soybean, the proposed mechanism for allantoate degradation is via allantoate amidohydrolase to NH4+, CO2, and ureidoglycolate (Tajima et al., 2004; Todd et al., 2006). Degradation of ureidoglycolate to glyoxylate and urea is catalyzed by ureidoglycolate urea-lyase (Todd and Polacco 2004; Todd et al., 2006; Witte, 2011; Alamillo et al., 2010). Urea is eventually metabolized into NH4+ and CO2; the resulting ammonia is then incorporated into organic acids needed for growth (Todd et al., 2006). The specific pathway to NH4+ and CO2 from allantoate in Phaseolus, however, is still under scrutiny (Díaz-Leal et al., 2012). Nodule biosynthesis of purines is not the only source of ureides, as non-nodulating legumes have been reported to accumulate ureides (Thomas et al., 1980; Alamillo et al., 2010

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and Díaz-Leal et al., 2012). Díaz-Leal et al. (2012) showed accumulation of high levels of ureides in non-nodulated Phaseolus vulgaris L. at early stages of development and at the onset of the reproduction. The accumulation was attributed to remobilization of nitrogen from older vegetative tissues. Furthermore, Alamillo et al. (2010) showed that non-nodulating Phaseolus vulgaris L. lines accumulated ureides under drought stress conditions. They concluded ureides were recycled from tissues undergoing senescence or from the catabolism of purines (Thomas et al., 1980). If so, total tissue ureide levels in plant tissues could include contributions from N2 fixation, purine catabolism, and remobilization (Aveline et al., 1995). It is essential then to include comparisons against non-nodulating lines when evaluating common bean germplasm for superior BNF or proportion of tissue N derived from N2 fixation. The impact of drought stress on N2 fixation has been associated with concentration of manganese (Mn) in leaves and accumulation of ureides in aerial plant tissues and nodules (Serraj et al., 1999; Purcell et al., 2000). Todd and Polacco (2004) illustrated in soybean the dependence of enzymes responsible for ureide catabolism on tissue Mn concentration. Furthermore, Purcell et al. (2000) showed that high concentration of Mn in plant tissues especially under drought conditions was correlated with a lower concentration of ureides in leaves. Lines with lower leaf Mn levels maintained higher rates of N2 fixation. Vadez and Sinclair (2001) also associated an accumulation of ureides in leaf tissues with depressed N2 fixation. They suggested selection of lines with a high rate of ureide degradation coupled with Mn-independent ureide degradation would improve N2 fixation under dry conditions. Similar studies have not been conducted in Phaseolus. Numerous studies have documented genotypic variability for symbiotic N2 fixation in Phaseolus vulgaris (St. Clair and Bliss 1991; Miranda and Bliss, 1991; Bliss, 1993; Hardarson et al., 1993; Peña-Cabriales and Castellanos, 1993; Kipe-Nolt, 1993 and Vadez et al., 1999; Devi et al., 2013). For example, Puebla 152 (Barrona et al., 1999) and BAT 477 (Miklas et al., 2006) have been used as parental lines to select progeny for improved BNF. Some of the plant characteristics that have been used to determine superiority for N2 fixation are early plant vigor, total biomass accumulation, grain yield, nodulation, total N, and ureide content in various plant tissues (Pedalino et al., 1992; Kipe-Nolt and Giller, 1993; Peña-Cabriales and Castellanos, 1993). Because ureides are a known product of N2 fixation, ureide accumulation in plant tissues

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has been suggested as an indirect indicator of the rate of nitrogen fixation in legumes (Purcell et al., 2000 and King and Purcell, 2005). If so, efforts to understand ureide accumulation, partitioning, and regulation in lines with varying characteristics related to N2 fixation would be beneficial in improving BNF in common beans. The objectives of this study were to identify phenotypic traits related to variation in BNF and N accumulation from BNF, and to determine if these traits could be transferred between genotypes. To address these objectives, ureide and N content, ureide and N concentration, biomass accumulation, and nodulation were evaluated in selected nodulated and non-nodulated Phaseolus vulgaris lines.

Hypotheses 1. Accumulation of ureides in aerial plant tissues could be used to identify bean genotypes superior for BNF. 2. Shoot and root control of nodulation can be transferred to alter ureide and total nitrogen accumulation and partitioning in common bean.

Two greenhouse studies were conducted to test these hypotheses. In both studies, Phaseolus vulgaris L. lines were selected based on differences in their phenotypic characteristics associated with N2 fixation. Plants were cultivated under controlled conditions favoring nodulation and active nitrogen fixation and harvested during flowering/early pod formation. Stem, leaf, petiole, pod, and root tissues were analyzed for biomass, ureide concentration and content, nitrogen concentration and content, and the number of nodules. The primary objectives of the first study were to identify phenotypic traits consistently associated with variation in capacity for BNF and to identify individual or sets of traits useful for selecting genotypes with superior capacity for N assimilation from BNF. The objectives of the second study was to determine shoot and/or root control of ureide accumulation and partitioning among four bean genotypes, and the extent to which that control could be transferred via intra-species grafting.

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Expected Outcomes Because N2 fixation and ureide synthesis take place in nodulated plants, there should be predictable relationships between nodulation, N accumulation, ureide accumulation, and percent N derived from BNF. These relationships should be evident and consistent among genotypes with varying capacity for nodulation. Grafting super-nodulating shoots onto normally nodulating and non-nodulating root stocks is expected to increase nodulation resulting in increased sites for N2 fixation and ureide synthesis. Also, greater nodulation is expected to promote and increase in tissue ureide content and percent N derived from N2 fixation. If so, these phenotypic traits will be particularly useful for selecting genotype with superior BNF and N assimilation.

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REFERENCES Abaidoo, R. C., T. George, B. B. Behlol, and P. W. Singleton. 1990. Influence of elevation and applied nitrogen on rhizosphere colonization and competition for nodule occupancy by different rhizobial strains on field grown soybean and common bean. Canadian J. Microbiol. 36: 92-96. Abd-Alla, M. H. 2011. Nodulation and nitrogen fixation in interspecies grafts of soybean and common bean is controlled by isoflavonoid signal molecules translocated from shoot. Plant, Soil, Environ. 57: 453-458. Atkins, C. A. 1991. Ammonia assimilation and export of nitrogen from the legume nodule. Studies in Plant Sci. 1: 293-319. Albrecht, S. L., J. M. Bennett, and K. J. Boote. 1984. Relationship of nitrogenase activity to plant water stress in field-grown soybeans. Field Crops Research 8: 61-71. Alamillo, M. J., J. L. Díaz-Leal, M. V. Sánchez-Moran, and M. Pineda. 2010. Molecular analysis of ureide accumulation under drought stress in Phaseolus vulgaris L. Plant Cell & Environ. 33: 1828-1837. Alva, A. K., C. J. Asher, and D. G. Edwards. 1990. Effect of solution pH, external calcium concentration and aluminum activity on nodulation and early growth of cowpea. Australian J. Agric. Res. 41: 359-365. Arrese-Igor, C., J. I. García-Plazaola, A. Hernández, and P. M. Aparicio-Tejo. 1990. Effect of low nitrate supply to nodulated lucerne on time course of activities of enzymes involved in inorganic nitrogen metabolism. Physiologia Plantarum 80: 185-190. Aveline, A., Y. Crozat, X. Pinochet, A. M. Domenach, and J. C. Cleyet-Marel. 1995. Early remobilization: a possible source of error in the ureide assay method for nitrogen fixation measurement by early maturing soybean. Soil Science Plant Nutrition 41: 737-751. Barrona, J. E., R. J. Pasini, D. W. Davis, D. D. Stuthman, and P. H. Graham. 1999. Response to selection for seed yield and nitrogen (N2) fixation in common bean (Phaseolus vulgaris L.). Field Crops Research 62: 119-128.

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Beebe, S., I. M. Rao, C. Mukankusi, and R. Buruchara. 2012. Improving resource use efficiency and reducing risk of common bean production in Africa, Latin America and the Caribbean. In: C. Hershey (ed), Issues in Tropical Agriculture. I. Eco-efficiency: From Vision to Reality. CIAT, Cali, Colombia (In Press). Bhatia, C. R., K. Nichterlein, and M. Maluszynski. 2001. Mutations affecting nodulation in grain legumes and their potential in sustainable cropping systems. Euphytica 120: 415-432. Bliss, F. A. 1993. Breeding common bean for improved biological nitrogen fixation. Plant Soil 152: 71–79. Boddey, R. M., J. C. De Moraes Sá, B. J. Alves, and S. Urquiaga. 1997. The contribution of biological nitrogen fixation for sustainable agricultural systems in the tropics. Soil Biology Biochemistry 29: 787-799. Boldt, R., and R. Zrenner. 2003. Purine and pyrimidine biosynthesis in higher plants. Physiologia Plantarum 117: 297-304. Bordeleau, L. M., and D. Prevost. 1994. Nodulation and nitrogen fixation in extreme environments. Plant Soil 161:115-125. Brockwell, J., A. Pilka, and R. A. Holliday. 1991. Soil pH is a major determinant of the numbers of naturally-occurring Rhizobium meliloti in non-cultivated soils of New South Wales. Australian. J. Exp. Agric. 31:211-219. Burdman, S., J. Kigel, and Y. Okon. 1997. Effects of Azospirillum brasilense on nodulation and growth of common bean (Phaseolus vulgaris L.) Soil Biology Biochemistry 29: 923-929. Busse, M. D., and P. J. Bottomley. 1989. Growth and nodulation responses of Rhizobium meliloti to water stress induced by permeating and non-permeating solutes. Applied Environ. Microbiol. 55: 2431-2436. Buttery, B. R., and S. J. Park. 1990. Effects of nitrogen, inoculation and grafting on expression of supernodulation in a mutant of Phaseolus vulgaris L. Canadian J. Plant Science 70: 375-381.

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Buttery, B. R., and S. J. Park. 1993. Characterization of some non-fixing mutants of common bean (Phaseolus vulgaris L.). Canadian J. Plant Science 73: 977-983. Caetano-Anolles, G., and P. M. Gresshoff. 1991. Plant genetic control of nodulation. Annual Reviews in Microbiol. 45: 345-382. Caetano-Anolles, G., A. Lagares, and G. Favelukes. 1989. Adsorption of Rhizobium meliloti to alfalfa roots: dependence on divalent cations and pH. Plant Soil 117: 67-74. Campo, R. J. 1995. Residual effects of aluminum on Bradyrhizobium japonicum in defined medium and soil solution. Ph.D. Thesis. The University of Reading, Reading. Correa, O. S., and A. J. Barneix. 1997. Cellular mechanisms of pH tolerance in Rhizobium loti. World J. Microbiol. Biotechnol. 13:153–157. Dakora, F. D., C. A. Appleby, and C. A. Atkins. 1991. Effect of pO2 on the formation and status of leghemoglobin in nodules of cowpea and soybean. Plant Physiology 95: 723-730. Dasharath, P. L., and K. A. VandenBosch. 2005. Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity. J. Exp. Botany 56: 1643-1650. Delgado, M. J., F. Ligero, and C. Lluch. 1994. Effects of salt stress on growth and nitrogen fixation by pea, faba-bean, common bean and soybean plants. Soil Biology Biochemistry 26: 371-376. Devi, M. J., T. R. Sinclair, S. E. Beebe, and I. M. Rao. 2013. Comparison of common bean (Phaseolus vulgaris L.) genotypes for nitrogen fixation tolerance to soil drying. Plant Soil 364: 29–37. Díaz-Leal, J. L., G. Gálvez-Valdivieso, J. Fernández, M. Pineda, and J. M. Alamillo. 2012. Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in Phaseolus vulgaris L. J. Exp. Botany 63: 4095-106. Gage, D. J. 2004. Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes. Microbiol. Molecular Biology 68: 280-300.

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N-determined

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Streeter, J. G. 1984. Nitrate inhibition of legume nodule growth and activity. Plant Physiol. 77: 325-328. Tajima S., M. Nomura, and H. Kouchi. 2004. Ureide biosynthesis in legume nodules. Frontiers Bioscience 9:1374-1381. Thomas, R. J., U. Feller and K. H. Erismann. 1980. Ureide metabolism in non-nodulated Phaseolus vulgaris L. J. Exp. Botany 31: 409-417. Todd, C. D., and J. C. Polacco. 2004. Soybean cultivars ‘Williams 82’and ‘Maple Arrow’ produce both urea and ammonia during ureide degradation. J. Exp. Botany 55: 867-877. Todd, C. D., P. A. Tipton, D. G. Blevins, P. Piedras, M. Pineda, and J. C. Polacco. 2006. Update on ureide degradation in legumes. J. Exp. Botany 57: 5-12. Vadez, V., and T. R. Sinclair. 2001. Leaf ureide degradation and N2 fixation tolerance to water deficit in soybean. J. Exp. Botany 52: 153-159. Vadez, V., J. H. Laso, D. P. Beck, and J. J. Drevon. 1999. Variability of N2-fixation in common bean (Phaseolus vulgaris L.) under P deficiency is related to P use efficiency. Euphytica 106: 231– 242. Vance, C. P. 1997. Enhanced agricultural sustainability through biological nitrogen fixation. NATO ASI Series G, Ecological Sciences 39: 179-186. Walsh, K. B. 1995. Physiology of the legume nodule and its response to stress. Soil Biology Biochemistry 27: 637–55. Walsh, K. B., M. J. Canny, and D. B. Layzell. 1989. Vascular transport and soybean nodule function: II. A role for phloem supply in product export. Plant, Cell, Environ. 12: 713723. Webb, M. A., and J. S. Lindell. 1993. Purification of allantoinase from soybean seeds and production and characterization of anti-allantoinase antibodies. Plant Physiol. 103: 12351241.

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CHAPTER 2. GENOTYPIC VARIATION IN UREIDE CONTENT IN PHASEOLUS VULGARIS L. 1

Mercy K. Kabahuma1 and Mark E. Westgate1 Iowa State University, Ames, Iowa, College of Agriculture and Life Sciences Department of Agronomy A paper to be submitted to Journal of Crop Science

Abstract Ureidic legumes, such as common beans (Phaseolus vulgaris L.), transport N derived from N2 fixation in root nodules to the shoot as ureides. As such, ureide accumulation in aerial plant tissues could be used to identify bean lines with superior biological nitrogen fixation (BNF). There has been limited characterization, however, of bean genotypes for phenotypic traits associated with variation in BNF. The objective of this study, therefore, was to identify phenotypic traits of common bean consistently associated with variation in BNF. Lines selected for study and their phenotypic characteristics included: Puebla which exhibits rapid biomass and N accumulation, Eagle which has small root system and low percent N from BNF, the supernodulating R32, and non-nodulating R99. Plants were cultivated under controlled conditions favoring nodulation and active nitrogen fixation and harvested during flowering/early pod formation. Plants were analyzed for root, stem, leaf, petiole and pod biomass, ureide concentration, nitrogen concentration, and nodule numbers. There was significant variation in ureide accumulation across plant tissues and genotypes. The nodulated lines (R32, Puebla, and Eagle) accumulated more ureide and N than the non-nodulated line R99. Genotypic rankings for ureide content (Eagle ~ R32 > Puebla > R99), total N (Puebla ~ Eagle ≥ R32 ≥ R99), biomass (Puebla ≥ Eagle ≥ R32 ~ R99), nodule number (R32 > Puebla > Eagle > R99) were not consistently related. Ureide concentrations also varied among tissues and were not consistently related with tissue N values. Leaves accumulated the greatest biomass and total N and could provide information needed to select for high BNF.

Puebla, which had moderate nodule

numbers and lowest tissue ureide concentration, derived the greatest percentage of plant N from N2 fixation. These results indicate a combination of phenotypic traits should be evaluated when selecting common bean germplasm for improved capacity for BNF. Moderate nodule number,

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leaf ureide content, and total biomass at flowering were consistent with greater BNF. Nodule effectiveness also should be considered for increasing %N derived from N2 fixation.

Introduction Legumes are noted for their ability to fix atmospheric dinitrogen through symbiotic relationships with nitrogen-fixing bacteria (Winkler et al., 1988). Furthermore, ureidic legumes transport nitrogen derived from nitrogen fixation to shoots in form of ureides (allantoin and allantoic acid) (Boldt and Zrenner, 2003; Todd and Polacco, 2004; Todd et al., 2006 and Zrenner et al., 2006). However, ureides are also derived from remobilized N in older vegetative tissues (Díaz-Leal et al., 2012) or recycled from tissues undergoing senescence (Alamillo et al., 2010). Numerous studies have focused on establishing the relationship between tissue ureide concentration and nodule capacity for nitrogen fixation. For example differences in response of N2 fixation to drought associated with ureide accumulation have been demonstrated in common beans (Phaseolus vulgaris L.) (Serraj and Sinclair, 1998), soybeans (Glycine max L.), cowpeas (Vigna unguiculata L.), peanuts (Arachis hypogaea L.), and chickpeas (Cicer arietinum L.) (Sinclair and Serraj, 1995). Low ureide accumulation in aerial plant tissues has been associated with less sensitivity of N2 fixation to drought (Sinclair and Serraj, 1995 and 1998). Alamillo et al. (2010), however, explored ureide accumulation in common bean tissues under drought stress conditions and reported a lack of relationship between ureide accumulation in drought-stressed tissues and ureide biosynthesis in nodules and nitrogen fixation. Several studies have revealed genetic variability in N2 fixation activity and total fixed nitrogen in common bean. Several cultivars were reported to accumulate high percentages of N from N2 fixation under water deficit conditions (Hardarson et al., 1993 and Peña-Cabriales and Castellanos, 1993). Additionally, superiority in symbiotic nitrogen fixation under phosphorus deficient conditions has been observed in some cultivars (Vadez et al., 1999). Therefore, cultivars of common bean with superior capacity for nitrogen fixation are available for selection and breeding purposes. Great value has been placed on identifying measurable and inheritable traits associated with greater capacity for N2 fixation (Bliss, 1993). In this respect, Miranda and Bliss (1991) used total seed N to select lines with greater N fixation when grown on N-deficient soils. Furthermore, St. Clair and Bliss (1991) used

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N dilution to identify progeny lines with

superior N2 fixation from a cross of high and low N-fixing parents.

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Since ureides are the primary transport product of nitrogen fixation from nodules (King and Purcell, 2005), selecting for genotypes with a greater capacity for ureide accumulation in shoot tissues could be useful as a selection strategy to improve N assimilation. It is not clear, however, whether this phenotypic characteristic might be correlated with greater N2 fixation and N accumulation in common bean. Therefore, the objectives of this study were: (1) to identify phenotypic traits of common bean consistently associated with variation in capacity for BNF, and (2) to identify individual or sets of traits useful for selecting genotypes that are superior in assimilating N from BNF.

Materials and Methods Plant Materials and Growth Conditions Four common bean genotypes (R99, R32, Puebla 152, and Eagle) were selected for this study (provided by Dr. Karen Cichy, USDA-ARS, East Lansing Michigan). The main criterion for selection was variability in biological nitrogen fixation. R99 is a non-nodulation mutant (Park and Buttery, 1988 and 2006), while R32 is a nitrate-tolerant super-nodulating mutant (Park and Buttery, 1989; Buttery and Park, 1990, 1993). R99 and R32 genotypes were both developed in Canada through chemical (Ethyl Methane Sulphonate) mutagenesis of OAC Rico (Park and Buttery, 1988, 1989, 2006; Buttery and Park, 1990, 1993). Eagle is an Andean snap bean commercial cultivar developed in 1971 by Seminis Vegetable Seeds (Navarro et al., 2007). Puebla 152 is a black bean landrace from Mexico known for its superiority in symbiotic nitrogen fixation (Barrona et al., 1999). Because R99 is non-nodulating, it was used as a control to estimate BNF against R32, Puebla and Eagle. Puebla, Eagle, R32 and R99 seeds were germinated on paper towels moistened with sterile distilled water in the dark at 20 C. After two days, germinated seeds were transferred to hydrated peat pellets (Jiffy Products (NB) Ltd., Canada) for five days in a controlled environment chamber at 24 C, light photon flux of 150-200 µmol-2s-1 PAR and 16h photoperiod. Vigorous seedlings with healthy unfolded primary leaves were transplanted to 1-L free draining pots filled with commercial potting soil mixture (Sun Gro Horticulture Distribution Inc., Bellevue, WA containing 15-25% Canadian spagum peat moss, composted bark, perlite,

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vermiculite, dolomite lime and blue chip) and transferred to a greenhouse set at 25 C and 16h photoperiod. One week after transplanting, pots were inoculated with a peat-based BioStacked® inoculant (Becker Underwood, Ames, IA) in a water suspension according to manufacturer’s instructions. A week after inoculation, 25 mL of fertilizer solution containing 100ppm N (1.1% ammoniacal N, 11.8% nitrate N and 2.1% urea N; Peter’s Excel Water Soluble Fertilizer, Everris Inc., Dublin, OH) was added to each pot. This amount of N was enough to enhance growth in R99 (non-nodulating) without inhibiting nodulation in R32, Puebla and Eagle. Three replicate pots of each line each with a single plant were arranged in a completely randomized design. At flowering/early pod set, plants were harvested and dried to measure plant biomass, nodule number, tissue ureide concentration, and N concentration. Leaves, petioles, stems and pods (except Puebla which lacked pods at the time of harvesting) and roots including nodules were dried at 60°C, weighed, and ground to pass through a 1-mm sieve. Samples were stored in tightly sealed glass vials at 20 C until they were analyzed for ureides and N. Nodule Counts Shoots were detached at the soil surface. Roots and soil were removed intact from pots and soaked in water to facilitate removal of soil and minimize loss of root nodules. Cleaned roots with nodules were stored in plastic bags at 4 C until nodules were counted manually. Nodules were not examined for color to determine whether or not they were active. Nitrogen and Ureide Analysis Tissue N concentration was measured in ~125 mg of stems, leaves, petioles, pods and roots as percent (%) N using Dumas complete combustion technique (Costech Analytical Technologies, Inc., Valencia, CA). Ureide concentration was determined according to Young and Conway (1942) with modifications (de Silva et al., 1996). Ureides were extracted from 25 mg of dried, ground tissue (leaves, petioles, stem, pods and roots). Samples were homogenized in 1 mL 0.2N sodium hydroxide (NaOH), boiled at 100°C for 30 min, cooled and centrifuged at 10,000g for 5 min. The supernatant was pipetted into 1.5 mL microfuge tubes and stored at 10 C until ureide determination. Tissue extract was boiled in 0.5N NaOH for 8 min, cooled, boiled in 0.74N

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hydrochloric acid (HCl) and 0.33% phenylhydrazine-HCl for 2 min, and cooled. Color was developed with the addition of concentrated HCl and 1.67% potassium ferricyanide (KFeCN). Ureide concentration was determined spectrophotometrically (Phoenix Equipment, Inc., Rochester, NY) against an allantoin standard at 520 nm within 15 min of addition of KFeCN. Ureide concentrations are presented in µmol per g dry weight, biomass in g, ureide content in µmoles, N concentration in mg per g dry weight tissue N and total N in mg. Statistical Analysis Single plants from each genotype were considered replicates with five tissues (stems, leaves, petioles, pods and roots). The results represent the means ± SE of three plants. Data were analyzed by ANOVA and means were compared using Fisher’s LSD test at a probability level of 0.1 (Williams and Abdi, 2010).

Results and Discussion Comparison between Genotypes To study the differences in nitrogen assimilation, ureide concentration and content was determined in stems, leaves, petioles, pods and roots of four genetically different common bean genotypes. Plants were cultivated under conditions favoring nodule formation and active nitrogen fixation. Plants were harvested at flowering/early pod set to ensure differences in BNF and total N assimilation were well established. Genotypes were ranked for nodule number, biomass, tissue ureide concentration, total ureide content, tissue nitrogen concentration, and total N with particular emphasis on identifying relationships between these phenotypic characteristics associated with BNF. Genetic Differences in Nodule Number The number of nodules per plant ranged from 0 to more than 2400 (Fig. 1). As expected, no (or few) nodules formed on R99, the non-nodulating line. Eagle supported a relatively small number of nodules per plant, while Puebla plants carried more than 300 nodules. Roots of the

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super-nodulating line R32 had a

well over

200 times more nodules than Eagle. Biomass Accumulation Genotype ranking for total biomass accumulation was Puebla ≥ Eagle ≥ R32 ~ R99 (Fig. 2). The greatest accumulation of biomass across R32, R99, Puebla and Eagle tissues was measured in leaves and was closely followed by stems. Leaves accounted for approximately 46% of the total biomass in R32, approximately 41% of total biomass in R99, approximately 56% of Puebla’s total biomass and approximately 36% of Eagle’s total biomass.

Puebla

accumulated significantly greater leaf and stem biomass than R32, R99 and Eagle (Table 1). This might reflect the late reproductive and longer flowering phase of this line, which had not as yet set pods at the time of harvesting (40 days after planting). It was interesting to note the supernodulating line R32 accumulated about the same biomass as the non-nodulating mutant R99 (Fig. 2). Evidently, R99’s capacity to absorb and assimilate mineral N was sufficient to support biomass accumulation. It is possible that the large number of nodules initiated by R32 diverted assimilates that would otherwise be used for shoot biomass accumulation (Voisin et al., 2003). This conclusion is supported by Day et al., (1986) who reported accumulation of dry matter in the super-nodulating mutant (nts382) was less than that in its normally-nodulating parent (cv. Bragg). Biomass accumulation varied across genotypes (Table 1). Among stems and leaves, genotypic ranking was Puebla > R32 ~ R99 ~ Eagle and Puebla ≥ R99 ~ Eagle ≥ R32 in petioles. There was no significant difference in pod biomass among R32, R99 and Eagle and root biomass accumulation across the four genotypes. Additionally, Puebla was more vegetative with greater growth compared to R32, R99 and Eagle. These results show that R32, Eagle and R99 were further along in development than Puebla. Ureide Concentration Ureide concentration varied by tissue (Fig. 3). In R32, the ureide concentration in shoot tissues was six or more times greater than the concentration in roots. However, ureide concentration in roots of R99 was about 4 times more than the concentration in its shoot tissues. Furthermore, Puebla registered the greatest concentration of ureides in roots (16.8 µmol/gDW)

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followed by 13.1 µmol/gDW in leaves and 11.5 µmol/gDW in stems and the least ureide concentration was noted in petioles. With 87.4 µmol/gDW, stems accounted for the greatest concentration of ureides in Eagle. Higher ureide concentration levels in stems and petioles of R32 than Puebla could reflect earlier translocation of ureides from roots to shoots of R32 since Puebla lacked pods at the time of harvesting. Ranking also varied with genotype (Table 2). For stems and pods, Eagle ranked first followed by R32 and lastly Puebla and R99. Ureide concentration in leaves and petioles, however, was dominated by R32 with 39.1 µmol/gDW in leaves and 52.4 µmol/gDW in stems. Ureide concentration ranking in roots was R99 ≥ Puebla ≥ Eagle > R32. All R99 tissues had detectable levels of ureides with the concentration in roots among the highest of all (Table 2). Nodule biosynthesis is not the only source of ureides in ureidic legumes especially in non-nodulating common bean. Remobilized N in older vegetative tissues (DíazLeal et al., 2012) and nucleic acids or proteins in tissues experiencing drought-induced senescence (Alamillo et al., 2010) have been reported as potential sources of ureide nitrogen. This was attributable to concentration of ureides in tissues of non-nodulating R99. There was little correspondence between nodulation level and ureide concentrations. Ureide Content Nodulated lines accumulated more ureide than non-nodulated lines on a whole plant basis (Fig. 4). Genotypic ranking for ureide content was Eagle ~ R32 > Puebla > R99. Since plant biomass of R99, Eagle, and R32 were similar, the difference in total ureide content was attributable primarily to N2 fixation in the nodulated lines. Excluding pods from this calculation did not change genotypic ranking. These results therefore, suggest the difference in whole plant ureide content in the nodulated plants Puebla, R32 and Eagle was not due to differences in developmental stage. Ureide content varied by tissue (Table 3). Both tissue biomass and ureide concentration impacted total ureide content, but the relative importance varied with tissue and genotype. The high level of ureide content in leaves of R32 and Puebla was attributable primarily to greater biomass accumulation.

Furthermore, high levels of ureide content in roots of R99 were

associated mainly with the high concentration of ureides. Eagle’s high level of ureide content in

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stems was also attributed mainly to a high ureide concentration. These results underscore the importance of genotypic differences in ureide content and partitioning. Ranking also varied with genotype (Table 3). Variation in development among R32, Eagle, R99 and Puebla may have influenced ureide content of individual plant tissues. Among stem tissues, ranking for genotypes was Eagle > R32 ≥ Puebla ≥ R99. In leaves, the ranking for genotypes was R32 > Puebla > Eagle > R99. Genotypic ranking in petioles, however, was Eagle ≥ R32 ~ Puebla ≥ R99. Furthermore, ranking for genotypes in pods was Eagle ≥ R32 ≥ R99. In roots, ranking was Puebla ~ R99 ≥ Eagle ≥ R32 (Table 3). The higher ureide content level of R32 among leaf tissues and least among root tissues, and Eagle among stem tissues was associated primarily with higher ureide concentration levels in these genotypes. For the most part, Puebla lagged behind Eagle and R32 in genotypic ranking of ureide content. This could be associated with R32 and Eagle being further ahead in development than Puebla therefore translocating ureides to stems and leaves earlier than Puebla. These results are consistent with data reported by Herridge and Peoples (1990) showing early-maturing genotypes had higher levels of relative ureide-N than those of later maturing genotypes. Nitrogen Concentration N concentration varied dramatically among tissues. Leaves had the highest concentration of N across all tissues (Fig. 5). Except among Eagle tissues, leaves were closely followed by roots in ranking. Variation of tissue N concentration also was observed across genotypes (Table 4). Nodulated lines had greater N concentration levels in their tissues than the non-nodulated line. Among stem and pod tissues, Eagle had the highest level of N concentration compared to R32, Puebla and R99. R32 had higher levels of N concentration in roots than Puebla, Eagle and R99. R99 had considerable amounts of N indicating N assimilation from inorganic sources. Nitrogen concentration in roots paralleled nodule number. Total Nitrogen Nodulated lines accumulated more N than the non-nodulated line (Fig. 6). Genotypic ranking for total N accumulation was Puebla ~ Eagle ≥ R32 ≥ R99 on a whole plant basis.

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Nodulated lines accumulated approximately 2.3 times as much N as the non-nodulating line. The difference in N accumulation was attributable primarily to N2 fixation. Tissue N varied within each genotype. However, leaves accumulated the highest N content among tissues of R32, R99, Puebla and Eagle (Table 5). This was attributed primarily to highest concentration level of N and greatest biomass accumulation in leaves. The largest plant Puebla also had the most N in the leaves. Genotypic ranking varied across tissues except in roots where no significant difference was noted in N accumulated (Table 5). Impact of tissue biomass and N concentration on N content varied depending on genotype and tissue. Interestingly, the greatest amount of plant N was associated with low nodule number in Eagle. % N derived from BNF Puebla derived the greatest percentage of N from N2 fixation at 62.2%, followed by Eagle at 56.9%, and R32 at 47.5% (Table 6). This calculation assumes the N accumulated by R99 represents the non-symbiotic accumulation in these lines. R32 had a higher number of nodules than Puebla and Eagle yet derived the least %N from BNF. This result evidently reflects ineffectiveness of most nodules formed on R32 roots. Since Eagle and R32 were slightly ahead of Puebla in development, a greater percentage of N from N2 fixation might have been expected (Diaz-Leal, 2012). These results suggest little impact of variation among these lines in development on the %N they derived from N2 fixation. Nodule Effectiveness Nodule effectiveness based on the amount of total plant N accumulated per nodule varied more than 300-fold among nodulated lines (Table 7). This range was much greater than the 200fold variation in nodule number. Eagle had the most effective nodules by far, while R32 had the least effective nodules. These results indicated a major contribution of BNF to total plant N can be achieved with a moderate number of effective nodules. And supernodulation does not necessarily lead to a high percentage of plant N derived from BNF.

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Conclusions The objectives of our study were: (1) to identify phenotypic traits of common bean consistently associated with variation in BNF, and (2) to identify individual or sets of traits useful for selecting genotypes with superior capacity for N assimilation from BNF. Leaves consistently accumulated the greatest biomass and N across the four genotypes. This result suggests that leaves would be the ideal tissue to use for N uptake and assimilation studies in common bean. Differences in N accumulation generally were associated closely with biomass accumulation. As such, total biomass and N accumulation should be measured together as criteria for selecting for superior BNF. Differences in ureide and N accumulation among common bean genotypes examined were not associated with nodulation. In fact, the greatest accumulation of total N and ureide content was observed in Eagle, which had only 11 nodules per plant, on average. These nodules, however, were highly effective for BNF as nearly 57% of the plant N was derived from them. Nodule effectiveness (plant N/nodule number) should be considered as a trait for increasing %N derived from N2 fixation. Further studies should be conducted with Eagle to determine why it exhibits relatively low BNF under field conditions. In this study, Eagle displayed superior nodule effectiveness, growth, total ureide, and total plant N accumulation. These studies should focus on identifying the physiological and environmental factors that limit BNF in this commercial variety.

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REFERENCES Alamillo, M. J., J. L. Díaz-Leal, M. V. Sánchez-Moran, and M. Pineda. 2010. Molecular analysis of ureide accumulation under drought stress in Phaseolus vulgaris L. Plant, Cell, Environ. 33: 1828-1837. Barrona, J. E., R. J. Pasini, D. W. Davis, D. D. Stuthman, and P. H. Graham. 1999. Response to selection for seed yield and nitrogen (N2) fixation in common bean (Phaseolus vulgaris L.). Field Crops Research 62: 119-128. Bliss, F. A. 1993. Breeding common bean for improved biological nitrogen fixation. Plant Soil 152: 71–79. Boldt, R., and R. Zrenner. 2003. Purine and pyrimidine biosynthesis in higher plants. Physiologia Plantarum 117: 297-304. Buttery, B. R., and S. J. Park. 1990. Effects of nitrogen, inoculation and grafting on expression of supernodulation in a mutant of Phaseolus vulgaris L. Canadian J. Plant Science 70: 375-381. Buttery, B. R., and Park, S. J. 1993. Characterization of some non-fixing mutants of common bean (Phaseolus vulgaris L.). Canadian J. Plant Science 73: 977-983. Day, D. A., H. Lambers, J. Bateman, B. J. Carroll, and P. M. Gresshoff. 1986. Growth comparisons of a supernodulating soybean (Glycine max) mutant and its wild-type parent. Physiologia Plantarum 68: 375-82. De Silva, M., L. C. Purcell, and C. A. King. 1996. Soybean petiole ureide response to water deficits and decreased transpiration. Crop Science 36: 611-616. Díaz-Leal, J. L., G. Gálvez-Valdivieso, J. Fernández, M. Pineda, and J. M. Alamillo. 2012. Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in Phaseolus vulgaris L. J. Exp. Botany 63: 4095-106. Hardarson, G., F. A. Bliss, M. R. Cigales-Rivero, R. A. Henson, J. A. Kipe-Nolt, L. Longer, A. Manrique, J. J. Peña Cabriales, P. A. A. Pereira, C. A. Sanabria, and S. M. Tsai. 1993. Genotypic variation in biological nitrogen fixation by common bean. Plant Soil 152: 59– 70.

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Herridge, D. F., and M. B. Peoples. 1990. Ureide assay for measuring nitrogen fixation by nodulated soybean calibrated by N methods. Plant Physiol. 93: 495-503. King, C. A., and L. C. Purcell. 2005. Inhibition of N2 fixation in soybean is associated with elevated ureides and amino acids. Plant Physiol. 137: 1389-1396. McClure, P. R., D. W. Israel, and R. J. Volk. 1980. Evaluation of the relative ureide content of xylem sap as an indicator of N2 fixation in soybeans: greenhouse studies. Plant Physiol. 66: 720-725. Miranda, B. D., and F. A. Bliss. 1991. Selection for increased seed nitrogen accumulation in common bean: implications for improving dinitrogen fixation and seed yield. Plant Breeding 106: 301–311. Navarro, F., P. Skroch, G. Jung, and J. Nienhuis. 2007. Quantitative trait loci associated with bacterial brown spot in Phaseolus vulgaris L. Crop Science 47: 1344-1353. Park, S. J., and B. R. Buttery. 1988. Nodulation mutants of white bean (Phaseolus vulgaris L.) induced by ethyl methane sulphonate. Canadian J. Plant Science 68:199–202. Park, S. J., and B. R. Buttery. 1989. Inheritance of nitrate tolerant nodulation in EMS induced mutants of common bean (Phaseolus vulgaris L.). J. Heredity 80: 486–488. Park, S. J., and B. R. Buttery. 2006. Registration of ineffective nodulation mutant R69 And nonnodulation Mutant R99 common bean genetic stocks. Crop Science 46: 1415-1417. Peña-Cabriales, J. J., and J. Z. Castellanos. 1993. Effects of water stress on N2-fixation and grain yield of Phaseolus vulgaris L. Plant Soil 152:151–155. McClure, P. R., and W. I. Israel. 1979. Transport of Nitrogen in the xylem of soybean plants. Plant Physiol. 64: 411-416. Serraj, R., and T. R. Sinclair, T. R. 1998. N2 Fixation response to drought in common bean (Phaseolus vulgaris L.). Annals Botany 82: 229-234. Shirtliffe, S. J., J. K. Vessy, B. R. Buttery, and S. J. Park. 1996. Comparison of growth and N accumulation of common bean (Phaseolus vulgaris L. cv. OAC Rico) and its nodulation mutants R69 and R99. Canadian J. Plant Science 76: 73–83.

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Sinclair, T. R., and R. Serraj. 1995. Dinitrogen fixation sensitivity to drought among grain legume species. Nature 378: 344. St. Clair, D. A., and F. A. Bliss. 1991. Intrapopulation recombination for

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dinitrogen fixation ability in common bean. Plant Breeding 106: 215–225. Thomas, R. J., U. Feller, and K. H. Erismann. 1979. The effect of different inorganic nitrogen sources and plant age on the composition of bleeding sap of Phaseolus vulgaris. New Phytologist 82: 657-669. Todd, C. D., and J. C. Polacco. 2004. Soybean cultivars ‘Williams 82’and ‘Maple Arrow’ produce both urea and ammonia during ureide degradation. J. Exp. Botany 55: 867-877. Todd, C. D., P. A. Tipton, D. G. Blevins, P. Piedras, M. Pineda, and J. C. Polacco. 2006. Update on ureide degradation in legumes. J. Exp. Botany 57: 5-12. Vadez, V., J. H. Laso, D. P. Beck, and J. J. Drevon. 1999. Variability of N2 fixation in common bean (Phaseolus vulgaris L.) under P deficiency is related to P use efficiency. Euphytica 106: 231– 242. Van Berkum, P., C. Sloger, D. F. Weber, P. B. Cregan, and H. H. Keyser. 1984. Relationship between ureide N and N2 fixation, aboveground N Accumulation, acetylene reduction, and nodule mass in greenhouse and field studies with Glycine max L. (Merr). Plant Physiol. 77: 53-58. Viosin, A. S., C. Salon, C. Jeudy, and F. R. Warembourg. 2003. Root and Nodule Growth in Pisum sativum L. in Relation to Photosynthesis: Analysis using 13C labeling. Annals Botany 92: 557-563 Winkler, R. G., D. G. Blevins, J. C. Polacco, and D. D. Randall. 1988. Ureide catabolism in nitrogen-fixing legumes. Trends Biochemical Sciences 13: 97-100. Williams, L. J., and H. Abdi. 2010. Fisher’s Least Significant Difference (LSD) Test. In: N. Salkind (ed.), Encyclopedia of Research Design, Sage Publications, Thousand Oaks, CA. Young, E. G., and C. F. Conway. 1942. On the estimation of allantoin by the Rimini-Schryver reaction. J. Biol. Chem. 142: 839-853.

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Zrenner, R., M. Stitt, U. Sonnewald, and R. Boldt. 2006. Pyrimidine and purine biosynthesis and degradation in plants. Annual Review Plant Biology 57: 805-836.

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Figure 1. Number of nodules on R32, R99, Puebla, and Eagle roots of greenhouse-grown bean lines sampled at flowering. The soil was carefully washed from the roots to minimize loss of nodules. Nodules were counted manually. There was no attempt made to distinguish between active/non-active nodules. The values are the mean ± SE of three plants. Means with the same letter above bars indicate nodule number in genotypes is not significantly different at P=0.1 using Fisher’s LSD test.

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Figure 2. Biomass of tissues collected from R32, R99, Puebla, and Eagle bean lines during flowering/early pod set. Tissues were dried at 60 C until a constant weight. Stems (blue bars), leaves (red bars), petioles (green bars), pods (purple bars) and roots (brown bars). The values are the mean ± SE of three plants. Same letters above bars indicate total mass of the genotypes are not significantly different at P=0.1 using Fisher’s LSD test.

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Figure 3. Ureide concentration in plant tissues of R32, R99, Puebla, and Eagle bean lines during flowering/early pod set. Ureide concentrations were determined spectrophotometrically against an allantoin standard. Stems (blue bars), leaves (red bars), petioles (green bars), pods (purple bars) and roots (brown bars). Puebla lacked pods at the time of harvesting. The values are the mean ± SE of three plants. Same letters above bars indicate tissue ureide concentration values within genotypes are not significantly different at P= 0.1 using Fisher’s LSD test.

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Figure 4. Ureide content in tissues of R32, R99, Puebla, and Eagle bean lines during flowering /early pod set. Stems (blue bars), leaves (red bars), petioles (green bars), pods (purple bars) and roots (brown bars). The values are the mean ± SE of three plants. Same letters above bars indicate total ureide content of the genotypes are not significantly different at P=0.1 using Fisher’s LSD test.

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Figure 5. N concentration in tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set. N concentration was determined using Dumas complete combustion technique. Stems (blue bars), leaves (red bars), petioles (green bars), pods (purple bars) and roots (brown bars). The values are the mean ± SE of three plants. Puebla lacked pods at the time of harvesting. Same letters above bars indicate tissue N values within genotypes are not significantly different at P=0.1 using Fisher’s LSD test.

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Figure 6. Total N accumulation in tissues of R32, R99, Puebla, and Eagle bean lines harvested during flowering/early pod set. Total N was determined using Dumas complete combustion technique. Stems (blue bars), leaves (red bars), petioles (green bars), pods (purple bars) and roots (brown bars). The values are the mean ± SE of three plants. Same letters above bars indicate total N of the genotypes is not significantly different at P=0.1 using Fisher’s LSD test.

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Table 1. Biomass of tissues from R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set. They were dried under 60 C until a constant weight was reached to obtain their biomass. The values are the mean ± SE of three plants. Using Fisher’s LSD test, means in rows followed by the same lower case letter are not significantly different at P= 0.1. Means in columns followed by the same Upper Case letter are not significantly different at P= 0.1. Phaseolus vulgaris Lines R32 R99 Puebla Eagle

Stem

Leaves

1.4 b B 1.4 b B 3.3 b A 1.7 bc B

3.6 a 3.2 a 7.4 a 3.3 a

B B A B

Tissue Biomass (g) Petioles Pods 0.3 b 0.7 c 0.9 c 0.8 c

B AB A AB

1.0 b A 1.1 b A 0.0 d B 2.2 ab A

Roots 1.5 b A 1.5 b A 1.6 c A 1.1 bc A

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Table 2. Ureide concentration in tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set. Ureide concentrations were determined spectrophotometrically against an allantoin standard. The values are the mean ± SE of three plants. Puebla lacked pods at the time of harvesting. Using Fisher’s LSD test means in rows followed by the same lower case letter are not significantly different at P= 0.1. Means in columns followed by the same Upper Case letter are not significantly different at P= 0.1. Phaseolus vulgaris Lines R32 R99 Puebla Eagle

Stem

Tissue Ureide Concentration (µmol/gDW) Leaves Petioles Pods

Roots

53.0 a B 4.3 c C 11.5 b C 87.4 a A

39.1 b A 5.1 c D 13.1 b C 19.5 c B

6.0 c C 18.6 a A 16.8 a AB 15.4 c B

52.4 a A 4.1 c C 6.5 c C 19.2 c B

41.2 b AB 7.0 b B 0.0 d C 57.4 b A

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Table 3. Ureide content in tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set. Ureide concentration and biomass were used to calculate ureide content. The values are the mean ± SE of three plants. Using Fisher’s LSD test, means followed by the same lower case letter across the same row are not significantly different at P= 0.1. Means in columns followed by the same Upper Case letter are not significantly different at P= 0.1. Phaseolus vulgaris Lines R32 R99 Puebla Eagle

Stem

Leaves

70.8 b B 5.8 c C 39.3 b BC 149.1 a A

163.5 a A 13.6 b D 96.1 a B 62.5 bc C

Tissue Ureide Content (µmoles) Petioles Pods 13.5 c AB 3.1 d B 5.9 c AB 16.3 c A

39.2 bc AB 7.6 c B 0.0 d C 110.3 ab A

Roots 9.1 c B 27.3 a A 28.0 bc A 16.8 c AB

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Table 4. N concentration in tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set. N concentration was determined using Dumas complete combustion technique. The values are the mean ± SE of three plants. Puebla lacked pods at the time of harvesting. Using Fisher’s LSD test, means in rows followed by the same lower case letter are not significantly different at P= 0.1. Means in columns followed by the same Upper Case letter are not significantly different at P= 0.1. Phaseolus vulgaris Lines R32 R99 Puebla Eagle

Stem

Leaves

16.1 c B 6.0 c C 16.8 c B 23.2 c A

47.1 a A 24.9 a B 39.9 a A 48.8 a A

Nitrogen concentration (mg/gDW tissue N) Petioles Pods 18.4 c AB 9.0 c C 16.2 c B 25.0 c A

0.0 d C 17.3 b B 0.0 d C 36.7 b A

Roots 35.0 b A 18.8 b C 25.4 b B 21.7 c BC

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Table 5. Total N accumulation in tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set. Total N was determined using Dumas complete combustion technique. The values are the mean ± SE of three plants. Using Fisher’s LSD test, means in rows followed by the same lower case letter are not significantly different at P= 0.1. Means in columns followed by the same Upper Case letter are not significantly different at P= 0.1. Phaseolus vulgaris Lines R32 R99 Puebla Eagle

Stem

Leaves

Total Nitrogen (mg) Petioles Pods

21.9 ab BC 8.2 c C 51.9 b A 37.0 bc AB

196.3 a AB 87.2 a C 282.3 a A 163.7 a BC

5.2 b B 9.1 c AB 13.9 b AB 21.4 c A

0.0 c B 9.1 c B 0.0 d B 92.3 b A

Roots 54.1 ab A 35.5 b A 36.9 b A 23.8 c A

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Table 6. Percent plant ureide and N derived from N2 fixation. Non-nodulating R99 was used as control for calculating % ureide and % N from N2 fixation. Both shoots and roots were considered in this calculation. Total ureides in nodulated line – Total ureides in non-nodulated line = % Ureide from N2 fixation Total ureides in nodulated line Total N in nodulated line – Total N in non-nodulated line = % N from N2 fixation Total N in nodulated line Phaseolus vulgaris genotypes R99 R32 Puebla Eagle

% ureide from N2 fixation 0 80.6 68.2 84.9

% N from N2 fixation 0.0 47.5 62.2 56.9

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Table 7. Nodule effectiveness. Non-nodulating R99 was used as control for calculating total N from BNF. The values are the averages of three plants. Phaseolus vulgaris lines

Total N from BNF (mg)

Nodule number per plant

Nodule effectiveness (mg N per nodule)

Puebla Eagle R32

231 184 124

333 11 2400

0.69 16.73 0.05

R99

0

0

N/A

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CHAPTER 3: SHOOT AND ROOT CONTROL OF UREIDE ACCUMULATION AND PARTITIONING IN PHASEOLUS VULGARIS L. 1

Mercy K. Kabahuma1 and Mark E. Westgate1 Iowa State University, Ames, Iowa, College of Agriculture and Life Sciences Department of Agronomy A paper to be submitted to Journal of Crop Science

Abstract Ureides are manufactured predominantly in legume root nodules and are the primary form of organic N transported to the shoots in common bean (Phaseolus vulgaris L.). How the production, transport, and accumulation of ureides are regulated in common bean is not understood, but is a fundamental aspect of improving nitrogen fixation in this crop. We used a grafting technique to determine shoot and/or root control of ureide accumulation and partitioning among four genotypes of common beans that have been noted for variation in nitrogen fixation related phenotypic traits (R99, Eagle, Puebla, and R32). Effect of shoot and/or root on ureide accumulation and partitioning was verified by analyzing differences in root, stem, leaf, and pod biomass, ureide concentration, nitrogen concentration, and nodule numbers among intra-species grafts of these four genotypes. The greatest accumulation of nitrogen was in leaves while the least accumulation was observed in petioles. Ureide content and total N were greater in R32, Puebla, and Eagle (nodulating) than in R99 (non-nodulating). Super-nodulation was observed when R32 (a super-nodulator) scion was grafted onto normal nodulating rootstocks. Conversely, grafting normal scions onto R32 roots suppressed nodulation. Grafting normal scions onto R99 rootstocks did not affect nodulation, total ureide content, or total nitrogen. The extent of nodulation only indirectly affected ureide and N accumulation, as increasing nodule number did not result in greater accumulation of nitrogen. These results suggest shoot regulation of nodulation, ureide metabolism, and nodule effectiveness would be ideal physiological targets for further investigations aimed at improving BNF and yield in common bean.

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Introduction Many studies have used ureide concentration in aerial plant tissues as a parameter for assessing genetic and environmental effects on nitrogen fixation in legumes (King and Purcell, 2005 and Alamillo et al., 2010). King and Purcell (2005) noted a feedback inhibition of N2 fixation by nodule ureides in soybean (Glycine max L.). Alamillo et al. (2010) reported accumulation of ureides in aerial tissues was independent of ureide synthesis in nodules and N2 fixation in drought stressed common bean (Phaseolus vulgaris L.). Whether there are consistent relationships between tissue ureide accumulation and N2 fixation in common bean under favorable conditions has not been established. If found, ureide accumulation could be a useful parameter for identifying genotypes superior in biological nitrogen fixation (BNF). Regulation of ureide accumulation and partitioning could be addressed using manipulative techniques such as grafting. Several studies have used grafting to investigate various physiological processes such as increasing both seed and clones for breeding purposes (Gurusamy et al., 2009), investigating shoot or root control of nodulation (Buttery and Park, 1989; Hamaguchi et al., 1993; Harper et al., 1997; Abd-Alla, 2011), and yield (White and Castillo, 1989; 1992). In their studies White and Castillo (1989; 1992) found that root traits were more important in drought tolerance of common bean than shoot traits. Hamaguchi et al. (1993) used grafting to determine control of super-nodulation in soybean and common bean. They reported a difference between the mechanism controlling nodulation or substances involved in auto-regulation between soybean and common bean. Harper et al. (1997) highlighted the control of hyper-nodulation by a shoot-transmissible factor common to soybean, mungbean (Vigna radiata L.) and hyacinth bean (Lablab purpureus L.). Since ureides are synthesized primarily in nodules (Boldt and Zrenner 2003; Todd et al., 2005 and Zrenner et al., 2006) and both shoot and root tissues participate in the control of nodulation, it might be possible to enhance BNF and ureide accumulation by manipulating shoot and root complements. To this end, we tested the impact of reciprocal grafting of shoot scions and rootstocks of bean genotypes varying in their ability to fix atmospheric nitrogen. Our goal was to determine the role played by shoots and roots in regulating nodulation, BNF, ureide accumulation, and N assimilation in common bean.

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Materials and Methods Plant Materials and Growth Conditions Four Phaseolus vulgaris genotypes (R99, R32, Puebla 152, and Eagle) were selected for this study (provided by Dr. Karen Cichy, USDA-ARS, East Lansing Michigan). The main criterion for selection was variability in biological nitrogen fixation. R99 is a non-nodulation mutant (Park and Buttery, 1988 and 2006); while R32 is a nitrate-tolerant supernodulation mutant (Park and Buttery, 1989; Buttery and Park, 1990, 1993). R99 and R32 genotypes were both developed in Canada through chemical (Ethyl Methane Sulphonate) mutagenesis of OAC Rico (Park and Buttery, 1988, 1989, 2006; and Buttery and Park, 1990, 1993). Eagle is an Andean snap bean commercial cultivar developed in 1971 by Seminis Vegetable Seeds (Navarro et al., 2007). Puebla 152 is a black bean landrace from Mexico known for its superiority in symbiotic nitrogen fixation (Barrona et al., 1999). Because R99 is nonnodulating, it was used as a control to estimate BNF against R32, Puebla and Eagle. Grafting Puebla, Eagle, R32 and R99 seeds were placed in paper towels moistened with sterile distilled water. These seeds germinated in the dark at room temperature (20 C). After two days, germinated seeds were transferred to hydrated peat pellets (Jiffy Products (NB) Ltd, Canada) for five days in a controlled environment chamber at 24 C, light photon flux of 150-200 µmol-2s-1 and a 16h photoperiod. Vigorous seedlings with healthy unfolded primary leaves were selected and reciprocal grafts using the wedge graft technique were made. Hypocotyls of each seedling were detached at mid-point with a razor. The base of the upper hypocotyls (with cotyledons) was cut on opposite sides to form a V-shape that would fit in a 1-cm deep vertical split on the lower half of the hypocotyl. The graft unions were secured using grafting tape. Grafted and non-grafted seedlings were returned to the growth chambers for three days and covered with transparent plastic covers to minimize transpiration from the leaves. The plants were uncovered after three days. A week later, vigorous seedlings were then transplanted to 1-L free draining pots filled with commercial soil mixture (Sun Gro Horticulture Distribution Inc., Bellevue, WA) containing 15-25% Canadian spagum peat moss, composted bark, perlite, vermiculite, dolomite lime and blue chip in a greenhouse set at 25 C with a 16h photoperiod. One week after transplanting, pots

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were inoculated with a peat-based BioStacked® inoculant (Becker Underwood, Ames, IA) in a water suspension according to manufacturer’s instructions. A week after inoculation, 25 mL of fertilizer solution containing 100ppm N (1.1% ammoniacal N, 11.8% nitrate N and 2.1% urea N; Peter’s Excel Water Soluble Fertilizer, Everris Inc., Dublin, OH) was added to each pot. This amount of N was enough to enhance growth in R99 (non-nodulating) without inhibiting nodulation in R32, Puebla and Eagle. Three replicate pots of each line, each with a single plant, were arranged in a completely randomized design. Harvest At flowering/early pod set, plants were harvested and dried to measure plant biomass on a shoot/root basis and ureide concentration. Leaves, petioles, stems and pods (except for Puebla which lacked pods at the time of harvesting) and roots (including nodules) were dried at 60°C, weighed, and ground to pass through a 1-mm sieve. Samples were stored in tightly sealed glass vials at 20 C until they were analyzed for ureides and N. Nodule Counts Shoots were detached at the soil surface. Roots and soil were removed intact from pots and soaked in water to facilitate removal of soil and minimize loss of root nodules. Cleaned roots with nodules were stored in plastic bags at 4 C until nodules were counted manually. Nodules were not examined to determine whether they were active or not. Nitrogen and Ureide Analysis Tissue N concentration was measured in ~125 mg of stems, leaves, petioles, pods and roots as percent (%) N using Dumas complete combustion technique (Costech Analytical Technologies, Inc., Valencia, CA). Ureide concentration was determined according to Young and Conway (1942) with modifications from Purcell (de Silva et al, 1996). Ureides were extracted from 25 mg of dried, ground tissue (leaves, petioles, stem, pods and roots). Samples were homogenized in 1mL 0.2N sodium hydroxide (NaOH), boiled at 100°C for 30 min, cooled and centrifuged at 10,000g for 5min. The supernatant was pipetted into 1.5 ml microfuge tubes and stored at 10 C until ureide determination. Tissue extract was boiled in 0.5N NaOH for 8 min, cooled; boiled in 0.74N

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hydrochloric acid (HCl) and 0.33% phenylhydrazine-HCl for 2 min, cooled, and color was developed with the addition of concentrated HCl and 1.67% potassium ferricyanide (KFeCN). Ureide concentration was determined spectrophotometrically (Phoenix Equipment, Inc., Rochester, NY) against an allantoin standard at 520 nm within 15 min of addition of KFeCN. Ureide concentrations are presented in µmol per g dry weight, biomass in g, ureide content in µmoles, N concentration in mg per g dry weight tissue N and total N in mg. Statistical Analysis Single plants from each genotype and graft were considered replicates with five tissues (stems, leaves, petioles, pods and roots). The results represent the means ± SE of three plants. Data were analyzed by ANOVA and means were compared using Fisher’s LSD test at a probability level of 0.1 (Williams and Abdi, 2010).

Results and Discussion Impact of Grafting on Nodulation Number of nodules ranged between 0 and 2,400. The ranking of number of nodules formed from least to greatest was R99, a non-nodulating line with no nodules, as reported by Shirtliffe et al. (1996) or a few small whitish nodules (Buttery and Park, 1993; and Park and Buttery, 2006) followed by Eagle, Puebla, and R32, a super-nodulating line with the greatest number of nodules (Table 1). R32 shoots did not induce nodulation when grafted onto R99 roots. However, R32’s supernodulating trait was expressed when its shoots were grafted to normally nodulating common bean genotypes (Puebla and Eagle). A seven-fold increase in nodule number in Puebla and a more than 100-fold increase in Eagle was registered when R32 shoots were grafted onto Puebla and Eagle roots. This confirms control of nodulation by a shoot translocatable signal (Park and Buttery, 1989 and Buttery and Park, 1993; Harper et al., 1997 and Abd-Alla, 2011). Grafting Eagle shoots onto R32 roots was unsuccessful as roots penetrated through the graft union.

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Park and Buttery (1989) and Buttery and Park (1993) showed that R99 roots did not produce nodules with any scion including R32. Additionally, R32 scions induced increased numbers of nodules on itself and its parental line (OAC Rico). Park and Buttery (1992) reported formation of small ineffective nodules on R99 roots which are similar to the small pale white nodules we noted on some R99 roots in our study. It is possible that these were pseudo- nodules. Whether the few nodules on R99 roots were an effect of grafting or rhizobial strains in the inoculum applied is unknown. Grafting R99 and Puebla shoots onto R32 root stocks significantly suppressed number of nodules formed on R32 roots (Table 1). It is possible that the autoregulatory effect on nodulation in R32 was restored by grafting Puebla and R99 shoots on R32 roots. This possibility is supported by results from the study carried out by Hamaguchi et al. (1993). They hypothesized shoots of the super-nodulating mutants (En6500 and RBS15) lacked the autoregulatory factor, which was probably restored by grafting shoots of their respective wild type cultivars. Furthermore, Eagle shoots were associated with a decrease in the number of nodules on Puebla roots, when self-grafted and no nodules on R99 roots. While changing the shoot of Eagle resulted in a more than 10-fold increase in nodule number, changing shoots in R99 had no effect on nodulation. This signifies shoot control of nodulation in R32, Puebla and Eagle genotypes and root control of non-nodulation in the R99 mutant as reported by Pedalino et al. (1992) and Buttery and Park (1993). Because R99 shoots did not completely inhibit nodulation on R32 roots and in fact increased nodulation in Puebla and Eagle signifies some root control of nodulation. Total Plant Biomass Accumulation In some cases, grafting resulted in significant differences in biomass accumulation across (Fig. 1) and within genotypes (Table 2). Changing shoots of the R99 mutant did not have a significant impact on whole plant biomass accumulation and yet self-grafting R99 resulted in a significantly smaller root leading to a higher shoot to root ratio (Table 3). Furthermore, whole plant biomass accumulation was significantly depressed when R99 and R32 shoots were grafted onto Puebla roots in comparison to Puebla controls (Fig. 1).

Day et al. (1986) found no

significant differences in accumulation of shoot biomass, whole plant biomass and whole plant N content between un-inoculated nts382 and cv. Bragg, but found significant differences between inoculated nts382 and cv. Bragg. They associated the retardation of plant biomass accumulation

52

and overall growth in inoculated (nodulated) plants to the prolific number of nodules formed on their roots. This result is in line with results from our study, which showed that super-nodulation of Puebla roots caused by R32 shoots (Table 1) could have been at the expense of shoot and (Table 3) and whole plant biomass accumulation in the Puebla-R32 graft combination (rootshoot) (Fig. 1). This resulted in a lesser shoot to root ratio than the Puebla control. This could in part explain why suppression of nodulation on R32 roots by R99 shoots (Table 1) resulted in significantly greater accumulation of whole plant biomass than R32 controls (Fig. 1). However, grafting R32 shoots onto Eagle root stocks contradicts with this. Greater induction of nodulation on Eagle roots by R32 shoots (Table 1) increased biomass accumulation (Fig. 1). In fact, changing shoots of Eagle stimulated a significant increase in whole plant biomass accumulation. Self-grafting Eagle depressed shoot biomass accumulation, which was associated with decrease in whole plant biomass accumulation and a lower shoot to root ratio than the control. The consistent negative response of Eagle scions to self-grafting on Eagle rootstocks was unique among reciprocal grafts.

Evidently, excising the Eagle shoot initiated a self-incompatible

response that was not present or effective with other scions or rootstocks. The nature of this incompatibility is not known, but might be related to the suppression of nodule formation in this genotype. Puebla as a shoot and its controls lacked pods at the time of harvesting (40 days after planting). This result indicates slower development of Puebla relative to R32, Eagle, and R99. Changing shoots of Puebla resulted in production of pods (Table 2). It is possible that grafting accelerated growth by shortening the vegetative period and advancing Puebla plants into reproductive phase earlier than the control and self-grafted plants. Leaves followed by stems accumulated the greatest biomass across all treatments (Table 2). Ureide Concentration Genotypes with R32, Puebla, and Eagle (nodulating genotypes) had higher levels of ureide concentration than genotypes with R99 (non-nodulating) rootstocks (Table 4). Puebla and R99 shoots on R32 rootstocks resulted in a decrease in ureide concentration in stems, leaves and petioles compared to the control (Table 4). Self-grafting R99 significantly increased ureide concentration in stems, petioles and pods and decreased concentration in roots. Even with the lack of nodules, it was interesting to note that R99 had ureides accumulating in its tissues. A study carried out by Díaz-Leal et al. (2012) indicated that at the onset of the reproductive phase,

53

common bean plants obtain ureides by remobilizing nitrogen from the oldest vegetative tissues. In relation to this, self-grafting R99 significantly increased biomass accumulation in pods (Table 2) indicating that R99 self-grafts were further in development compared to R99 controls (ungrafted). It is possible that self-grafting R99 stimulated its rate of development which shortened the vegetative stage and thus advanced to the reproductive stage earlier. Remobilization of nitrogen from the oldest vegetative tissues, therefore, could in part be the source of ureides in R99 especially in self-grafts of R99. Among the graft combinations in which Puebla was the rootstock, using R32 as a shoot onto Puebla roots registered the greatest increase in ureide concentration in stems, leaves, petioles, pods and roots. Puebla has been identified for its superiority in symbiotic nitrogen fixation (Barrona et al., 1999). Ureides are primarily synthesized in root nodules and transported to the shoot where they accumulate in various shoot tissues (Boldt and Zrenner, 2003; Todd et al., 2006 and Zrenner et al., 2006). Increase in nodule number (sites for ureide synthesis) when R32 shoots were grafted onto Puebla roots coupled with Puebla’s greater ability to symbiotically fix nitrogen could account for the significantly greater ureide and N concentration observed in this graft combination compared to Puebla controls. Ureide Content There was great variation in the accumulation of ureides across the treatments (Fig. 2). Ureide content was greater in R32, Puebla and Eagle (nodulating) than in R99 (non-nodulating). This was attributable to greater number of nodules in R32, Puebla and Eagle than R99. Significantly greater ureide content was accumulated when R99 (non-nodulating) shoots were grafted onto R32 (super-nodulating) roots (Fig. 2). Conversely, Matsumoto et al. (1978) reported allantoin content was less in shoots of an A62-2 (non-nodulating) soybean line grafted onto A621 (nodulating) root than in ungrafted A62-1. They concluded allantoin production was not dependent on the type of shoot and that an increase in nodule number led to an increase in allantoin level. Our results, however, indicate a decrease in nodule number (Table 1) and an increase in ureide content in stems, pods and roots (Table 5) when R99 shoots were grafted onto R32 rootstocks compared to R32 controls. This increase in ureide content could be attributed to the greater accumulation of biomass in stems, pods and roots in this graft combination. Furthermore, the lack of significant difference in whole plant ureide content and biomass between R99 controls and grafts in which shoots of R99 shoots were replaced with R32, Puebla,

54

or Eagle scions demonstrates limited control of shoots over ureide content in R99 mutants (Fig. 2). Moreover R32 shoots did not induce nodulation when grafted onto R99 roots (Table 1), but resulted in biomass accumulation in pods greater than that of R99 control plants (Table 2). Significantly greater ureide content noted when R32 shoots were grafted onto Puebla and Eagle roots (Fig. 2) could be associated with the greater number of nodules formed on Puebla and Eagle roots when a super-nodulating genotype R32 was used as a scion (Table 1). A large increase in the number of nodules formed on Puebla and Eagle roots could have increased sites for ureide synthesis thereby increasing ureide concentration in plant tissues of Puebla-R32 and Eagle-R32 (root-shoot) grafts (Table 4). Importantly, the scions of these grafts supported the additional sink demand created by these additional nodules. Significantly greater whole plant ureide content was noted in R99 self-grafts. Moreover, changing the shoots of R99 did not have a significant effect on ureide content compared to R99 controls (Fig. 2). Self-grafted Eagle genotypes accumulated the least amount of ureide content among Eagle grafts. Grafting Eagle shoot onto its root, therefore, interfered with ureide accumulation. For R32 rootstocks, leaves and stems accumulated the greatest amount of ureides while petioles and roots accumulated the least amount (Table 5). Grafting R32 caused ureide content to decrease in leaves and increase in roots (Table 5). It is possible that grafting interfered with the translocation of ureides from roots to shoots leading to a decrease in concentration of ureides in stems and leaves and an increase in concentration in R32 roots (Table 4). Whereas Eagle and R32 shoots accumulated greater ureides in leaves and roots, Puebla shoots accumulated the greatest levels in roots when grafted onto R99 stocks. Nonetheless, self-grafting R99 depressed ureide content in roots and significantly increased them in stems and pods. This was attributable to high concentration of ureides in stems (Table 4), and high biomass accumulation in pods (Table 2). Grafting Puebla, R99 and R32 shoots onto Puebla roots accumulated the greatest amount of ureides in leaves (Table 5). This was associated with the greater accumulation of biomass in leaves than stems, petioles, pods and roots in these graft combinations (Table 2). Petioles generally accumulated the least amount of ureides across all graft combinations reflecting their small biomass. This result implies that petiole ureide concentration or content would not be the best indicators of BNF in common beans.

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Nitrogen Concentration Irrespective of scion, R32 and Puebla rootstock grafts maintained the greatest concentration of nitrogen in leaves and the least concentration of nitrogen in petioles and stems (Table 6). Grafting depressed N concentration in stems, leaves and petioles across all graft combinations in which Eagle was used as a root stock. Grafting R99 and Puebla shoots onto R32 rootstocks resulted in a significant decrease in concentration of ureides in leaves and roots. On the other hand, self-grafting R99 resulted in a significant increase in nitrogen concentration in stems, petioles and pods. Grafting R32 shoots onto Puebla root stocks led to a rise in concentration of N in R32 stems, leaves, petioles, pods and roots. The increased number of nodules on the Puebla rootstocks was supported by the R32 scions, which enabled the observed increase in tissue N concentration and content. Thus, Puebla’s superiority in BNF (Barrona et al., 1999) apparently could be enhanced further with support from the shoot. Total Nitrogen Accumulation Save for Eagle self-grafts, total nitrogen accumulation in R32, Puebla and Eagle rootstock grafts was greater than that in R99 rootstock grafts (Fig 3). This was partly attributable to the N derived from nitrogen fixation (Table 8) and greater nitrogen concentration in R32, Puebla and Eagle tissues than R99.

Self-grafting R99 significantly increased total N

accumulation over the control. Similar to Buttery and Park’s (1993) findings, R99 self-grafts formed very few, small and white nodules which were ineffective in N2 fixation. It is therefore possible the increase in total N partly resulted from grafting having a stimulatory effect on the capacity of R99 to absorb and assimilate mineral N. Many plants utilize assimilated N from mineral N before flowering and remobilize it from older tissues after flowering (Hirel et al., 2007). This could have been the source of N and ureides observed in the non-nodulating R99 mutants (Fig. 2). Save for self-grafting Eagle which had a significantly decreased total N accumulation (whole plant basis), changing shoots of Eagle plants did not have a significant effect on accumulation of total N. Self-grafting Eagle did not significantly affect its biomass accumulation and nodule formation compared to the Eagle controls. Lower N content in self-grafts of Eagle

56

was attributed partly to the least nitrogen concentration reported in the tissues of this graft combination (Table 6). Despite the lack of pods in the Puebla control and self-grafts, grafting did not have a significant effect on whole plant N accumulation but changed partitioning and accumulation of N in the stems, petioles and pods when Eagle, R99 and R32 shoots were grafted onto Puebla roots. Grafts of R32 scions on Puebla roots were noteworthy in that N concentration in all tissues was greater than in the non-grafted Puebla control (Table 6). This result shows that the stage of development was not a major inhibitor to nitrogen accumulation in Puebla. Grafting R99 shoots onto R32 roots significantly increased total plant N accumulation (Fig. 3). This grafting combination produced significantly fewer nodules (Table 1), lowered N concentration in leaves and roots, and maintained nitrogen concentration in stems and petioles (Table 6). Thus, the calculated increase in total N accumulation was attributable to the greater plant biomass at flowering. The contribution of total plant N from BNF, however, increased from 44.7% in the control to 54.8% in the R32/R99 rootstock/scion combination (data not shown). As in the previous study (Chapter 2), leaves of grafted scions consistently accumulated the greatest amount of N and biomass among the tissues sampled (stems, leaves, petioles, pods and roots). Ureide accumulation was highly variable, with no discernible pattern among tissues or graft combinations (Table 5). As such, the results of this study on grafted plants confirm the combination of leaves and biomass as the most appropriate phenotypic traits to identify improvements in common bean BNF.

Conclusions Puebla has been identified for its superior capacity for nitrogen fixation (Barrona et al., 1999). Eagle is known for its relatively low capacity for nitrogen fixation. These lines are being used as parental lines to produce progeny that vary in biological nitrogen fixation in an attempt to identify phenotypic and genotypic markers associated with variation in BNF. We used grafting to identify shoot and root traits associated with BNF that might be transferred among genotypes to improve common bean BNF and yield. Our results show that R32 and Puebla scions on Eagle rootstocks increased nodulation, ureide content, and biomass accumulation. Additionally, the

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increase in nodulation and ureide content associated with R32 shoots on Puebla and Eagle rootstocks indicates superiority of R32 shoots in terms of supporting ureide accumulation and nodulation. These characteristics could be utilized to improve nitrogen fixation in Puebla and Eagle. Further investigations should focus on the cause of reduced nodulation, overall development, ureide concentration, ureide content, and total nitrogen of self-grafted Eagle. This information could help improve BNF and productivity of this genotype. Although few consistent trends in nodule number, biomass, ureide, and N accumulation were associated with specific graft combinations, extent of nodulation and %N derived from N2 fixation are transferable traits that can be manipulated to improve N accumulation in common beans. Harvesting was targeted after onset of flowering yet some plants had formed pods while others lacked pods. Although this difference in development did not appear to give podded plants an advantage in terms of ureide and nitrogen accumulation, future plant sampling should be carried out at the same developmental stage to avoid the potential for developmental bias.

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REFERENCES Abd-Alla, M. H. 2011. Nodulation and nitrogen fixation in interspecies grafts of soybean and common bean is controlled by isoflavonoid signal molecules translocated from shoot. Plant Soil Environ.-UZEI 57: 453-458. Alamillo, M. J., J. L. Díaz-Leal, M. V. Sánchez-Moran, and M. Pineda. 2010. Molecular analysis of ureide accumulation under drought stress in Phaseolus vulgaris L. Plant Cell & Environ. 33: 1828-1837. Barrona, J. E., R. J. Pasini, D. W. Davis, D. D. Stuthman, and P. H. Graham. 1999. Response to selection for seed yield and nitrogen (N2) fixation in common bean (Phaseolus vulgaris L.). Field Crops Research 62: 119-128. Bliss, F. A. 1993. Breeding common bean for improved biological nitrogen fixation. Plant Soil 152: 71–79. Boldt, R., and R. Zrenner. 2003. Purine and pyrimidine biosynthesis in higher plants. Physiologia Plantarum 117: 297-304. Buttery, B. R., and S. J. Park. 1990. Effects of nitrogen, inoculation and grafting on expression of supernodulation in a mutant of Phaseolus vulgaris L. Canadian J. Plant Science 70: 375-381. Buttery, B. R., and Park, S. J. 1993. Characterization of some non-fixing mutants of common bean (Phaseolus vulgaris L.). Canadian J. Plant Science 73: 977-983. Day, D. A., H. Lambers, J. Bateman, B. J. Carroll, and P. M. Gresshoff. 1986. Growth comparisons of a supernodulating soybean (Glycine max) mutant and its wild-type parent. Physiol. Plant 68: 375-82. Díaz-Leal, J. L., G. Gálvez-Valdivieso, J. Fernández, M. Pineda, and J. M. Alamillo. 2012. Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in Phaseolus vulgaris L. J. Exp. Botany 63: 4095-106. Gurusamy, V., K. E. Bett, and A. Vandenberg. 2010. Grafting as a tool in common bean breeding. Canadian J. Plant Science 90: 299-304.

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Hamaguchi, H., M. Kokubun, T. Yoneyama, A. P. Hansen, and S. Akao. 1993. Control of supernodulation in intergeneric grafts of soybean and common bean. Crop Science 33: 794-797. Harper, J. E., K. A. Corrigan, A. C. Barbera, and M. H. Abd-Alla. 1997. Hypernodulation of soybean, mung bean, and hyacinth bean is controlled by a common shoot signal. Crop Science 37: 1242-1246. Hirel, B., J. Le Gouis, B. Ney, and A. Gallais. 2007. The challenge of improving nitrogen use efficiency in crop plant: towards a more central role for genetic variability and quantitative genetics within integrated approaches. J. Exp. Botany 58: 2369-2387. King, C. A., and L. C. Purcell. 2005. Inhibition of N2 fixation in soybean is associated with elevated ureides and amino acids. Plant Physiol. 137: 1389-1396. Matsumoto, T., M. Yatazawa, and Y. Yamamoto. 1978. Allantoin metabolism in soybean plants as influenced by grafts, a delayed inoculation with Rhizobium, and a late supply of nitrogen-compounds. Plant Cell Physiol. 19:1161-1168. Navarro, F., P. Skroch, G. Jung, and J. Nienhuis. 2007. Quantitative Trait Loci Associated with Bacterial Brown Spot in Phaseolus vulgaris L. Crop Science 47: 1344-1353. Park, S. J., and B. R. Buttery. 1988. Nodulation mutants of white bean (Phaseolus vulgaris L.) induced by ethyl methane sulphonate. Canadian J. Plant Sci. 68:199–202. Park, S. J., and B. R. Buttery. 1989. Inheritance of nitrate tolerant nodulation in EMS induced mutants of common bean (Phaseolus vulgaris L.). J. Hered. 80: 486–488. Park, S. J., and B. R. Buttery. 1992. Ethyl-methane sulphonate (EMS) induced nodulation mutants of common bean (Phaseolus vulgaris L.). Plant Soil 139: 295-298. Park, S. J., and B. R. Buttery. 1994. Inheritance of non-nodulation and ineffective nodulation mutants in common bean (Phaseolus vulgaris L.). J. Hered. 85: 1-3. Park, S. J., and B. R. Buttery. 2006. Registration of ineffective nodulation mutant R69 and nonnodulation mutant R99 common bean genetic stocks. Crop Science 46: 1415-1417.

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Pedalino, M., K. E. Giller, and J. Kipe-Nolt. 1992. Genetic and Physiological Characterization of the Non-nodulating Mutant of Phaseolus vulgaris L.-NOD125. J. Exp. Botany 43: 843849. Shirtliffe, S. J., J. K. Vessy, B. R. Buttery, and S. J. Park. 1996. Comparison of growth and N accumulation of common bean (Phaseolus vulgaris L. cv. OAC Rico) and its nodulation mutants R69 and R99. Canadian J. Plant Sci. 76: 73–83. Todd, C. D., P. A. Tipton, D. G. Blevins, P. Piedras, M. Pineda, and J. C. Polacco. 2006. Update on ureide degradation in legumes. J. Exp. Botany 57: 5-12. White, J. W., and J. A. Castillo. 1989. Relative effect of root and shoot genotypes on yield of common bean under drought stress. Crop Science 29: 360-362. White, J. W., and J. A. Castillo. 1992. Evaluation of diverse shoot genotypes on selected root genotypes of common bean under soil water deficits. Crop Science 32: 762-765. Young, E. G., and C. F. Conway. 1942. On the estimation of allantoin by the Rimini-Schryver reaction. J. Biol. Chem. 142: 839-853. Zrenner, R., M. Stitt, U. Sonnewald, and R. Boldt. 2006. Pyrimidine and purine biosynthesis and degradation in plants. Annual Review Plant Biology 57: 805-836.

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Figure 1. Biomass of tissues collected from R32, R99, Puebla, and Eagle controls and grafted plants during flowering/early pod set. Tissues were dried at 60 C until a constant weight. Stems (blue bars), leaves (red bars), petioles (green bars), pods (purple bars) and roots (brown bars). The values are the mean ± SE of three plants. Same letters above bars indicate total biomass of the genotypes are not significantly different at P=0.1 using Fisher’s LSD test.

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Figure 2. Ureide content in tissues of R32, R99, Puebla, and Eagle controls and grafts harvested during flowering/early pod set. Stems (blue bars), leaves (red bars), petioles (green bars), pods (purple bars) and roots (brown bars). The values are the mean ± SE of three plants. Same letters above bars indicate total ureide content of the genotypes are not significantly different at P=0.1 using Fisher’s LSD test.

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Figure 3. Total N accumulation in tissues of R32, R99, Puebla, and Eagle controls and grafts harvested during flowering/early pod set. Total N was determined using Dumas complete combustion technique. N concentration and biomass were used to calculate total N content in the tissues. Stems (blue bars), leaves (red bars), petioles (green bars), pods (purple bars) and roots (brown bars). The values are the mean ± SE of three plants. Same letters above bars indicate total N of the genotypes are not significantly different at P=0.1 using Fisher’s LSD test.

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Table 1. Number of nodules on controls, self- and intra-species grafts of R32, R99, Puebla, and Eagle roots sampled at flowering. The soil was carefully washed from the roots to minimize loss of nodules. Nodules were counted manually. Nodules were not analyzed for activity. The values are the mean ± SE of three plants. Using Fisher’s LSD test, means in columns followed by the same letter are not significantly different at P= 0.1. Phaseolus vulgaris lines Root/Shoot R32 control R32/R32 R32/R99 R32/Puebla

Number of nodules

R99 control R99/R99 R99/Eagle R99/Puebla R99/R32

0 9 0 3 0

Puebla control Puebla/Puebla Puebla/Eagle Puebla/R99 Puebla/R32

333 bc 339 bc 185 c 480 b 2400 a

Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32

11 c 9 c 716 b 640 b 1194 a

2400 a 2833 a 840 b 910 b b a b ab b

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Table 2. Biomass of controls, self- and intra-species graft tissues from R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set. They were dried at 60 C until a constant weight was reached to obtain their biomass. The values are the mean ± SE of three plants. Puebla lacked pods at the time of harvesting. Using Fisher’s LSD test, means in rows followed by the same lower case letter within the same rootstock are not significantly different at P= 0.1. Means in columns followed by the same Upper Case letter within the same tissue are not significantly different at P= 0.1. Phaseolus vulgaris lines Root/Shoot

Leaves

Biomass (g) Petioles

Stems

Pods

Roots

R32 control R32/R32 R32/R99 R32/Puebla

1.4 b C 2.1 b BC 4.1 b A 3.2 a AB

3.6 a B 4.9 a B 7.0 a A 4.2 a B

0.3 b C 0.6 c B 0.9 c A 0.8 b A

1.0 b BC 1.8 b AB 3.0 b A 0.0 c C

1.5 b B 1.9 b B 2.9 b A 1.9 b B

R99 control R99/R99 R99/Eagle R99/Puebla R99/R32

1.4 b AB 0.8 b B 1.8 b A 1.6 b AB 2.1 ab A

3.2 a AB 2.3 a B 4.5 a A 3.8 a A 3.7 a AB

0.5 c AB 0.3 b B 0.7 d A 0.6 c A 0.6 b A

1.1 b B 2.5 a A 1.1 c B 0.0 c C 1.6 b AB

1.5 b A 0.3 b B 1.4 c A 1.7 b A 1.8 b A

Puebla control Puebla/Puebla Puebla/R99 Puebla/Eagle Puebla/R32

3.3 b A 3.5 b A 2.5 b B 2.9 b AB 1.6 b C

7.4 a A 7.4 a A 4.9 a BC 6.1 a AB 4.5 a C

0.9 c BC 1.4 d A 0.8 c BC 1.2 c AB 0.4 c C

0.0 d B 0.0 e B 1.5 c A 1.0 c A 1.2 b A

1.6 c A 1.9 c A 1.5 c A 1.4 c A 1.6 b A

Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32

1.7 bc B 1.5 b B 3.2 b A 3.9 b A 3.6 bc A

3.3 a C 2.4 a C 5.5 a B 7.8 a A 5.8 a B

0.8 c AB 0.4 c C 0.7 d BC 1.1 c A 0.8 d AB

2.2 ab AB 0.6 c BC 1.5 c BC 0.0 d C 3.8 b A

1.1 bc B 1.6 b AB 1.8 c AB 2.1 bc A 1.9 cd A

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Table 3. Shoot: root ratios of controls, self- and intra-species graft of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set. They were dried at 60 C until a constant weight was reached to obtain their biomass. The values are the mean ± SE of three plants. Puebla lacked pods at the time of harvesting. Using Fisher’s LSD test, means in columns followed by the same Upper Case letter within the same tissue are not significantly different at P= 0.1. Phaseolus vulgaris lines Root/Shoot

Shoot

Biomass ratios (g) Root

Shoot: root ratio

R32 control R32/R32 R32/R99 R32/Puebla

6.3 B 9.4 B 14.9 A 8.2 B

1.5 B 1.9 B 2.9 A 1.9 B

4.2 A 5.0 A 5.1 A 4.3 A

R99 control R99/R99 R99/Eagle R99/Puebla R99/R32

5.3 A 5.7 A 7.8 A 6.2 A 7.5 A

1.5 A 0.3 B 1.4 A 1.7 A 1.8 A

3.5 19.0 5.6 3.7 4.2

Puebla control Puebla/Puebla Puebla/R99 Puebla/Eagle Puebla/R32

11.6 A 10.9 A 9.7 AB 11.2 A 7.6 B

1.6 A 1.9 A 1.5 A 1.4 A 1.6 A

7.3 AB 5.7 BC 6.5 ABC 8.0 A 4.8 C

Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32

8.0 C 4.9 D 9.9 B 12.8 AB 13.8 A

1.1 B 1.6 AB 1.8 AB 2.1 A 1.9 A

7.3 A 3.1 C 5.5 BC 6.1 AB 7.3 A

B A B B B

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Table 4. Ureide concentration in controls, self- and intra-species graft tissues of R32, R99, Puebla, and Eagle lines collected during flowering/early pod set. Ureide concentrations were determined spectrophotometrically against an allantoin standard. The values are the mean ± SE of three plants. Puebla lacked pods at the time of harvesting. Using Fisher’s LSD test, means in rows followed by the same lower case letter within the same rootstock are not significantly different at P= 0.1. Means in columns followed by the same Upper Case letter within the same tissue are not significantly different at P= 0.1. Phaseolus vulgaris lines Root/Shoot

Ureide concentration (µmol./gDW) Petioles Pods

Stems

Leaves

R32 control R32/R32 R32/R99 R32/Puebla

53.0 a A 47.5 a B 46.8 a B 22.9 b C

39.1 a A 22.9 b C 18.3 d C 32.8 a B

52.4 a A 56.3 a A 20.5 d B 17.1 c B

41.2 a A 45.0 a A 33.1 b A 0.0 c B

6.0 b C 23.4 b B 28.0 c A 20.4 bc B

R99 control R99/R99 R99/Eagle R99/Puebla R99/R32

4.3 c B 89.4 a A 4.6 d B 4.7 b B 3.8 c B

5.1 5.7 7.3 4.4 6.9

c BC c ABC cA bC b AB

4.1 c B 20.3 b A 3.7 d B 4.0 b B 3.8 c B

7.0 b C 22.2 b A 11.7 b B 0.0 c E 4.8 c D

18.6 a A 2.4 c C 14.8 a B 18.1 a A 16.0 a B

Puebla control Puebla/Puebla Puebla/R99 Puebla/Eagle Puebla/R32

11.5 b C 16.0 b C 14.7 b C 30.4 a B 49.0a A

13.1 b BC 15.1 b B 13.8 bc BC 11.3 c C 25.8 b A

6.5 c B 9.1 c B 6.9 d B 10.1 c B 48.5 a A

0.0 d D 0.0 d D 12.1 c C 21.1 b B 28.9 b A

16.8 a BC 18.2 a BC 21.7 a B 12.8 c C 32.7 b A

Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32

87.4 a A 4.6 c C 22.6 c B 21.5 b B 80.6 a A

19.5 b C 5.7 bc D 32.8 b B 49.0 a A 34.0 b B

19.2 c B 3.6 c C 26.0 bc B 19.4 b B 81.0 a A

57.4 b A 8.0 b C 28.6 bc B 0.0 c C 31.8 b B

15.4 c C 14.9 a C 40.9 a A 20.0 b B 14.7 c C

Roots

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Table 5. Ureide content in controls, self- and intra-species graft tissues of R32, R99, Puebla, and Eagle bean lines collected during flowering/early pod set. Ureide concentration and biomass were used to calculate ureide content. The values are the mean ± SE of three plants. Puebla lacked pods at the time of harvesting. Using Fisher’s LSD test, means followed by the same lower case letter across the same row within the same rootstock are not significantly different at P= 0.1. Means in columns followed by the same Upper Case letter within the same tissue are not significantly different at P= 0.1. Phaseolus vulgaris lines Root/shoot

Ureide content (µmoles) Petioles Pods

Stems

Leaves

R32 control R32/R32 R32/R99 R32/Puebla

70.8 b B 99.9 a B 191.1 a A 70.3 b B

163.5 a A 105.8 a B 129.1 b AB 139.2 a AB

13.5 c B 31.0 c A 18.3 d B 13.0 c B

39.2 bc B 78.2 b A 98.1 bc A 0.0 d C

9.1 c C 45.7 c B 81.3 c A 38.3 c B

R99 control R99/R99 R99/Eagle R99/Puebla R99/R32

5.8 c B 72.3 a A 7.4 c B 6.7 c B 8.0 b B

13.6 b B 12.5 b B 29.9 a A 17.6 b AB 27.7 a A

3.1 4.3 3.0 2.2 2.2

7.6 c B 54.7 a A 13.3 b B 0.0 d C 7.8 b B

27.3 a A 0.7 c B 26.4 a A 27.2 a A 28.6 a A

Puebla control Puebla/Puebla Puebla/Eagle Puebla/R99 Puebla/R32

39.3 b B 56.7 b B 88.8 a A 37.1 b C 76.6 b A

96.1 a A 97.2 a A 68.0 b B 67.7 a B 115.9 a A

5.9 c BC 10.1 d BC 12.0 c B 5.5 d C 20.0 d A

0.0 d B 0.0 e B 21.2 c A 17.8 c A 34.7 dc A

28.0 bc BC 35.6 c AB 18.2 c C 32.1 b BC 50.0 c A

Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32

149.1 a B 6.9 c D 72.7 bc C 80.5 b C 268.8 a A

62.5 bc C 13.8 b D 181.7 a B 386.0 a A 197.5 b B

16.3 c B 1.4 c C 16.8 d B 19.8 c B 63.6 d A

110.3 ab A 4.0 c C 42.4 cd B 0.0 d D 113.8 c A

16.8 c C 24.7 a BC 87.9 b A 42.5 bc B 27.8 e BC

d AB bA c AB dB bB

Roots

69

Table 6. N concentration in controls, self- and intra-species graft tissues of R32, R99, Puebla, and Eagle plants collected during flowering/early pod set. N concentration was determined using Dumas complete combustion technique. The values are the mean ± SE of three plants. Puebla lacked pods at the time of harvesting. Using Fisher’s LSD test, means in rows followed by the same lower case letter within the same rootstock are not significantly different at P= 0.1. Means in columns followed by the same Upper Case letter within the same tissue are not significantly different at P= 0.1. Phaseolus vulgaris lines Root/Shoot

Stems

Nitrogen concentration (mg/g) Leaves Petioles Pods

Roots

R32 control R32/R32 R32/R99 R32/Puebla

16.1 c A 15.5 c A 15.4 d A 17.0 c A

47.1 a A 49.3 a A 39.7 a C 42.9 a B

18.4 c A 18.9 c A 15.5 d A 17.7 c A

0.0 d B 33.9 b A 31.6 b A 0.0 d B

35.0 b A 31.2 b AB 25.4 c B 27.2 b B

R99 control R99/R99 R99/Eagle R99/Puebla R99/R32

6.0 b B 22.3 b A 8.0 c B 6.3 d B 8.7 b B

34.6 a A 45.1 a A 15.5 b B 14.1 b B 16.6 a B

14.5 b B 22.5 b A 6.6 c C 8.7 c BC 8.0 b BC

17.3 b C 36.5 a A 22.1 a B 0.0 e D 16.9 a C

18.8 b A 16.5 b AB 13.6 b B 18.4 a A 17.0 a AB

Puebla control Puebla/Puebla Puebla/Eagle Puebla/R99 Puebla/R32

16.8 c AB 15.8 c AB 16.9 b AB 14.2 c B 18.6 d A

39.9 a BC 43.4 a B 32.3 a C 36.2 a BC 53.8 a A

16.2 c B 17.5 c B 16.9 b B 13.1 c B 23.4 c A

0.0 d B 0.0 d B 32.5 a A 26.1 b B 33.5 b A

25.4 b B 24.8 b B 21.2 b C 23.2 b BC 32.9 b A

Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32

23.2 c A 12.7 ab BC 11.2 c C 12.7 b BC 17.9 c AB

48.8 a A 16.4 ab C 34.5 a B 33.4 a B 47.2 a A

25.0 c A 10.0 b D 13.3 c CD 15.2 b C 20.3b B

36.7 b A 17.2 ab B 30.6 ab A 0.0 c C 32.0 b A

21.7 c BC 17.8 a C 27.5 b AB 29.6 a A 32.8 b A

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Table 7. Total N accumulation in controls, self- and intra-species graft tissues of R32, R99, Puebla, and Eagle plants collected during flowering/early pod set. N concentration and biomass were used to calculate total N content in the tissues. The values are the mean ± SE of three plants. Puebla lacked pods at the time of harvesting. Using Fisher’s LSD test, means in rows followed by the same lower case letter within the same rootstock are not significantly different at P= 0.1. Means in columns followed by the same Upper Case letter within the same tissue are not significantly different at P= 0.1. Phaseolus vulgaris lines Root/Shoot

Total nitrogen (mg) Petioles Pods

Stems

Leaves

R32 control R32/R32 R32/R99 R32/Puebla

21.9 b B 32.7 bc B 61.7 b A 54.2 b A

196.3 a B 236.8 a AB 276.5 a A 182.6 a B

5.2 b B 10.4 c A 13.8 c A 13.2 b A

0.0 c B 62.3 b A 90.7 b A 0.0 c B

54.1b A 61.7 b A 73.1 b A 52.8 b A

R99 control R99/R99 R99/Eagle R99/Puebla R99/R32

8.2 c B 20.1 b A 12.7 c AB 9.8 c B 17.3 bc AB

87.2 a A 111.9 a A 68.6 a A 53.3 a A 60.3 a A

9.1 bc A 7.7 b A 5.2 d A 4.9 c A 4.8 c A

13.5 bc B 89.4 a A 25.2 b B 0.0 d C 25.8 bc B

35.5 b A 6.4 b B 22.0 b AB 30.5 b A 36.0 b A

Puebla control Puebla/Puebla Puebla/Eagle Puebla/R99 Puebla/R32

51.9 b AB 55.7 b A 48.8 b AB 35.8 b BC 29.7 cd C

282.3 a A 264.9 a AB 191.6 a BC 177.8 a C 239.5 a ABC

13.9 c AB 19.4 c A 19.5 b A 10.5 c B 9.8 d B

0.0 d C 0.0 d C 31.9 b B 42.7 b B 38.4 bc B

36.9 b A 47.2 bc A 31.0 b A 34.6 b A 53.5 b A

Eagle control Eagle/Eagle Eagle/R99 Eagle/Puebla Eagle/R32

37.0 bc AB 18.7 bc C 25.2 cd BC 47.9 b A 59.8 c A

163.7 a B 39.3 a C 188.6 a AB 259.1 a A 274.1 a A

21.4 c A 3.9 c C 8.8 d BC 15.9 c AB 16.2 c AB

92.3 b A 9.9 c B 47.3 bc AB 0.0 d C 119.6 b A

23.8 c B 30.6 ab B 59.0 b A 61.7 b A 57.8 c A

Roots

71

Table 8. Percent N derived from N2 fixation. Non-nodulating R99 was used as control for calculating % N from N2 fixation. Averages of total N accumulated by R99 were subtracted from averages of total N accumulated by R32, Puebla and Eagle plants. Phaseolus vulgaris Lines used as scions R99 R32 Puebla Eagle

% N from N fixation 0.0 60.3 69.2 27.6

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CHAPTER 4: GENERAL CONCLUSIONS Nitrogen uptake and assimilation are contributors to plant growth and development. Studies described in this thesis were aimed at determining genotypic variation in BNF and N assimilation from BNF and determining the transferability traits related to BNF and N assimilation from BNF. Identification of a trait or sets of traits associated with superiority for BNF and N assimilation from BNF is important in improving BNF. However, the variability in phenotypic traits among genotypes and plant tissues made it complex for identification of a trait or traits related to N assimilation from BNF. Nevertheless, biomass accumulation was a vital contributor to N accumulation and this was associated with high capacity for BNF. Biomass and N accumulation, nodule effectiveness might be useful for selecting genotypes with superior capacity for BNF. Also nodule number as low as 11, leaf total N content, and total biomass at flowering were consistent with greater BNF in this study. Results from this study will be used to focus phenotypic analysis of an Andean Diversity Panel (400+ lines) for genomic marker development in a related USAID-funded project.

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ACKNOWLEDGEMENTS To God Almighty for the wisdom, knowledge, endurance, strength, love, mercy, grace and favour He has blessed me with and for bringing me thus far. I am in awe. Appreciation goes to Dr. Mark Westgate, my major advisor without whom my success at Iowa State University would not have been realized. For the academic and technical advice, encouragement, words of wisdom and always seeing potential in me, I will forever be grateful. My program of study committee members, Dr. Gwyn Beattie and Dr. Kathleen Delate for the academic and technical assistance rendered during the course of my stay at Iowa State University. Dr. Karen Cichy (Michigan State University) for providing seed and literature for the research study. Steven Beebe (CIAT International Center for Tropical Agriculture) for providing seed for research. Dr. Larry Purcell (University of Arkansas) for allowing us to learn from his laboratory and providing literature related to our study. Special thanks go to Whitney Bouma (Iowa State University) for the technical assistance offered during the course of my research. Thanks also go to Nicholas Howell (ISU Horticulture farm) and the Agronomy Farm manager (Iowa State University) for all the help in the field studies. To my family and friends both in Uganda and the US, I will forever be indebted. You stood with me physically and emotionally enabling me to finish this race even stronger than I started. Gratitude goes to the funders, USAID, for the financial assistance.

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