Europ. J. Agronomy 22 (2005) 131–139

Plant responses of quinoa (Chenopodium quinoa Willd.) to frost at various phenological stages S.-E. Jacobsen a,∗ , C. Monteros a,1 , J.L. Christiansen b , L.A. Bravo c , L.J. Corcuera c , A. Mujica d b

a International Potato Center (CIP), Apartado 1558, Lima 12, Peru Department of Agricultural Sciences, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark c Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Casilla 160-C, Concepción, Chile d Proyecto Quinua CIP-DANIDA, Universidad Nacional del Altiplano, Puno, Peru

Received 4 April 2003; received in revised form 4 December 2003; accepted 21 January 2004

Abstract Frost is one of the principal limiting factors for agricultural production in the high Andean region. One of the most important grain crops in that region, quinoa (Chenopodium quinoa Willd.), is generally less affected by frost than most other crop species, but little is known about its specific mechanisms for frost resistance. This study was undertaken to help understand quinoa’s response to various intensities and durations of frost under different levels of relative humidity (RH). The effect of frost on seed yield and plant death rate was studied, and content of soluble sugars, proteins, and free proline, was analyzed, in order to develop criteria for the selection of cultivars with improved resistance to frost. On the basis of greenhouse and phytotron experiments, it was concluded that at the two-leaf stage, cultivars from the altiplano of Peru, 3800 m above sea level, tolerated −8 ◦ C for 4 h, whereas a cultivar from the Andean valleys tolerated the same temperature for only 2 h. At −4 ◦ C, plant death rate increased from 25% at high relative humidity to 56% at low RH After a frost treatment of −4 ◦ C applied at the two-leaf stage, final seed yield was reduced by 9% compared to control plants not exposed to frost. For the same treatment applied at the 12-leaf and flowering stages, yield reductions were 51 and 66%, respectively, indicating that frost for 2 h or more during anthesis caused significant damage to the plants. In general, an increased level of soluble sugars implied a greater tolerance to frost, resulting in higher yields. © 2004 Elsevier B.V. All rights reserved. Keywords: Quinoa; Soluble sugar; Proline; Freezing tolerance; Frost duration; Frost intensity; Phytotron

∗ Corresponding author. Present address: Department of Agricultural Sciences, Royal Veterinary and Agricultural University, Højbakkegaard Alle 9, DK-2630 Taastrup, Denmark. Tel.: +45-35283388; fax: +45-35283384. E-mail address: [email protected] (S.-E. Jacobsen). 1 Present address: Instituto Nacional de Investigaciones Agropecuarias (INIAP), Apartado Postal 17-17-1362, Quito, Ecuador.

1161-0301/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.eja.2004.01.003

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1. Introduction Agriculture in the Andean highlands is characterized by a high degree of risk due to a range of adverse climatic factors such as drought, frost, wind, hail, and soil salinity (Mujica and Jacobsen, 1999; Jensen et al., 2000; Jacobsen et al., 2003; Garcia et al., 2003). A certain degree and duration of frost is lethal to most organisms, due to dehydration of the intracellular environment and physical damage by ice crystals. This adverse factor is of great importance in the Andes with significant diurnal temperature variations, and frost at night up to 200 days a year. Temperatures below 0 ◦ C normally occur in the Andean region for a period of time between 12 p.m. and 6 a.m. (Grace, 1985; Capelo, 1993). The two most common types of frost are radiative and convective, also known as white and black frost, respectively. White frost, which causes relatively little damage in nature, occurs under high relative humidity (RH) and a relatively high dew point temperature (Ruiz, 1995). With this type of frost, water vapor condenses and freezes on the leaf surface, causing the release of heat and a gradual cooling of the environment. Black frost occurs when the air is dry, with temperatures not reaching the dew point temperature. In this case, the water in the leaf tissue freezes rather than the water vapor. Because there is no atmospheric vapor to alleviate this phenomenon, the air temperature drops rapidly. At sunrise the following morning, the ice evaporates quickly, leaving necrotic spots in the foliage (Ruiz, 1995). In the Andean highland adverse factors such as frost and drought affect the length of the growth season and the crops and varieties to grow. The process of acclimation, for instance to freezing temperature, is important in many crops. Some crops, e.g. maize, rice, cotton, cucumber and tomato, are commonly chilling-sensitive, due to their tropical and subtropical origin. When the temperature drops slightly below the growth optima, the photosynthesis rate decreases, and the critical chilling stress temperature leads to a marked reduction of photosynthesis (Wise and Naylor, 1987). In wintersown crops, such as cereals and oil seed rape, a cold hardening (acclimatization) takes place under natural conditions in the autumn when the temperature gradually decreases to 0 ◦ C over several weeks. Temperatures of 2–5 ◦ C and photoperiods of about 12 h are considered to be optimal for

cold hardening. During cold acclimation a complex of responses in plants at the cellular, physiological and developmental levels take place. The adaptation processes related to frost resistance are regulated in a polygenic manner (Galiba et al., 2001). Freezing tolerance is composed of at least two independent genetic components, non-acclimated freezing tolerance and acclimation capacity (Stone et al., 1993). The strategy of adaptation consisting of autumn accumulation of reserves and their subsequent utilization during hibernation (wintersleep) may be considered common for even remote organisms. This becomes possible as a result of higher thermoresistance of photosynthesis, as compared to respiration, and low light requirements for photosynthesis saturation at low positive or negative temperatures (Klimov, 2003). Klimov (2003) also mentioned that the absolute values of the photosynthesis/respiration ratio in the hardened plants was 1.5–2 times higher than non-hardened plants. Even in frost tolerant winter varieties a certain period of growth at low, but non-freezing temperature is required for the development of frost hardiness (Janda et al., 2003). This cold acclimation includes several physical and biochemical processes, including changes in membrane composition (Nishida and Murata, 1996) and the accumulation of protective compounds, such as carbohydrates, abscisic acid (ABA), free amino acid and polyamines (Racz et al., 1996). Studies on potato (Solanum tuberosum L.) (Stone et al., 1993) and Brassica rapa (Teutonico et al., 1995) showed that inherent and acclimation-specific freezing tolerance are under separate genetic control. This was also shown for oil seed rape (Brassica napus L.) (Hawkins et al., 2002). During acclimation to drought stress, it has been demonstrated that several metabolic and physiological changes that may increase resistance to desiccation occur, and that some of these changes may be common to various adverse factors. An important common response to drought and cold stress seems to be the increased accumulation of sugars, for instance in oat (Avena sativa L.), rye (Secale cereale L.) and other crops (Alberdi et al., 1993; Koster and Lynch, 1992; Alberdi and Corcuera, 1991). Rapacz (1999) showed that the progress of the cold acclimation process may lead to an increase in soluble sugar content in oil seed rape. Proline and sucrose contents increased with stress duration, while the content of

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glycine betaine increased only after 12 h. at −2.0 MPa. The root, on the other hand, is a critical part of the plant with respect to cold resistance. It has no ability to cold hardening, so its cold resistance is mechanical (Huyghe, 1988). Changes in glucose, fructose and sucrose contents were related to variations in freezing tolerance of seedlings of cabbage (Brassica oleracea L.) (Sasaki et al., 1998). The amounts of organic acids and carbohydrates in water-stressed cotton plants were two to five times greater than those in irrigated plants (Timpa et al., 1986). Proline (Vartanian et al., 1992), amino acids (Good and Zaplachinski, 1994), and betain (Hanson and Hitz, 1982) also accumulated in plants subjected to drought stress. In general, solutes lower the osmotic potential, which in fact seems to be related to freezing tolerance of plants. Besides the colligative effects, sugars protect the plasma membranes and proteins from freezing and dehydration (Sakai and Yoshida, 1968; Santarius, 1973; Steponkus, 1984). Proline accumulation has often been observed to occur in plants subjected to environmental stresses (Aspinall and Paleg, 1981). Rapid accumulation of free proline in tissues of many plant species as a response to salt, drought or temperature stress, has been associated with the ability of proline to act as an osmolyte (Steward and Lee, 1974; Voetberg and Sharp, 1991), as protective agent for cytoplasmatic enzymes (Paleg et al., 1984), as reservoir of nitrogen and carbon sources for post-stress growth (Fukutaku and Yamada, 1984), or as a stabilizer of protein synthesis (Kardpal and Rao, 1985). Quinoa is a highly nutritious seed crop (RepoCarrasco et al., 2003) mainly cultivated in the Andean region at altitudes from 2500 to 3800 m, indicating a risk of night frost over the entire growth period (Gandarillas, 1979). However, the periods of major frost risk are in the beginning of the growth season, and towards the end when the winter starts. The minimum temperature which quinoa cultivars can resist in various phenological stages therefore may define its adaptation to specific agroecological zones. Canahua and Rea (1979) mentioned that in the vegetative stage from five leaves quinoa does not suffer frost damage, but at flower bud formation and anthesis it is susceptible. Catacora and Canahua (1991) showed that quinoa selected for resistance to frost tolerated temperatures down to −16 ◦ C in the vegetative stage. Tapia (1979) found that even a mild frost

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of −2 ◦ C at anthesis caused serious damage to the crop. The objectives of the present study were to determine (1) the damage caused to quinoa by various intensities and durations of frost under different relative humidities, (2) the effect of frost intensity at different phenological stages, and (3) the solute content in a valley and altiplano cultivars of quinoa as a response to frost treatment.

2. Materials and methods All experiments were carried out in a greenhouse and phytotron at the International Potato Center (CIP) in La Molina, Lima, Peru (arid lowland climate, altitude 280 m, latitude 12◦ 05 S) during the winter season (temperate to cool) in 1999. The quinoa plants were grown in greenhouse, except for the period of the frost treatment, under natural light conditions. Sowing took place in March, at an average temperature of 23.9 ◦ C, and harvest was performed in October. Minimum average temperature occurred in July with 16.5 ◦ C, and relative humidity ranged between 76 and 89%. As frost in nature, where quinoa is grown, always occurs at night, the phytotron was kept in darkness under the frost treatment. No major temperature variations were detected inside the pot, thus simulating field conditions where only gradual variations in temperature occur in the soil. 2.1. Experiment 1 Frost was applied in the two-leaf stage at 90% RH Plant death rate at three temperatures (−2, −4, and −8 ◦ C) and three frost durations (2, 4, and 6 h) was studied in a Peruvian valley cultivar (Quillahuaman), adapted to altitudes from 2500 to 3000 m above sea level, and four cultivars from the Peruvian altiplano, situated 3800 m above sea level, three of them local landraces (Wariponcho, Witulla, Ayara), and one selected line (LP-4B). The levels and durations of frost were selected according to natural conditions, prevalent on the altiplano during the growth season (Grace, 1985). Death rate was evaluated 1 week after frost treatment. Plants were regarded as dead when they showed strong plasmolysis of leaves and stem, with the upper part of the plant hanging down. Data were

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analyzed using a factorial design with 45 treatments and three replications (5 cultivars × 3 tempeartures × 3 duration). 2.2. Experiment 2 In the second experiment, the same valley cultivar (Quillahuaman) and one of the altiplano cultivars (Witulla) were studied at two phenological stages (12 leaves and anthesis) at different temperatures (−2 and −4 ◦ C), frost durations (2, 4, and 6 h), and RH (60 and 90%). Data were analyzed using a factorial design with 48 treatments and three replications (2 cultivars × 2 RH × 2 phenological stages × 2 temperatures × 3 durations). Plant death was assessed, and the content of soluble sugars, proline, and soluble protein was analyzed. 2.3. Experiment 3 The third experiment was conducted at 75% RH, and the temperature treatment was applied for 4 h. The same two cultivars as in experiment 2 were exposed to three temperatures (19, −2, and −4 ◦ C), at three phenological stages (2-leaf and 12-leaf stage, and anthesis). Data were analyzed using a factorial design with 18 treatments and three replications (2 cultivars × 3 phenological stages × 3 temperatures). Seed yield and content of soluble sugars, proline, and soluble protein was measured.

stem region. The material was lyophilized, crusted in a mortar, and 0.1 g of material was weighed out. 2.5.1. Soluble sugar analysis The plant material was homogenized in a mortar with 1 ml of 85% ethanol, and centrifuged for 15 min at 8500 rpm. The 100 ␮l of the supernatant were mixed with 3 ml of anthrone reagent and allowed to react in boiling water for 10 min. When the sample was cooled down to ambient temperature, absorbance was measured by spectrophotometer at 625 nm, according to Bravo et al. (1998). 2.5.2. Proline analysis The plant material was crushed in a mortar with 10 ml sulfosalicylic acid and centrifuged at 8500 rpm for 15 min. The 3 ml of the supernatant was mixed with 3 ml ninhydrin and 3 ml acetic acid, and incubated for 1 h at 100 ◦ C. The 6 ml toluene was added, and absorbance was determined by a spectrophotometer at 520 nm. Proline content was derived from a standard curve obtained with pure proline (Merck KGaA, Darmstadt, Germany), according to Bates et al. (1973). 2.5.3. Soluble protein analysis The plant material was homogenized in a mortar with 1 ml 0.5 M Tris buffer (pH 6.8) and centrifuged for 15 min at 8500 rpm. The 50 ␮l of the supernatant was added 2.5 ml of Bradford reagent (Bradford, 1976), and the mixture was shaken for 30 s. Absorbance was measured at 595 nm.

2.4. Experimental unit 2.6. Statistical analyses In all experiments, the experimental unit comprised of two pots of 5 kg soil, each containing four plants. The soil was prepared with two parts mineral soil, one part compost, and one part sand. Soil moisture was kept at 75% of field capacity, which has shown to be optimal for growing quinoa (Jensen et al., 2000; Jacobsen et al., 2003). Pots were irrigated with a nutrient solution (Pioneer NPK Macro 14-3-23 + Mg combined with Pioneer Micro; pH = 5.5; EC = 1.3).

For the statistical analyses were used analysis of variance. The Duncan test was applied to determine if the means of two treatments were different (SAS, 1988). Percentage values were transformed for normalization.

3. Results 3.1. Plant death

2.5. Chemical analyses For chemical analyses, the leaves and the corresponding part of the stem were removed from the mid

In experiment 1, the five cultivars were not damaged at the two-leaf stage when they were exposed to −2 and −4 ◦ C for up to 6 h (i.e., there were no

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Table 1 Plant death rate (%) in five quinoa cultivars, exposed to −8 ◦ C for different durations of time (experiment 1) Time (h)

Quillahuaman

Wariponcho

LP-4B

Witulla

Ayara

2 4 6

12.50d 25.00b 50.00a

8.33e 12.50d 20.83bc

4.17f 12.50d 20.83bc

0.30g 4.17f 16.67cd

0.30g 4.17f 12.50d

Note: a–g indicate significant differences between treatments (P < 0.01). Table 2 Plant death (%) caused by different combinations of relative humidity (RH), temperature and cultivar, in 12-leave stage (experiment 2) Temperature (◦ C)

RH 60%

90%

2h

4h

Mean

6h

2h

4h

6h

2h

4h

16.7ef 8.3fg

0g 0g

0g 0g

0g 0g

0 0

0 0

6h

−2

Quillahuaman Witulla

0g 0g

−4

Quillahuaman Witulla

0g 0g

33.3cd 8.3fg

50b 25de

0g 0g

25de 8.3fg

25de 16.7ef

0 0

29.2 8.3

37.5 20.8

Mean

Quillahuaman Witulla

0 0

16.7 4.2

33.3 16.7

0 0

12.5 4.2

12.5 8.8

0 0

14.6 4.2

22.9 12.5

0g 0g

8.34 4.17

Note: a–g indicate significant differences between treatments (P < 0.01).

dead plants). At −8 ◦ C, death rate in all cultivars increased with frost exposure, but the valley cultivar was consistently more sensitive than the altiplano cultivars (Table 1). Experiment 2 showed that the severity of frost was influenced by RH, with a stronger effect under dry conditions. Death rate was significantly affected by the duration of the frost (Table 2). Experiment 2 showed the effect of time of frost exposure, and with both cultivars suffering a greater plant death rate at flowering

at 4 h and −4 ◦ C (75 and 63% for the valley and altiplano cultivar, respectively) (Table 3) compared with the 12-leaf stage (29 and 8% for the valley and altiplano cultivar, respectively) (Table 2). In general, the valley cultivar Quillahuaman was twice as much negatively affected by frost than the altiplano cultivar Witulla. At −2 ◦ C there was almost no damage at the 12 leaf stage, whereas in anthesis there was some damage to be seen in the valley cultivar. At −4 ◦ C, on the other hand, there was some damage at

Table 3 Plant death (%) caused by different combinations of relative humidity (RH), temperature and cultivar, in anthesis (experiment 2) Tempearture (◦ C)

Cultivation/duration

RH 60% 2h

90% 4h

−2

Quillahuaman Witulla

0g 0g

50b 0g

−4

Quillahuaman Witulla

100a 41.7bc

100a 100a

Mean

Quillahuaman Witulla

50 20.8

75 50

6h 50b 16.7ef 100a 100a 75 58.3

Note: a–e indicate significant differences between treatments (P < 0.01).

2h

Mean 4h

6h

2h

4h

6h

0g 0g

25de 0g

33.3cd 8.3fg

0 0

37.5 0

41.7 12.5

16.7ef 0g

50b 25de

100a 33.3cd

58.3 20.8

75 62.5

100 66.7

8.3 0

37.5 12.5

66.7 20.8

29.2 10.4

56.3 31.3

70.8 39.6

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Table 4 Content of soluble sugars, proline, and soluble protein at different phenological stages and at two temperatures, after exposure to frost (mean of −2 and −4 ◦ C), means of two RH and three frost durations, in two cultivars of quinoa (experiments 2 and 3)

Phenological stage 12 leaf Flowering Temperature (◦ C) −4 19

Cultivar

Soluble sugar (mg/g dry weight)

Proline (␮g/g dry weight)

Soluble protein (mg/g dry weight)

Quillahuaman Witulla

2.9b 3.2a

23.1b 18.3c

14.7b 16.6a

Quillahuaman Witulla

2.6c 2.7bc

27.7a 22.3b

5.5c 6.3c

Quillahuaman Witulla

3.1b 3.3a

32.3a 25.0b

10.6b 12.1a

Quillahuaman Witulla

2.4d 2.6c

18.5c 15.6d

9.6b 10.9a

Note: a–d indicate significant differences between treatments (P < 0.01). Ser´ıa bueno poner los valores ± S.E., adem´as de las letras de significancia estad´ıstica.

the 12 leaf stage. At anthesis almost complete death of Quillahuman was observed. Witulla resisted frost at high RH even in anthesis. In the 12-leaf stage there was no effect of a 2 h frost (Tables 2 and 3). 3.2. Sugar, proline, and protein content The content of soluble sugars was higher in the altiplano cultivar than in the valley cultivar, both at ambient temperature and after frost treatment (experiments 2 and 3, Table 4). There were highly significant differences for proline content for all factors studied, being higher at flowering than 12-leaf stage, and higher in the valley than in the altiplano cultivar, and higher with exposure to frost (Table 4). Soluble protein content was higher at the 12 leaf than at the flowering stage, and it was higher in the altiplano cultivar. Protein content did not increase significantly when exposed to frost (Table 4).

3.3. Seed yield and seed size There were highly significant differences for cultivar, temperature, and phenological stage with respect to seed yield in experiment 3. When comparing the effect of frost damage at different phenological stages, it was found that plants at the two-leaf stage were only slightly affected by −4 ◦ C, but at the twelve-leaf stage and at anthesis yield decrease was as high as 51 and 66%, respectively (Table 5). Frost treatment (−4 ◦ C) seriously affected the valley cultivar, which yielded 56% less than the control. The altiplano cultivar was less affected by this temperature, yielding 27% less than the control (Table 5). Seed size was little affected by frost at either the two-leaf or the twelve-leaf stage, whereas at flowering both −2 and −4 ◦ C reduced size significantly (Table 6). The valley cultivar had a higher percentage of large seeds than the altiplano cultivar.

Table 5 The effect of temperature treatment for 4 h on seed yield in three phenological stages, as an average between two cultivars (Quillahuaman and Witulla), and for each of the cultivars, as an average over phenological stages (experiment 3) Temperature (◦ C)

−2 −4 19

Seed yield (mg/pl.) 2 Leaves

12 Leaves

Flowering

1026ab 998ab 1109a

953ab 529c 1084a

869b 369c 1073a

Note: a–d indicate significant differences between treatments (P < 0.01).

Quillahuaman

Witulla

884bc 492d 1121a

1014ab 772c 1056a

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Table 6 The effect of short-term temperature treatment of plant at three phenological stages on seed size (percentage >1.7 mm) Temperature (◦ C)

−2 −4 19

Seed size (%) 2 Leaves

12 Leaves

Flowering

76.2a 76.3a 76.7a

74.9a 75.5a 77.2a

67.4b 60.3c 77.0a

Quillahuaman

Witulla

89.9b 86.2c 94.4a

55.8e 55.2e 59.4d

Note: a–e indicate significant differences between treatments (P < 0.01), Temperature treatments lasted 4 h. Results are expressed as an average between two cultivars (Quillahuaman and Witulla), and for each of the cultivars, as an average over phenological stage (experiment 3).

4. Discussion The risk of cellular tissue death due to extracellular ice formation and dehydration of the tissue may increase with longer frost periods and lower temperatures. At the two-leaf stage, the five quinoa cultivars were damaged only when exposed to −8 ◦ C. Limache (1992) found that at the six-leaf stage 16 ecotypes suffered a damage rate of 3–27% at that temperature, which fits well with the data presented here. At −4 ◦ C and 60% RH, as an average over phenological stage, cultivar and frost duration, just over half of the plants died, while at the same temperature but at 90% RH the death rate was only 25%. Phenological stage seems to be important regarding the degree of frost damage, as plants were much more affected at the twelve-leaf stage and at anthesis than at the two-leaf stage. Canahua and Rea (1979) found that in the cotyledoneous, two- and five-leaf stage, quinoa demonstrated frost resistance, with no damage from low temperatures, whereas frost exposure during flower bud formation and anthesis had a serious negative effect. Limache (1992) concluded that quinoa resists frost without major damage before the flower bud formation phase, but is susceptible to frost during and after anthesis. The results presented here have confirmed that quinoa is most susceptible to frost from the flower bud formation stage onwards, and much less susceptible in the vegetative stages. The variation in frost tolerance between the valley and the altiplano cultivars of quinoa could be due to differences in the level of soluble sugar, proline and protein content, and thus frost tolerance may be partly attributed to an increased level of solutes, which protect and support cellular structures under frost stress.

An accumulation of compatible solutes, abscisic acid, soluble proteins, and specific proteins, may help to avoid the alteration of the permeability of the cell membranes, caused by dehydration when extracellular ice is forming (Levitt, 1980; Guy, 1990). Under frost, the two cultivars accumulated soluble sugars and proteins. However, it should be stated that the solute contents have been calculated on a dry matter basis, not in relation to the symplastic water. It means that the small differences in sugar and protein concentration, although significant, between the two cultivars, might be removed when considered on a plant water basis. The altiplano cultivar has a slightly higher water content than the valley cultivar. This should be further investigated. Thus, high sugar content, whether due to treatment or cultivar effects, was associated with greater hardiness. It is suggested that the level of soluble sugar may be used as an indicator of frost resistance, as the content of soluble sugars was positively correlated to yield (r = 0.688). The variation in tolerance to freezing in the two quinoa cultivars could be explained partly by increased content of soluble sugars and proteins in the altiplano landrace Witulla, compared to the valley cultivar Quillahuaman. Unexpectedly, basal proline content was higher in the valley than in the altiplano cultivar. It would be of interest to study the capacity of different cultivars to accumulate proline at low temperature. Frost was more detrimental during anthesis than during the vegetative stage, that is, frost at the end of the growth season is of higher risk to the crop than in the early stages of growth. This emphasizes the importance of earliness when selecting plant material for new varieties, as this is a mechanism of frost escape.

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