Journal of Experimental Botany, Vol. 48, No. 307, pp. 255-263, February 1997

Journal of Experimental Botany

Sugar beet guard cell protoplasts demonstrate a remarkable capacity for cell division enabling applications in stomatal physiology and molecular breeding Robert D. Hall1'5, Tjitske Riksen-Bruinsma1, Guy Weyens2, Marc Lefebvre2, Jim M. Dunwell3'4, Arjen Van Tunen1 and Frans A. Krens1 1

Department of Cell Biology, DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Postbox 16, 6700 AA Wageningen, The Netherlands 2

SES Europe NV/SA, Industriepark 15, B-3300 Tienen, Belgium Zeneca Seeds, Jealott's Hill Research Station, Bracknell, Berkshire RG126EY, UK

3

Received 28 May 1996; Accepted 30 August 1996

Abstract A highly-efficient protocol for the large-scale isolation of guard cell protoplasts from sugar beet (Beta vulgaris L.) has been developed. Optimization of conditions for culturing these protoplasts resulted in extensive cell division and colony formation, at frequencies exceeding 50%. Plants can subsequently be regenerated from these guard cell-derived colonies. This provides definitive confirmation that, in sugar beet leaf protoplast populations, only guard cells are the source of totipotent protoplasts. These findings are the outcome of a directed, non-empirical approach to overcoming plant cell recalcitrance which was initiated by exploiting computer-assisted microscopy to couple in vitro response to cell origin. The results reaffirm the conclusion that, in plants, extreme degrees of cytodifferentiation need not entail terminal specialization. The responsive nature of this system can be ascribed to the unique use of cultures essentially comprising a single in vivo cell type. A uniform model system has thus been created with potential for widespread application. Their distinct morphological (and mechanical) features make guard cells a valuable choice for studying various fundamental aspects, not only of stomatal physiology, but also of plant cell (de)differentiation, differential gene expression etc. Furthermore, an applied value for such a system can also be envis4 5

aged. Results indicate that these cells are highly amenable to genetic manipulation techniques. The importance of these observations to our understanding of plant cell function and behaviour is discussed. Key words: Beta, guard cells, stomatal physiology, totipotency, transformation.

Introduction Only a detailed knowledge and understanding of in vitro cell culture systems permits a denned and directed approach towards the improvement of cellular responses in vitro to produce reliable, uniform and reproducible experimental systems. However, the in vitro response of plant cells and tissues is, even in non-recalcitrant systems, distinctly heterogeneous. In protoplast populations it is usual that only a minority of cells proceed to divide and form viable colonies (for references, see Roest and Gilissen, 1993). Countless empirical attempts have been made to improve this situation but with limited success. In a previous publication, the novel application of computer-assisted microscopy to track down and subsequently identify the origin of rare totipotent cells in heterogeneous protoplast populations isolated from sugar beet leaves was reported (Hall el cil., 1995). In this highly recalcitrant system only a tiny proportion ( 2 % of cells were GUS + with 83% of these being guard cell protoplasts.

Discussion By designing a protocol specifically around stomatal guard cells, recalcitrance in sugar beet to protoplast-based technology has been overcome. In addition, the results detailed here represent the successful development of a single system which can also function both as an invaluable tool for carrying out fundamental research into the processes of stomatal physiology and plant cell (de)differentiation and, additionally, as an ideal starting point in the development of a genetic manipulation protocol for what is presently one of the most recalcitrant crop species. It has proven possible to isolate sugar beet guard cell protoplasts with high yields and at high degrees of purity with remarkable ease. Under optimum conditions these protoplasts can be induced to undergo division at frequencies of > 50% and ultimately can also proceed to regenerate plants. These results compare favourably with those reported by Tallman for Nicotiana glauca which, presently, is the only other species for which guard cell totipotency has been successfully demonstrated (Sahgal et al., 1994). The previous report that protoplast division in this system was specifically linked to a single tissue in the source material (Hall et al., 1995) paved the way for this directed approach to overcoming the limited response of these cultures. Exploiting this knowledge has enabled improvements to be introduced into the existing protocol in a defined manner to instigate an overall enhancement in culture efficiency. In so doing it has been possible to avoid the usual empiricism frequently associated with this type of research. Division frequency in these cultures increased in parallel with the proportion of guard cells present. This, combined with a total failure to demonstrate division in virtually every other cell type (Hall et al., 1995), provides the definitive confirmation that these cells are the sole progenitors of totipotent callus in this system. This is doubly remarkable considering that decades of research into guard cell division, which began in the 1920s (Thielman, 1925), proved fruitless (Dehnel, 1960) while division of epidermis or mesophyll cells is commonplace (Pillai et al., 1991, Park and Wernicke, 1993; D'Ultra-Vaz et al., 1993; Creemers-Molenaar et al., 1994). The obvious question arising from these observations is 'why guard cells?'.

Table 4. Transient expression of the GUS gene in sugar beet cells after transforming protoplasts with the plasmid pPG5 Histochemical staining was performed 1. 2 or 3 d after transformation. Protoplasts were plated in the standard way with 62 500 cells/dish, of which 65% were guard cell protoplasts. Calf thymus DNA was used as carrier Results are the means of three repicates. n.a. = result not available. Genotype

NF

^g DNA/5 x 105 protoplasts

Blue-stained GUS + cells (guard cells iin parentheses)/dish

pPG5

Carrier DNA

di

d2

d3

10 50

40 0

11 (5) n.a.

108(86) 1267 (1045)

4(2) 298(276)

Sugar beet guard cell protoplasts

The culture conditions used are, in general terms, quite standard and ought to be suitable for most species, tissues and cell types (Wei and Xu, 1990; Bhadra et al., 1994). However, in developing a culture protocol for a recalcitrant system, it may be that only a highly-specialized cell type can ultimately respond. In this sugar beet system, the cause underlying the general non-responsive nature of leaf protoplasts may be so dominant that cells with unique properties are required before the barriers determining in vitro behaviour are overcome. There are a number of specific features of stomatal guard cells which may enable their survival under conditions which remain inappropriate for that of their neighbours. Guard cells lack plasmodesmata and thus have, in vivo, a more isolated existence than most plant cells. They are also the only cells in the leaf which are accustomed to regular fluctuations in osmotic potential—this in accordance with the key role which osmotic pressure plays in stomatal function (Willmer, 1993, Hedrich et al., 1994). As such, guard cells may, inherently, be better equipped to deal with the inevitable osmotic shocks associated with protoplast isolation and purification. However, this cannot be the entire explanation as in mixed protoplast populations, cells derived from, for example, mesophyll or the epidermis, while not dividing, also do not die, but remain viable for considerable periods (Hall et al., 1995). Nevertheless, it may be that during preparation, their physiology is disturbed to such an extent that they are subsequently unable to respond effectively to the culture environment. Furthermore, cell division in cambial protoplasts has been observed in this system but, despite intensive efforts over a number of years, it has never proven possible to achieve even an indication of a regenerative response from the calli obtained (Pedersen et al., 1993; Schlangstedt et al., 1994; Hall et al., 1995). It is perhaps more likely that it is the typical physiological state of guard cells which makes them most amenable to expressing their full potential for cell division and totipotency. Despite their specialized function (Sack, 1987; Mansfield et al., 1990), guard cells are particularly versatile in their responsiveness to both environmental factors and signals from within the plant (Assmann, 1993; Mansfield, personal communication). This must entail a dependance on many more genes remaining active than in most (or all) other cells in the leaf. As such, guard cells may paradoxically, be well suited to re-expressing full genetic potential. It would, therefore, be fruitful to investigate this point using other recalcitrant and even non-recalcitrant systems to determine whether guard cells remain responsive in these cultures also. In this regard, computer-assisted microscopy, as described previously (Hall et al., 1995), could provide the ideal approach for such work as it would entail no extra requirement for guard cell isolation protocols prior to experimentation. The one distinguishing feature of the culture protocol

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for sugar beet protoplasts is the inclusion of the antioxidant nPG in all media. This was previously found to be essential for supporting cell division in these cultures (Krens et al., 1990; Hall et al., 1994). nPG inhibits lipoxygenase (Peterman and Siedow, 1983) which, through fatty acid oxidation, initiates free radical chain reactions in biological systems (Vliegenthart and Veldink, 1982). Resultant (differential) membrane damage could subsequently determine cellular response through altered uptake properties or the loss of critical (hormone) receptors. However, and perhaps more importantly, free radicals in plants lead to the formation of ethylene (Kacperska and Kubacka-Zebalska, 1989; Ievinsh, 1992) which has known wide-ranging, physiological effects on plant cells (Abeles et al., 1992). It has recently been demonstrated that nPG significantly inhibits the release of ethylene in sugar beet leaf protoplast systems (Krens et al., 1994). Guard cells are the first line of defence in plants regarding gaseous exchange. It is therefore feasible to suggest that they may have a greater ethylene tolerance. As such, this cell type may again be better equipped to deal with what might be an unusually hostile environment. The idea of an influential role for ethylene in guard cell development is not unprecedented. Recently, Roberts et al. (1995) proposed a key role for ethylene in determining the eventual cellular response of their tobacco guard cell protoplast system. It would be valuable to determine if the level of ethylene produced or the degree of sensitivity of sugar beet cells to it, is significantly different in this system in comparison to other recalcitrant and non-recalcitrant ones. Furthermore, now that a sugar beet guard cell protoplast isolation protocol is available, direct comparisons between mixed leaf protoplast populations (recalcitrant) and purified guard cell protoplasts (non-recalcitrant) can easily be made. Perhaps therefore, the more important question which should be addressed is not 'why guard cells?', but rather 'why not the expected cell types?'. It is quite surprising that while guard cells can be induced to divide at frequencies of up to 60% (Hall et al., 1995), virtually every other cell fails to divide. The central cause must be the inability of such cells to re-express or switch off critical genes. The factors influential in this process are likely to differ in different systems. Investigating a potential role for ethylene in determining the ultimate behaviour of cells in in vitro systems might shed some additional light on this problem. Guard cells in plants, despite their functional specialization, are clearly not irreversibly differentiated. Their uniqueness of form concerns not only morphological features such as cell wall structure and cytoplasmic organization, but also a wide range of physiological and metabolic processes (Zeiger, 1983; Sack, 1987). As such, a guard cell culture system lends itself to potential applications concerning gene expression, guard cell function and

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the general phenomenon of (de) differentiation in plant cells. The frequent observation of stomatal failure in in vitro-grown plants, which can cause extensive complications in the micropropagation industry (Kozai, 1991), could also be effectively investigated using this system. However, potentially the most fruitful application of a guard cell culture protocol is its use for studies on basic guard cell physiology. In this regard, a cautionary note is warranted concerning the observed effects of BSA as reported above. While BSA has generally become a standard ingredient in media used for isolating guard cell protoplasts destined for physiological studies (Weyers and Meidner, 1990), results presented here clearly indicate that despite the significant advantageous effect on protoplast recovery, BSA has a distinct detrimental effect on subsequent cell development. Consequently, the advisability of employing this compound in such studies is brought into serious question. The ability of guard cells actively to express alien genes has previously been shown in transgenic Arctbidopsis plants using the GUS reporter gene driven by putative guard cell specific promoters (Nakamura et al., 1995; Taylor et al., 1995). The results detailed here support these findings and indicate that a guard cell protoplast system could be exploited in transient expression studies concerning gene expression associated with guard cell physiology and metabolism. The high metabolic activity and great uniformity of such a system augur well for the development of such a procedure. Sugar beet breeders are still in search of an efficient genetic manipulation protocol for this difficult crop (Steen and Pedersen, 1993). On the basis of the results of the preliminary investigation described here, a commercial application for this stomatal guard cell system has also become feasible (Hall et al., 1996).

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