Journal of Plant Physiology 178 (2015) 10–16
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Expression of genes for the biosynthesis of compatible solutes during pollen development under heat stress in tomato (Solanum lycopersicum) E. Sangu a , F.I. Tibazarwa a , A. Nyomora a , R.C. Symonds b,∗ a b
University of Dar-es-Salaam, Botany Department, PO Box 35060, Dar-es-Salaam, Tanzania The World Vegetable Center, 60 Yi-Min Liao, Shanhua, Tainan 74151, Taiwan
a r t i c l e
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Article history: Received 16 October 2014 Received in revised form 13 February 2015 Accepted 13 February 2015 Available online 20 February 2015 Keywords: Compatible solute accumulation Gene expression Heat stress tolerance Pollen viability Solanum lycopersicon
a b s t r a c t Accumulation of compatible solutes is considered a key adaptation mechanism in many plants in response to abiotic stress. The expression of four genes, involved in sucrose metabolism (SPS and SuSy), biosynthesis of galactinol (GoLS1) and proline accumulation (P5CS) was compared: at meiosis (MM), vacuolated and mature stages of pollen development in heat tolerant and heat sensitive tomato genotypes. The results showed differences in gene expression across tomato genotypes and stages of pollen development. Three genes (P5CS, SPS and SuSy) were up regulated in heat tolerant genotype CLN1621L at the mature stage and one gene (P5CS) in genotype CLN5915-93D at the MM stage. Two genes (SPS and GoLS1) were down regulated in heat sensitive genotype CA4 and one gene (GoLS1) in genotype CLN2498E at the MM stage. Additionally, the continuous exposure of tomato genotypes to temperatures of 35 ◦ C/28 ◦ C day/night completely impaired flower development in genotypes CA4 and CLN2498E but not in genotypes CLN1621L and CLN5915-93D. Tomato genotypes CLN1621L and CLN5915-93D produced fully developed flowers containing mixture of non viable pollens and very few viable pollens grains. Membrane permeability was affected at all stages of development under heat stress with heat tolerant genotypes CL5915-93D4, CLN2498E and CLN1621L showing varying degrees of heat acclimation. Significant increases in total chlorophyll were seen in all genotypes in response to heat stress. The expression of compatible solute genes at MM is more critical than at mature stage for the development of viable pollen grain. © 2015 Elsevier GmbH. All rights reserved.
Introduction Heat stress is one of the most limiting environmental factors for agriculture, accounting for significant crop losses worldwide. Agricultural production and yield are predicted to be affected by increasing temperatures resulting from global warming (Ainsworth and Ort, 2010), with the rate of increase in the production of major crops decreasing (Fischer and Edmeades, 2010).
Abbreviations: FDA, fluorescein diacetate; GoLS1, galactinol synthase; KGMN, Kigamboni; LSD, least significant difference; MP, mature pollen; MM, meiosis stage; P5CS, pyrroline 5 carboxylate synthase; RCBD, randomized complete block design; REST, Relative Expression Software Tool; RI, relative injury; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; UDSM, The University of Dar-es-Salaam; VM, vacuolated stage. ∗ Corresponding author. Present address: School of Biosciences, The University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Darul Ehsan, Malaysia. Tel.: +60 38724 8138; fax: +60 38924 8018. E-mail address: [email protected] (R.C. Symonds). http://dx.doi.org/10.1016/j.jplph.2015.02.002 0176-1617/© 2015 Elsevier GmbH. All rights reserved.
Temperatures in the tropics and subtropics are predicted to exceed current maximums by the end of the 21st century (Battisti and Naylor, 2009) and heat wave events are set to become more frequent (Tebaldi et al., 2006). Substantial crop losses due to elevated temperatures have been predicted in both temperate and subtropical areas and there is an urgent need to mitigate against these losses through agricultural policy and crop adaptation (Teixeria et al., 2013). Sexual reproduction is highly sensitive to heat stress, affecting flower development and number, and pollen production and viability. This in turn results in reduced seed set and yield (Prasad et al., 2006; Das et al., 2014). It has been recognized as the most heat stress susceptible phase in cereals (Monterroso and Wien, 1990; Barnaba et al., 2008) and vegetables (Erickson and Markhart, 2002), with male reproductive stages more sensitive to heat stress than female or vegetative stages of growth (Sakata and Higashitani, 2008). Tomato (Solanum lycopersicum) is among the most popular and the second most consumed vegetable worldwide after potatoes (FAO, 2005). It grows under diverse environmental conditions worldwide with optimal temperatures of 25–30 ◦ C day and 20 ◦ C
E. Sangu et al. / Journal of Plant Physiology 178 (2015) 10–16
night (Camejo et al., 2005). Tomato production in tropical and subtropical areas is constrained by above optimal ( 0.001). All genotypes showed varying degrees of cell membrane sensitivity to heat stress across all stages of tomato development. CLN1621L exhibited relatively low cell membrane damage across 36 ◦ C and 41 ◦ C temperature treatment (grand mean of 3.856% and 13.409%, respectively) while CLN2498E exhibited high cell membrane damage (grand mean 4.82% and 21.029%), (Table 1a). Genotype CA4 exhibited low cell membrane damage at seedling and vegetative stages whilst the damage increased significantly at the flowering stage (P > 0.001). Genotypes CL591593D4, CLN1621L, and CLN2498E exhibited high cell membrane damage at the seedling stage which decreased through the vegetative and flowering stages. However, the damage in genotype CLN1621L increased slightly at the flowering stage at 41 ◦ C. The controlled growth room study showed a similar pattern with all genotypes exhibiting significant increases in cell membrane damage at the seedling stage (P > 0.001), with the largest increase seen in CA4 (Table 1b). However no genotype showed an increase in membrane damage at the flowering stage (F = 0.30 ns) (Table 1b). These observations indicate varying degrees of acclimatization response in CL5915-93D4, CLN2498E and CLN1621L but not in CA4,
E. Sangu et al. / Journal of Plant Physiology 178 (2015) 10–16
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Table 1a Mean percentage of electrolyte leakage for the four tomato genotypes sampled from field site USDM Tanzania, at seedling, vegetative and flowering stages. Measured after treating leaf samples with either 36 ◦ C or 41 ◦ C water bath temperature conditions for 1 h. Mean electrolyte leakages (%) Genotype
Seedling
Vegetative
◦
CLN1621L CL5915-93D4 CLN2498E CA4 Grand mean SEM F * **
◦
Flowering
◦
◦
◦
Grand mean ◦
◦
Grand mean
36 C
41 C
36 C
41 C
36 C
41 C
36 C
41 ◦ C
7.27 ± 1.37 6.39 ± 2.86 6.5 ± 2.18 4.46 ± 1.77 6.15 0.68 3.10*
12.66 ± 6.29 24.68 ± 21.43 27.39 ± 17.62 9.63 ± 3.62 18.59 4.68 3.50*
2.87 ± 1.26 4.47 ± 2.50 6.31 ± 3.44 3.67 ± 2.06 4.33 0.78 3.57*
12.37 ± 2.63 17.35 ± 6.70 17.79 ± 5.45 11.92 ± 3.96 14.860 1.60 3.84*
1.42 ± 0.65 2.30 ± 1.07 1.65 ± 0.55 5.96 ± 1.07 2.83 0.45 22.59**
15.19 ± 3.11 15.16 ± 4.15 17.90 ± 9.89 25.52 ± 20.34 18.44 3.77 1.68 ns
3.86 4.38 4.82 4.70 4.44
13.41 19.06 21.03 15.69 17.30
Indicates significant differences in electrolyte leakage across growth stages and genotypes. Indicates highly significant differences in electrolyte leakage across growth stages and genotypes.
Table 1b Mean percentage of electrolyte leakages for the four tomato genotypes measured from leaf samples collected from controlled temperature growth chambers set at 35 ◦ C/28 ◦ C day/night and 24 ◦ C/18 ◦ C day/night temperature conditions at the seedling and flowering stage. Mean electrolyte leakages (%) Genotype
Seedling ◦
CLN1621L CL5915-93D4 CLN2498E CA4 Grand mean SEM F * **
Flowering ◦
◦
◦
◦
◦
◦
◦
24 C/18 C
35 C/28 C
24 C/18 C
35 C/28 C
11.57 ± 2.51 4.88 ± 1.85 10.05 ± 2.59 6.50 ± 3.97 8.25 0.99 9.69**
7.18 ± 2.94 8.50 ± 1.63 10.16 ± 2.85 13.42 ± 3.67 9.92 1.25 4.58*
10.92 ± 2.52 11.04 ± 4.23 14.34 ± 2.09 17.26 ± 5.38 13.39 1.56 3.75*
12.16 ± 3.92 12.32 ± 0.94 14.80 ± 2.61 18.19 ± 2.84 14.02 1.41 2.45
Grand mean 10.46 9.18 12.34 13.84 11.45
Indicates significant differences in electrolyte leakage across growth stages and genotypes. Indicates highly significant differences in electrolyte leakage across growth stages and genotypes.
leading to a relative increase of cell membrane thermostability at the flowering stage.
Total chlorophyll content Total leaf chlorophyll content was measured at seedling, vegetative and flowering stages on plants subjected to control (24 ◦ C/18 ◦ C) and heat stress (35 ◦ C/28 ◦ C) conditions. At all stages measured; seedling, vegetative and flowering, there was a highly significant increase in chlorophyll content in the plants subjected to heat stress compared to the control (P > 0.001) in all genotypes (Table 2). Heat sensitive genotype CA4 had consistently higher leaf chlorophyll content when plants were subjected to heat stress at all stages compared to heat tolerant genotypes CLN1621, CL5915 and CLN2498E (P > 0.001) (Table 2).
Number of flowers, fruit and percentage fruit set As shown in Table 3, the tomato genotypes produced variable numbers of flowers across treatments and environments. More flowers were produced when plants were under heat stress (the hot season) than under normal conditions (the cool season) (P > 0.001), however this did not correspond to total number of fruit per plant, with significantly fewer fruit being produced under heat stress (P > 0.001). Heat sensitive CA4 produced average of 2 fruits per plant under heat stressed conditions and average of 20 fruits per plant under normal conditions (Table 3). The percentage fruit set averaged between 55 and 75% in all genotypes during the cool season but was significantly lower, ( 0.001). A significant decrease was noted in heat sensitive genotype CA4 with a fruit set percentage of 55% in the cool season as compared to 3.14–6.40% during the hot season. Heat stress
tolerant CLN 1621L exhibited the highest fruit set (46%) followed by CL 5915-93D4 (36%) in the hot season. Pollen number and viability The genotypes produced variable numbers of pollen grains of different degrees of viability in the control experiment. CLN2498E produced the largest amount of pollen (29,481) followed by CL591593D (11,522) then CLN1621L (10,007) and CA4 (6910) (Table 4). Pollen viability was highest in CL5915-93D (85.84%) and lowest in CA4 (64.91%) (Table 4). Two genotypes CLN1621L and CL5915-93D produced a mixture of viable and non viable pollen grains with non viable pollen grains being the majority under heat stress conditions. Viable pollen grains were not detected under the microscope in CLN1621L and CL5915-93D under conditions of heat stress but were inferred from seeded fruits that were produced (Table 4). The complete development of floral structures to full maturity suggests the presence of unique metabolic adjustments in the heat tolerant lines CL5915-93D and CLN1621L. Genotypes CA4 and CLN2498E failed to produce fully developed flowers and therefore no pollen grains were collected. It was further observed that the stamens in genotype CA4 were degraded. Gene expression in tomato lines and pollen development stages The expression of compatible solute genes SPS, SuSy, P5CS and GoLS1 were compared in terms of relative gene expression ratios. Only the normalized expression ratios are presented, however the non-normalized data produced a similar trend (data not shown). Three genes: SPS, P5CS and SuSy were up-regulated in two heat tolerant genotypes CL5915-93D and CLN1621L at different stages of pollen development. Three genes: SPS, P5CS, GoLS1 were down-regulated in heat sensitive genotypes CA4 (SPS and
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Table 2 Mean chlorophyll values for four tomato genotypes measured at the seedling, vegetative and flowering stages from controlled temperature growth chambers set at 35 ◦ C/28 ◦ C day/night and 24 ◦ C/1 ◦ C day/night temperature conditions. Seedling
Genotype
Vegetative
24 ◦ C/18 ◦ C CA4 CLN1621L CL5915-93D4 CLN2498
40.63 33.56 30.22 36.09
± ± ± ±
35 ◦ C/28 ◦ C
0.46 0.51 0.95 0.5
57.22 51.35 51.84 46.78
± ± ± ±
Flowering
24 ◦ C/18 ◦ C
3.84 2.74 3.45 1.99
43.86 37.74 30.68 40.87
± ± ± ±
35 ◦ C/28 ◦ C
1.30 1.98 0.62 0.29
60.98 48.05 43.58 47.03
± ± ± ±
4.22 1.82 3.32 1.94
24 ◦ C/18 ◦ C 45.13 41.08 30.68 41.12
± ± ± ±
35 ◦ C/28 ◦ C
3.61 1.92 1.52 1.26
60.81 48.01 45.02 48.22
± ± ± ±
5.63 4.42 2.27 1.93
Table 3 Mean comparisons of the number of flowers, number of fruit and percentage fruit set of four tomato genotypes across breeding seasons (cool and hot). Genotypes
CL5915-93D4 CLN1621L CA4 CLN2498E LSD
Number of flowers Season
Number of fruit Season
% Fruit set Season
Cool UDSM
Hot 1 Ruvu
Hot 2 KGMN
Cool UDSM
Hot 1 Ruvu
Hot 2 KGMN
Cool UDSM
Hot 1 Ruvu
Hot 2 KGMN
47.83 ± 9.62 46.17 ± 6.87 25.00 ± 3.19 31.92 ± 4.27 37.75b
45.00 ± 7.99 49.67 ± 16.43 32.83 ± 6.90 41.92 ± 5.00 42.50b
67.33 ± 10.24 58.67 ± 7.35 63.67 ± 5.87 47.67 ± 6.77 59.25a
34.33 ± 6.98 30.50 ± 7.13 13.92 ± 4.96 20.17 ± 3.33 24.75a
16.67 ± 4.36 22.08 ± 7.48 2.00 ± 1.04 15.25 ± 2.09 14.00b
23.83 ± 6.12 26.17 ± 3.97 2.00 ± 1.04 17.08 ± 2.02 17.25b
73.80 ± 16.25 66.24 ± 12.84 54.93 ± 16.31 63.57 ± 8.72 64.75a
37.22 ± 6.96 45.03 ± 7.70 6.40 ± 4.09 36.65 ± 5.29 31.25b
35.45 ± 6.88 45.98 ± 8.88 3.15 ± 1.62 36.47 ± 6.65 30.00b
NB: significance difference is indicated by the letter difference within group i.e. ‘a’ and non-significance difference by letter similarity within group i.e. ‘b’
Table 4 Response of tolerant and sensitive tomato genotypes to a continuous exposure to heat stress, 35 ◦ C/28 ◦ C, compared to control conditions, 24 ◦ C/18 ◦ C. Genotype 24 ◦ C/18 ◦ C CL5915-93D4 CLN1621L CLN2498E CA4 35 ◦ C/28 ◦ C CL5915-93D4 CLN1621L CLN2498E CA4
Number of trusses
Average number of flowers per plant
6 6 3 4
39 44 16 14
± ± ± ±
12 7 2 2.5
45 33 11 6
± ± ± ±
Average number of pollen grains per flower
%Pollen viability
2.08 4.41 3.28 2.02
11,520.67 ± 180.63 9875.03 ± 1883.63 18,419.33 ± 2360.33 6910.00 ± 367.37
85.84 ± 2.80 80.07 ± 4.65 69.49 ± 13.22 70.36 ± 4.67
2.73 4.58 1.20 1.63
2445 ± 1797.44 1500 ± 726.23 ND ND
ND ND 0 0
Table 5 The relative gene expression ratios of compatible solute genes (SPS, GoLS1, P5CS and SuSy) measured in heat tolerant and sensitive tomato genotypes under continuous exposure to heat stress. Pollen development stages are labeled MM, VM and MP. Selected genes
SuSy
Genotypes and development stage
Expression ratio
P
Expression ratio
P
Expression ratio
P
Expression ratio
P
CA4 (MM) CA4 (VM) CA4 (PM) CLN1621L (MM) CLN1621L (VM) CLN1621L (PM) CL5915-93D (MM) CL5915-93D (VM) CL5915-93D (PM) CLN2498E (MM) CLN2498E (VM) CLN2498E (PM)
0.663 1.016 0.617 0.380 1.230 3.286** 1.235 0.555 0.981 1.008 1.108 0.485
0.500 0.966 0.360 0.216 0.431 0.003 0.580 0.184 0.955 0.977 0.815 0.300
0.255* 1.342 0.939 0.305 0.636 0.741 0.423* 0.976 0.282* 0.196* 1.560 1.000
0.007 0.246 0.890 0.064 0.152 0.600 0.018 0.932 0.039 0.006 0.284 0.999
0.014* 1.383 0.768 0.404 1.241 10.758** 4.392 0.205 0.937 1.413 1.294 1.145
0.001 0.296 0.759 0.129 0.508 0.032 0.177 0.062 0.759 0.387 0.797 0.823
1.317 1.719 0.544 0.440 0.673 17.065** 12.059** 0.199* 0.827 2.114 2.402 1.289
0.788 0.259 0.444 0.176 0.312 0.000 0.031 0.047 0.556 0.103 0.329 0.722
* **
GoLS1
SPS
P5CS
Indicates significant down-regulation in heat stressed samples. Indicates significant up-regulation in heat stressed samples.
GoLS1), CLN2498E (P5CS) and one heat stress tolerant genotype CL5915-93D (GoLS1) at the MM stage (Table 5). The SPS gene was significantly up-regulated by a mean factor of 10.758 (P > 0.05) in CLN1621L at the mature stage and down-regulated in CA4 by the mean factor of 0.014 (P > 0.001) at MM stage of pollen development. No significant difference in SPS expression was observed in CLN2498E at any stage of development. The overall expression ratios of SPS gene were higher than that of SuSy and GoLS1 but lower than the expression ratios of P5CS (Table 5). The expression of P5CS was significantly up-regulated by a mean factor of 12.095 (P > 0.05) in CL5915-93D at the MM stage and CLN1621L by a mean factor of 17.065 (P > 0.001) at mature stage
of pollen development. The gene was significantly down-regulated in CL5915-94D by a mean factor of 0.199 (P > 0.05) at the vacuolated stage but showed no significant difference in genotypes CA4 and CLN2498E, at all the three stages of pollen development (Table 5). The expression of GoLS1 gene was relatively low at all the three stages of pollen development with minimum and maximum expression ratios of 0.196 and 1.56, respectively. The gene GoLS1 was significantly down-regulated by mean factors of 0.255 (P > 0.001) in CA4, 0.423 (P > 0.01) in CL5915-93D, and 0.196 (P > 0.01) in CLN2498E at the MM stage. The gene was also downregulated by a mean factor of 0.295 (P > 0.01) at the mature stage but not significant in CLN1621 in all the three stages of pollen
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development and at vacuolated stage in the other genotypes (Table 5). The gene SuSy was not significantly up-regulated in any genotype at any stage of pollen development, with the exception of expression of heat tolerant CLN1621L which was significantly up-regulated by a mean factor of 3.286 (P > 0.001) at the MP stage (Table 5). Discussion Tomato is sensitive to heat stress with temperatures above 27 ◦ C inhibiting pollen development and therefore seeded fruit production (Peet et al., 1997). High temperatures cause a reduction in pollen germination, ovule development and fruit set (Peet et al., 1988). Selection and breeding of plants with tolerance to heat stress is one way to mitigate the adverse effects of increasing global temperatures (Warner and Erwin, 2005). Cell membrane thermostability has been successfully used as a selection criterion for heat tolerant genotypes in wheat (Shanahan et al., 1990), cotton (Rahman et al., 2004) and tomato (Camejo et al., 2005). In agreement with these findings the present study shows a marked genotypic difference in the cell membrane thermostability in response to elevated temperatures, with low levels of cell membrane leakage observed in heat tolerant genotype CLN1621L and acclimation observed in genotypes CL5915-93D4 and CLN2498E. In contrast heat sensitive CA4 suffered cell membrane damage at all growth stages. Whilst cell membrane thermostability is a useful selection criteria for thermotolerance, Blum et al. (1990) cautioned that there are several factors contributing to thermotolerance such as heat avoidance, and therefore cell membrane thermostability should be used in conjunction with other selection criteria for selection of heat tolerant genotypes. There was a significant increase in the total leaf chlorophyll content under heat stress in all genotypes in the present study. The largest increases in chlorophyll content were seen in heat sensitive CA4, which also showed higher levels of leaf chlorophyll content under control conditions. The significantly larger increase in chlorophyll content in this genotype may therefore be a result of the higher basal levels under control conditions, however Camejo et al. (2005) showed a decrease in chlorophyll/carotenoid content in heat tolerant but not heat sensitive lines of tomato in response to heat stress. Previous studies have demonstrated a reduction in fruit setting percentage in tomato in response to heat stress (Dane et al., 1991; Abdul-Baki and Stommel, 1995). This reduction in tomato fruit set in response to heat stress has been attributed to the impairment of pollen development in anthers 8–13 d before anthesis (Iwahori, 1965, 1966). Hanson et al. (2002) observed a lower decrease in percentage fruit set in CL5915-93D4 when compared to a heat sensitive tomato line and concluded that CL5915-93D4 is a valuable source of heat tolerance genetic material. Similarly Comlekcioglu and Soylu (2010) observed that genotypes CL5915-93D4 and CLN1621L retained high levels of fruit set under moderate heat stress, and although percentage fruit set was reduced under high heat stress conditions, levels were higher compared to heat sensitive genotypes. Reductions in percentage fruit set were seen in all genotypes in the present study; however the largest decrease was seen in the heat sensitive genotype CA4. This corresponded with a very low level of pollen viability under heat stress in this genotype. A lower percentage reduction in both fruit set and pollen viability was seen in CLN1621L and Cl5915-93D4 in agreement with Comlekcioglu and Soylu (2010). In the present study it was hypothesized that the expression of the compatible solute genes differ in tomato genotypes at various stages of pollen development with high gene expression corresponding to the production of viable pollen grain and
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consequently high fruit set under heat stress. The up-regulation of P5CS, SPS and SuSy genes in heat tolerant genotypes and not in heat sensitive genotypes suggest the presence of sucrose and proline biosynthesis in the anther tissues during heat stress. The expression of compatible solute genes SPS, P5CS, SuSy, and GoLS1 during pollen development showed clear demarcation between heat tolerant tomato and sensitive tomato cultivars following continuous exposure to heat stress. Three genes (SPS, P5CS and SuSy) were up-regulated by heat stress in heat tolerant lines CLN1621L at the MP stage. Conversely two genes (SPS and GoLS1) were down-regulated in the heat sensitive cultivar CA4. Analysis of the expression patterns in heat tolerant lines showed that the genes were up-regulated at MM (P5CS) in cultivar CL5915-93D and at the MP (SPS, SuSy, and P5CS) in line CLN1621L corresponding to the anther growth phase and anther maturation phases respectively. These findings are in agreement with that of Datta et al. (2002) who reported a temporal change in the expression of carbohydrate genes during a transition from no starch (anther growth) to an active starch-filling (anther maturation) phase in male fertile but, not in male sterile maize genotypes during pollen development. Therefore, maintenance of high levels of soluble sugars is a prerequisite for production of high quality pollen grains (Pressman et al., 2002). According to Datta et al. (2002) the up-regulation of the hexose transporter gene in male fertile, but not in male sterile maize genotypes suggested a potential role for this gene transporting extracellular sugars from the nutrient rich locular fluid to the developing pollen. However, during heat stress the transportation of extracellular sugars and other compatible solutes from the nutrient rich locular fluid to the developing pollen by hexose transporter (Datta et al., 2002) and proline transporter (Sato et al., 2006) is disrupted. In cowpea, male sterility occurs if high temperatures coincide with floral development, due to premature degradation of the tapetum and a lack of endothecial development (Ahmed et al., 1992). In Arabidopsis, heat shock disrupts pollen development in a stage specific manner with critical effects at the meiotic stage of pollen mother cell development (Kim et al., 2001). In rice night temperatures above 35 ◦ C result in a significant reduction in pollen tube length and viability (Das et al., 2014). In the present study, similar effects were observed in tomato genotypes CA4 and CLN2498E which despite producing similar percentages of viable pollen grains under non stress conditions produced no pollen under heat stressed conditions. Tomato lines CLN1621L and CLN5915-93D produced a mixture of very few viable pollen grains and large number of non-viable pollen grains despite having morphologically sound flowers (data not shown). The observed phenomenon could partly be explained by the up-regulation of the three compatible solute genes SPS for sucrose synthesis, SuSy for sucrose cleavage and P5CS for proline synthesis. The observed increase in the number of mRNA transcripts in heat tolerant and not in heat sensitive cultivars suggests the potential for proline synthesis and sucrose re-synthesis in non-photosynthetic tissues. Im (2004), reported in maize, the possibility of sucrose re-synthesis in non-photosynthetic tissues such as anther, subsequent to the failure of the normal functioning of the hexose transporter gene (Datta et al., 2002). The failure of pollen formation and abnormal flower development could potentially be explained by the disruption of sugar metabolism and proline transport (Sato et al., 2006) which affected not only the pollens but also injured the anther cells and tissues (Sakata and Higashitani, 2008). The tolerant tomato cultivars (CL5915-93D and CLN1621L) showed an increased expression of compatible solute genes (SPS, SuSy, and P5CS) in at least in one stage of pollen development, and non-significant expression in either the previous or subsequent stages. These expression patterns suggest that heat tolerant lines have developed a unique metabolic switch
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which activates synthesis of compatible solutes just after the interference of sugar and proline transporter metabolism. Conclusions The data presented in this study emphasize the importance of maintaining appropriate levels of soluble sugars during pollen development under heat stress as exemplified by the increased expression of SPS, P5CS and SuSy genes in heat tolerant lines as opposed to heat sensitive lines. The ability of tomato genotype to achieve soluble sugar homeostasis during heat stress depends upon its ability to switch from metabolic sugar translocation in anther tissues to soluble sugar biosynthesis. However, it was not clear how this expression is regulated under heat stress. Acknowledgements This work was funded by GTZ project No: 07.7860.5-001.00 AVRDC project. The authors would like to thank the molecular biotechnology unit at AVRDC Headquarters, Taiwan and the Botany Department University of Dar es Salaam, Tanzania. References Abdul-Baki AA, Stommel JR. Pollen viability and fruit set of tomato genotypes under optimum and high-temperature regimes. Hortscience 1995;30:115–7. Ahmed FE, Hall AE, DeMason DA. Heat injury during floral development in Cowpea (Vigna Unguiculata, Fabaceae). Am J Bot 1992;79:784–91. Ainsworth EA, Ort DR. How do we improve crop production in a warming world. Plant Physiol 2010;154:526–30. Aloni B, Karni L, Zaidman Z, Schaffer AA. Changes of carbohydrates in pepper (Capsicum annuum L.) flowers in relation to their abscission under different shading regimes. Ann Bot 1996;78:163–8. Barnaba B, Jager K, Feher A. The effect of drought and heat stress on reproductive processes in cereals. Plant Cell Environ 2008;31:11–38. Battisti DS, Naylor RL. Historical warnings of future food insecurity with unprecedented seasonal heat. Science 2009;323:240–4. Bruinsma J. The quantitative analysis of chlorophyll a and b in plant extracts. Photochem Photobiol 1963;2:241–9. Camejo D, Rodriguez P, Morales MA, Dell’Amico JM. High temperature effects on photosynthetic activity of two tomato cultivars with different heat susceptibility. J Plant Physiol 2005;162:281–9. Comlekcioglu N, Kemal Soylu M. Determination of high temperature tolerance via screening of flower and fruit formation in tomato. J Agric Sci 2010;20:123–30. Dane F, Hunter AG, Chambliss OL. Fruit set, pollen fertility, and combining ability of selected tomato genotypes under high-temperature field conditions. J Am Soc Hortic Sci 1991;116:906–10. D’Aoust MA, Yelle S, Nguyen-Quoc B. Antisense inhibition of tomato fruit synthase decreases fruit setting and the sucrose unloading capacity of young fruit. Plant Cell 1999;11:2407–18. Das S, Krishnan P, Nayak M, Ramakrishnan B. High temperature stress effects on pollens of rice (Oryza sativa L.) genotypes. Environ Exp Bot 2014;101:36–46. Datta R, Chamusco KC, Chourey PS. Starch biosynthesis during pollen maturation is associated with altered patterns of gene expression in Maize. J Plant Physiol 2002;130:1645–56. Demnitz-King A, Ho LC, Baker DA. Activity of sucrose hydrolysing enzymes and sugar accumulation during tomato fruit development. Plant Growth Regul 1997;22:193–201. Erickson AN, Markhart AH. Flower developmental stage and organ sensitivity of bell pepper (Capsicum annuum L.) to elevated temperature. Plant Cell Environ 2002;25:123–30. Firon N, Shaked R, Peet MM, Pharr DM, Zamski E, Rosenfeld K, et al. Pollen Grains of heat tolerant tomato cultivars retain higher carbohydrate concentration under heat stress conditions. Sci Hort 2006;109:212–7. Fischer RA, Edmeades GO. Breeding and cereal yield progress. Crop Sci 2010;50:85–98. FAO. Food and Agriculture Organisation of United Nations report; April, 2005, Website database: http://www.fao.org. Giorno F, Wolters-Arts M, Mariani C, Rieu I. Ensuring reproduction at high temperatures: the heat stress response during anther and pollen development. Plants 2013;2:489–506.
Hanson PM, Chen JT, Kuo G. Gene action and heritability of high temperature fruit set in tomato line CL5915. Hortscience 2002;37:172–5. Heslop-Harrison J, Heslop-Harrison Y, Shivanna KR. The evolution of pollen quality and a further appraisal of the fluorochromatic (Fcr) test procedure. Theor Appl Genet 1984;67:367–75. Hong B, Ma C, Yang Y, Wang T, Yamaguchi-Shinozaki K, Gao J. Over-expression of AtDREB1 A in chrysanthemum enhances tolerance to heat stress. Plant Mol Biol 2009;70:239–40. Im KH. Expression of sucrose-phosphate synthase (SPS) in non-photosynthetic tissues of maize. Mol Cells 2004;17:404–9. Iwahori S. High temperature injuries in tomato. Development of normal flower buds and morphological abnormalities of flower buds treated with high temperature. J Jpn Soc Hortic Sci 1965;34:55–62. Iwahori S. High temperature injuries in tomato. V. Fertilization and development of embryo with special reference to the abnormalities caused by high temperature. J Jpn Soc Hortic Sci 1966;35:379–86. Kim SY, Hong CB, Lee I. Heat shock stress causes stage specific male sterility in Arabidopsis thaliana. J Plant Res 2001;114:301–7. Monterroso VA, Wien HC. Flower and pod abscission due to heat stress in beans. J Am Soc Hortic Sci 1990;115:631–4. Panikulangara TJ, Eggers-Schumacher G, Wunderlich M, Stransky H, Schöffl F. Galactinol synthase1. A novel heat shock factor target gene responsible for heat-induced synthesis of raffinose family oligosaccharides in Arabidopsis. Plant Physiol 2004;136:3148–58. Peet MM, Sato S, Gardner RG. Comparing heat stress on male-fertile and malesterile tomatoes to chronic, sub-acute high temperature stress. J Exp Bot 1988;21:225–31. Peet MM, Willits DH, Gardner RG. Responses of ovule development and post pollen production processes in male sterile tomatoes to chronic sub-acute high temperature stress. J Exp Bot 1997;48:101–11. Peet MM, Sato S, Gardner RG. Comparing heat stress effects on male-fertile and male-sterile tomatoes. Plant Cell Environ 1998;21:225–31. Pfaffi MW. A new mathematical model for relative quantification in real time PCR. Nucleic Acids Res 2001;29:2002–7. Porra RJ, Thompson WA, Kriedemann PE. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: Verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 1989;975:384–94. Prasad PVV, Boote KJ, Allen JLH. Adverse high temperature effects on pollen viability, seed-set, seed yield and harvest index of grain-sorghum (Sorghum bicolor (L) Moench) are more severe at elevated carbon dioxide due to higher tissue temperatures. Agric For Meteorol 2006;139:237–51. Pressman E, Peet MM, Pharr DM. The effects of heat stress on tomato pollen characteristics is associated with carbohydrate concentration in the developing anthers. Ann Bot 2002;90:631–6. Rahman H, Malik S, Saleem M. Heat tolerance of upland cotton during the fruiting stage evaluated using cellular membrane thermostability. Field Crops Res 2004;85:149–58. Sakata T, Higashitani A. Male sterility accompanied with abnormal anther development in plants, genes and environmental stresses with special reference to high temperature injury. Int J Plant Dev Biol 2008;2:42–51. Sato S, Peet MM, Thomas JF. Determining critical pre and post-anthesis periods and physiological processes in tomatoes exposed to moderately elevated temperatures. J Exp Bot 2002;53:1187–95. Sato S, Kamiyama M, Iwata T, Makita N, Furukawa H, Ikeda H. Moderate increase of daily mean temperature adversly affects fruit set of Lycopersicon esculentum by disrupting specific physiological processes in male reproductive development. Ann Bot 2006;97:731–8. Sawhney VK, Bhadula SK. Microsporogenesis in the normal and male-sterile stamenless-2 mutant of tomato (Lycopersicum esculentum). Can J Bot 1988;66:2013–21. Shanahan JF, Edwards IB, Quick JS, Fenwick JR. Membrane thermostability and heat tolerance of spring wheat. Crop Sci 1990;30:247–55. Suzuki N, Mittler R. Reactive oxygen species and temperature stresses: A delicate balance between signalling and destruction. Physiol Plantarum 2006;126:45–51. Tebaldi C, Hayhoe K, Arblaster J, Meehl G. Going to extremes: an intercomparison of model-simulated historical and future changes in extreme events. Clim Change 2006;79:185–211. Teixeria EI, Fischer G, Van Velthuizen H, Walter C, Ewert F. Global hot-spots of heat stress on agricultural crops due to climate change. Agric For Meteorol 2013;170:206–15. Verbruggen N, Hermans C. Proline accumulation in plants: a review. Amino Acids 2008;35:753–9. Warner RM, Erwin JE. Naturally occurring variation in high temperature induced floral bud abortion across Arabidopsis thaliana accessions. Plant Cell Environ 2005;28:1255–66.
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Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Expression of genes for the biosynthesis of compatible solutes during pollen development under heat stress in tomato (Solanum lycopersicum) E. Sangu a , F.I. Tibazarwa a , A. Nyomora a , R.C. Symonds b,∗ a b
University of Dar-es-Salaam, Botany Department, PO Box 35060, Dar-es-Salaam, Tanzania The World Vegetable Center, 60 Yi-Min Liao, Shanhua, Tainan 74151, Taiwan
a r t i c l e
i n f o
Article history: Received 16 October 2014 Received in revised form 13 February 2015 Accepted 13 February 2015 Available online 20 February 2015 Keywords: Compatible solute accumulation Gene expression Heat stress tolerance Pollen viability Solanum lycopersicon
a b s t r a c t Accumulation of compatible solutes is considered a key adaptation mechanism in many plants in response to abiotic stress. The expression of four genes, involved in sucrose metabolism (SPS and SuSy), biosynthesis of galactinol (GoLS1) and proline accumulation (P5CS) was compared: at meiosis (MM), vacuolated and mature stages of pollen development in heat tolerant and heat sensitive tomato genotypes. The results showed differences in gene expression across tomato genotypes and stages of pollen development. Three genes (P5CS, SPS and SuSy) were up regulated in heat tolerant genotype CLN1621L at the mature stage and one gene (P5CS) in genotype CLN5915-93D at the MM stage. Two genes (SPS and GoLS1) were down regulated in heat sensitive genotype CA4 and one gene (GoLS1) in genotype CLN2498E at the MM stage. Additionally, the continuous exposure of tomato genotypes to temperatures of 35 ◦ C/28 ◦ C day/night completely impaired flower development in genotypes CA4 and CLN2498E but not in genotypes CLN1621L and CLN5915-93D. Tomato genotypes CLN1621L and CLN5915-93D produced fully developed flowers containing mixture of non viable pollens and very few viable pollens grains. Membrane permeability was affected at all stages of development under heat stress with heat tolerant genotypes CL5915-93D4, CLN2498E and CLN1621L showing varying degrees of heat acclimation. Significant increases in total chlorophyll were seen in all genotypes in response to heat stress. The expression of compatible solute genes at MM is more critical than at mature stage for the development of viable pollen grain. © 2015 Elsevier GmbH. All rights reserved.
Introduction Heat stress is one of the most limiting environmental factors for agriculture, accounting for significant crop losses worldwide. Agricultural production and yield are predicted to be affected by increasing temperatures resulting from global warming (Ainsworth and Ort, 2010), with the rate of increase in the production of major crops decreasing (Fischer and Edmeades, 2010).
Abbreviations: FDA, fluorescein diacetate; GoLS1, galactinol synthase; KGMN, Kigamboni; LSD, least significant difference; MP, mature pollen; MM, meiosis stage; P5CS, pyrroline 5 carboxylate synthase; RCBD, randomized complete block design; REST, Relative Expression Software Tool; RI, relative injury; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; UDSM, The University of Dar-es-Salaam; VM, vacuolated stage. ∗ Corresponding author. Present address: School of Biosciences, The University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Darul Ehsan, Malaysia. Tel.: +60 38724 8138; fax: +60 38924 8018. E-mail address: [email protected] (R.C. Symonds). http://dx.doi.org/10.1016/j.jplph.2015.02.002 0176-1617/© 2015 Elsevier GmbH. All rights reserved.
Temperatures in the tropics and subtropics are predicted to exceed current maximums by the end of the 21st century (Battisti and Naylor, 2009) and heat wave events are set to become more frequent (Tebaldi et al., 2006). Substantial crop losses due to elevated temperatures have been predicted in both temperate and subtropical areas and there is an urgent need to mitigate against these losses through agricultural policy and crop adaptation (Teixeria et al., 2013). Sexual reproduction is highly sensitive to heat stress, affecting flower development and number, and pollen production and viability. This in turn results in reduced seed set and yield (Prasad et al., 2006; Das et al., 2014). It has been recognized as the most heat stress susceptible phase in cereals (Monterroso and Wien, 1990; Barnaba et al., 2008) and vegetables (Erickson and Markhart, 2002), with male reproductive stages more sensitive to heat stress than female or vegetative stages of growth (Sakata and Higashitani, 2008). Tomato (Solanum lycopersicum) is among the most popular and the second most consumed vegetable worldwide after potatoes (FAO, 2005). It grows under diverse environmental conditions worldwide with optimal temperatures of 25–30 ◦ C day and 20 ◦ C
E. Sangu et al. / Journal of Plant Physiology 178 (2015) 10–16
night (Camejo et al., 2005). Tomato production in tropical and subtropical areas is constrained by above optimal ( 0.001). All genotypes showed varying degrees of cell membrane sensitivity to heat stress across all stages of tomato development. CLN1621L exhibited relatively low cell membrane damage across 36 ◦ C and 41 ◦ C temperature treatment (grand mean of 3.856% and 13.409%, respectively) while CLN2498E exhibited high cell membrane damage (grand mean 4.82% and 21.029%), (Table 1a). Genotype CA4 exhibited low cell membrane damage at seedling and vegetative stages whilst the damage increased significantly at the flowering stage (P > 0.001). Genotypes CL591593D4, CLN1621L, and CLN2498E exhibited high cell membrane damage at the seedling stage which decreased through the vegetative and flowering stages. However, the damage in genotype CLN1621L increased slightly at the flowering stage at 41 ◦ C. The controlled growth room study showed a similar pattern with all genotypes exhibiting significant increases in cell membrane damage at the seedling stage (P > 0.001), with the largest increase seen in CA4 (Table 1b). However no genotype showed an increase in membrane damage at the flowering stage (F = 0.30 ns) (Table 1b). These observations indicate varying degrees of acclimatization response in CL5915-93D4, CLN2498E and CLN1621L but not in CA4,
E. Sangu et al. / Journal of Plant Physiology 178 (2015) 10–16
13
Table 1a Mean percentage of electrolyte leakage for the four tomato genotypes sampled from field site USDM Tanzania, at seedling, vegetative and flowering stages. Measured after treating leaf samples with either 36 ◦ C or 41 ◦ C water bath temperature conditions for 1 h. Mean electrolyte leakages (%) Genotype
Seedling
Vegetative
◦
CLN1621L CL5915-93D4 CLN2498E CA4 Grand mean SEM F * **
◦
Flowering
◦
◦
◦
Grand mean ◦
◦
Grand mean
36 C
41 C
36 C
41 C
36 C
41 C
36 C
41 ◦ C
7.27 ± 1.37 6.39 ± 2.86 6.5 ± 2.18 4.46 ± 1.77 6.15 0.68 3.10*
12.66 ± 6.29 24.68 ± 21.43 27.39 ± 17.62 9.63 ± 3.62 18.59 4.68 3.50*
2.87 ± 1.26 4.47 ± 2.50 6.31 ± 3.44 3.67 ± 2.06 4.33 0.78 3.57*
12.37 ± 2.63 17.35 ± 6.70 17.79 ± 5.45 11.92 ± 3.96 14.860 1.60 3.84*
1.42 ± 0.65 2.30 ± 1.07 1.65 ± 0.55 5.96 ± 1.07 2.83 0.45 22.59**
15.19 ± 3.11 15.16 ± 4.15 17.90 ± 9.89 25.52 ± 20.34 18.44 3.77 1.68 ns
3.86 4.38 4.82 4.70 4.44
13.41 19.06 21.03 15.69 17.30
Indicates significant differences in electrolyte leakage across growth stages and genotypes. Indicates highly significant differences in electrolyte leakage across growth stages and genotypes.
Table 1b Mean percentage of electrolyte leakages for the four tomato genotypes measured from leaf samples collected from controlled temperature growth chambers set at 35 ◦ C/28 ◦ C day/night and 24 ◦ C/18 ◦ C day/night temperature conditions at the seedling and flowering stage. Mean electrolyte leakages (%) Genotype
Seedling ◦
CLN1621L CL5915-93D4 CLN2498E CA4 Grand mean SEM F * **
Flowering ◦
◦
◦
◦
◦
◦
◦
24 C/18 C
35 C/28 C
24 C/18 C
35 C/28 C
11.57 ± 2.51 4.88 ± 1.85 10.05 ± 2.59 6.50 ± 3.97 8.25 0.99 9.69**
7.18 ± 2.94 8.50 ± 1.63 10.16 ± 2.85 13.42 ± 3.67 9.92 1.25 4.58*
10.92 ± 2.52 11.04 ± 4.23 14.34 ± 2.09 17.26 ± 5.38 13.39 1.56 3.75*
12.16 ± 3.92 12.32 ± 0.94 14.80 ± 2.61 18.19 ± 2.84 14.02 1.41 2.45
Grand mean 10.46 9.18 12.34 13.84 11.45
Indicates significant differences in electrolyte leakage across growth stages and genotypes. Indicates highly significant differences in electrolyte leakage across growth stages and genotypes.
leading to a relative increase of cell membrane thermostability at the flowering stage.
Total chlorophyll content Total leaf chlorophyll content was measured at seedling, vegetative and flowering stages on plants subjected to control (24 ◦ C/18 ◦ C) and heat stress (35 ◦ C/28 ◦ C) conditions. At all stages measured; seedling, vegetative and flowering, there was a highly significant increase in chlorophyll content in the plants subjected to heat stress compared to the control (P > 0.001) in all genotypes (Table 2). Heat sensitive genotype CA4 had consistently higher leaf chlorophyll content when plants were subjected to heat stress at all stages compared to heat tolerant genotypes CLN1621, CL5915 and CLN2498E (P > 0.001) (Table 2).
Number of flowers, fruit and percentage fruit set As shown in Table 3, the tomato genotypes produced variable numbers of flowers across treatments and environments. More flowers were produced when plants were under heat stress (the hot season) than under normal conditions (the cool season) (P > 0.001), however this did not correspond to total number of fruit per plant, with significantly fewer fruit being produced under heat stress (P > 0.001). Heat sensitive CA4 produced average of 2 fruits per plant under heat stressed conditions and average of 20 fruits per plant under normal conditions (Table 3). The percentage fruit set averaged between 55 and 75% in all genotypes during the cool season but was significantly lower, ( 0.001). A significant decrease was noted in heat sensitive genotype CA4 with a fruit set percentage of 55% in the cool season as compared to 3.14–6.40% during the hot season. Heat stress
tolerant CLN 1621L exhibited the highest fruit set (46%) followed by CL 5915-93D4 (36%) in the hot season. Pollen number and viability The genotypes produced variable numbers of pollen grains of different degrees of viability in the control experiment. CLN2498E produced the largest amount of pollen (29,481) followed by CL591593D (11,522) then CLN1621L (10,007) and CA4 (6910) (Table 4). Pollen viability was highest in CL5915-93D (85.84%) and lowest in CA4 (64.91%) (Table 4). Two genotypes CLN1621L and CL5915-93D produced a mixture of viable and non viable pollen grains with non viable pollen grains being the majority under heat stress conditions. Viable pollen grains were not detected under the microscope in CLN1621L and CL5915-93D under conditions of heat stress but were inferred from seeded fruits that were produced (Table 4). The complete development of floral structures to full maturity suggests the presence of unique metabolic adjustments in the heat tolerant lines CL5915-93D and CLN1621L. Genotypes CA4 and CLN2498E failed to produce fully developed flowers and therefore no pollen grains were collected. It was further observed that the stamens in genotype CA4 were degraded. Gene expression in tomato lines and pollen development stages The expression of compatible solute genes SPS, SuSy, P5CS and GoLS1 were compared in terms of relative gene expression ratios. Only the normalized expression ratios are presented, however the non-normalized data produced a similar trend (data not shown). Three genes: SPS, P5CS and SuSy were up-regulated in two heat tolerant genotypes CL5915-93D and CLN1621L at different stages of pollen development. Three genes: SPS, P5CS, GoLS1 were down-regulated in heat sensitive genotypes CA4 (SPS and
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Table 2 Mean chlorophyll values for four tomato genotypes measured at the seedling, vegetative and flowering stages from controlled temperature growth chambers set at 35 ◦ C/28 ◦ C day/night and 24 ◦ C/1 ◦ C day/night temperature conditions. Seedling
Genotype
Vegetative
24 ◦ C/18 ◦ C CA4 CLN1621L CL5915-93D4 CLN2498
40.63 33.56 30.22 36.09
± ± ± ±
35 ◦ C/28 ◦ C
0.46 0.51 0.95 0.5
57.22 51.35 51.84 46.78
± ± ± ±
Flowering
24 ◦ C/18 ◦ C
3.84 2.74 3.45 1.99
43.86 37.74 30.68 40.87
± ± ± ±
35 ◦ C/28 ◦ C
1.30 1.98 0.62 0.29
60.98 48.05 43.58 47.03
± ± ± ±
4.22 1.82 3.32 1.94
24 ◦ C/18 ◦ C 45.13 41.08 30.68 41.12
± ± ± ±
35 ◦ C/28 ◦ C
3.61 1.92 1.52 1.26
60.81 48.01 45.02 48.22
± ± ± ±
5.63 4.42 2.27 1.93
Table 3 Mean comparisons of the number of flowers, number of fruit and percentage fruit set of four tomato genotypes across breeding seasons (cool and hot). Genotypes
CL5915-93D4 CLN1621L CA4 CLN2498E LSD
Number of flowers Season
Number of fruit Season
% Fruit set Season
Cool UDSM
Hot 1 Ruvu
Hot 2 KGMN
Cool UDSM
Hot 1 Ruvu
Hot 2 KGMN
Cool UDSM
Hot 1 Ruvu
Hot 2 KGMN
47.83 ± 9.62 46.17 ± 6.87 25.00 ± 3.19 31.92 ± 4.27 37.75b
45.00 ± 7.99 49.67 ± 16.43 32.83 ± 6.90 41.92 ± 5.00 42.50b
67.33 ± 10.24 58.67 ± 7.35 63.67 ± 5.87 47.67 ± 6.77 59.25a
34.33 ± 6.98 30.50 ± 7.13 13.92 ± 4.96 20.17 ± 3.33 24.75a
16.67 ± 4.36 22.08 ± 7.48 2.00 ± 1.04 15.25 ± 2.09 14.00b
23.83 ± 6.12 26.17 ± 3.97 2.00 ± 1.04 17.08 ± 2.02 17.25b
73.80 ± 16.25 66.24 ± 12.84 54.93 ± 16.31 63.57 ± 8.72 64.75a
37.22 ± 6.96 45.03 ± 7.70 6.40 ± 4.09 36.65 ± 5.29 31.25b
35.45 ± 6.88 45.98 ± 8.88 3.15 ± 1.62 36.47 ± 6.65 30.00b
NB: significance difference is indicated by the letter difference within group i.e. ‘a’ and non-significance difference by letter similarity within group i.e. ‘b’
Table 4 Response of tolerant and sensitive tomato genotypes to a continuous exposure to heat stress, 35 ◦ C/28 ◦ C, compared to control conditions, 24 ◦ C/18 ◦ C. Genotype 24 ◦ C/18 ◦ C CL5915-93D4 CLN1621L CLN2498E CA4 35 ◦ C/28 ◦ C CL5915-93D4 CLN1621L CLN2498E CA4
Number of trusses
Average number of flowers per plant
6 6 3 4
39 44 16 14
± ± ± ±
12 7 2 2.5
45 33 11 6
± ± ± ±
Average number of pollen grains per flower
%Pollen viability
2.08 4.41 3.28 2.02
11,520.67 ± 180.63 9875.03 ± 1883.63 18,419.33 ± 2360.33 6910.00 ± 367.37
85.84 ± 2.80 80.07 ± 4.65 69.49 ± 13.22 70.36 ± 4.67
2.73 4.58 1.20 1.63
2445 ± 1797.44 1500 ± 726.23 ND ND
ND ND 0 0
Table 5 The relative gene expression ratios of compatible solute genes (SPS, GoLS1, P5CS and SuSy) measured in heat tolerant and sensitive tomato genotypes under continuous exposure to heat stress. Pollen development stages are labeled MM, VM and MP. Selected genes
SuSy
Genotypes and development stage
Expression ratio
P
Expression ratio
P
Expression ratio
P
Expression ratio
P
CA4 (MM) CA4 (VM) CA4 (PM) CLN1621L (MM) CLN1621L (VM) CLN1621L (PM) CL5915-93D (MM) CL5915-93D (VM) CL5915-93D (PM) CLN2498E (MM) CLN2498E (VM) CLN2498E (PM)
0.663 1.016 0.617 0.380 1.230 3.286** 1.235 0.555 0.981 1.008 1.108 0.485
0.500 0.966 0.360 0.216 0.431 0.003 0.580 0.184 0.955 0.977 0.815 0.300
0.255* 1.342 0.939 0.305 0.636 0.741 0.423* 0.976 0.282* 0.196* 1.560 1.000
0.007 0.246 0.890 0.064 0.152 0.600 0.018 0.932 0.039 0.006 0.284 0.999
0.014* 1.383 0.768 0.404 1.241 10.758** 4.392 0.205 0.937 1.413 1.294 1.145
0.001 0.296 0.759 0.129 0.508 0.032 0.177 0.062 0.759 0.387 0.797 0.823
1.317 1.719 0.544 0.440 0.673 17.065** 12.059** 0.199* 0.827 2.114 2.402 1.289
0.788 0.259 0.444 0.176 0.312 0.000 0.031 0.047 0.556 0.103 0.329 0.722
* **
GoLS1
SPS
P5CS
Indicates significant down-regulation in heat stressed samples. Indicates significant up-regulation in heat stressed samples.
GoLS1), CLN2498E (P5CS) and one heat stress tolerant genotype CL5915-93D (GoLS1) at the MM stage (Table 5). The SPS gene was significantly up-regulated by a mean factor of 10.758 (P > 0.05) in CLN1621L at the mature stage and down-regulated in CA4 by the mean factor of 0.014 (P > 0.001) at MM stage of pollen development. No significant difference in SPS expression was observed in CLN2498E at any stage of development. The overall expression ratios of SPS gene were higher than that of SuSy and GoLS1 but lower than the expression ratios of P5CS (Table 5). The expression of P5CS was significantly up-regulated by a mean factor of 12.095 (P > 0.05) in CL5915-93D at the MM stage and CLN1621L by a mean factor of 17.065 (P > 0.001) at mature stage
of pollen development. The gene was significantly down-regulated in CL5915-94D by a mean factor of 0.199 (P > 0.05) at the vacuolated stage but showed no significant difference in genotypes CA4 and CLN2498E, at all the three stages of pollen development (Table 5). The expression of GoLS1 gene was relatively low at all the three stages of pollen development with minimum and maximum expression ratios of 0.196 and 1.56, respectively. The gene GoLS1 was significantly down-regulated by mean factors of 0.255 (P > 0.001) in CA4, 0.423 (P > 0.01) in CL5915-93D, and 0.196 (P > 0.01) in CLN2498E at the MM stage. The gene was also downregulated by a mean factor of 0.295 (P > 0.01) at the mature stage but not significant in CLN1621 in all the three stages of pollen
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development and at vacuolated stage in the other genotypes (Table 5). The gene SuSy was not significantly up-regulated in any genotype at any stage of pollen development, with the exception of expression of heat tolerant CLN1621L which was significantly up-regulated by a mean factor of 3.286 (P > 0.001) at the MP stage (Table 5). Discussion Tomato is sensitive to heat stress with temperatures above 27 ◦ C inhibiting pollen development and therefore seeded fruit production (Peet et al., 1997). High temperatures cause a reduction in pollen germination, ovule development and fruit set (Peet et al., 1988). Selection and breeding of plants with tolerance to heat stress is one way to mitigate the adverse effects of increasing global temperatures (Warner and Erwin, 2005). Cell membrane thermostability has been successfully used as a selection criterion for heat tolerant genotypes in wheat (Shanahan et al., 1990), cotton (Rahman et al., 2004) and tomato (Camejo et al., 2005). In agreement with these findings the present study shows a marked genotypic difference in the cell membrane thermostability in response to elevated temperatures, with low levels of cell membrane leakage observed in heat tolerant genotype CLN1621L and acclimation observed in genotypes CL5915-93D4 and CLN2498E. In contrast heat sensitive CA4 suffered cell membrane damage at all growth stages. Whilst cell membrane thermostability is a useful selection criteria for thermotolerance, Blum et al. (1990) cautioned that there are several factors contributing to thermotolerance such as heat avoidance, and therefore cell membrane thermostability should be used in conjunction with other selection criteria for selection of heat tolerant genotypes. There was a significant increase in the total leaf chlorophyll content under heat stress in all genotypes in the present study. The largest increases in chlorophyll content were seen in heat sensitive CA4, which also showed higher levels of leaf chlorophyll content under control conditions. The significantly larger increase in chlorophyll content in this genotype may therefore be a result of the higher basal levels under control conditions, however Camejo et al. (2005) showed a decrease in chlorophyll/carotenoid content in heat tolerant but not heat sensitive lines of tomato in response to heat stress. Previous studies have demonstrated a reduction in fruit setting percentage in tomato in response to heat stress (Dane et al., 1991; Abdul-Baki and Stommel, 1995). This reduction in tomato fruit set in response to heat stress has been attributed to the impairment of pollen development in anthers 8–13 d before anthesis (Iwahori, 1965, 1966). Hanson et al. (2002) observed a lower decrease in percentage fruit set in CL5915-93D4 when compared to a heat sensitive tomato line and concluded that CL5915-93D4 is a valuable source of heat tolerance genetic material. Similarly Comlekcioglu and Soylu (2010) observed that genotypes CL5915-93D4 and CLN1621L retained high levels of fruit set under moderate heat stress, and although percentage fruit set was reduced under high heat stress conditions, levels were higher compared to heat sensitive genotypes. Reductions in percentage fruit set were seen in all genotypes in the present study; however the largest decrease was seen in the heat sensitive genotype CA4. This corresponded with a very low level of pollen viability under heat stress in this genotype. A lower percentage reduction in both fruit set and pollen viability was seen in CLN1621L and Cl5915-93D4 in agreement with Comlekcioglu and Soylu (2010). In the present study it was hypothesized that the expression of the compatible solute genes differ in tomato genotypes at various stages of pollen development with high gene expression corresponding to the production of viable pollen grain and
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consequently high fruit set under heat stress. The up-regulation of P5CS, SPS and SuSy genes in heat tolerant genotypes and not in heat sensitive genotypes suggest the presence of sucrose and proline biosynthesis in the anther tissues during heat stress. The expression of compatible solute genes SPS, P5CS, SuSy, and GoLS1 during pollen development showed clear demarcation between heat tolerant tomato and sensitive tomato cultivars following continuous exposure to heat stress. Three genes (SPS, P5CS and SuSy) were up-regulated by heat stress in heat tolerant lines CLN1621L at the MP stage. Conversely two genes (SPS and GoLS1) were down-regulated in the heat sensitive cultivar CA4. Analysis of the expression patterns in heat tolerant lines showed that the genes were up-regulated at MM (P5CS) in cultivar CL5915-93D and at the MP (SPS, SuSy, and P5CS) in line CLN1621L corresponding to the anther growth phase and anther maturation phases respectively. These findings are in agreement with that of Datta et al. (2002) who reported a temporal change in the expression of carbohydrate genes during a transition from no starch (anther growth) to an active starch-filling (anther maturation) phase in male fertile but, not in male sterile maize genotypes during pollen development. Therefore, maintenance of high levels of soluble sugars is a prerequisite for production of high quality pollen grains (Pressman et al., 2002). According to Datta et al. (2002) the up-regulation of the hexose transporter gene in male fertile, but not in male sterile maize genotypes suggested a potential role for this gene transporting extracellular sugars from the nutrient rich locular fluid to the developing pollen. However, during heat stress the transportation of extracellular sugars and other compatible solutes from the nutrient rich locular fluid to the developing pollen by hexose transporter (Datta et al., 2002) and proline transporter (Sato et al., 2006) is disrupted. In cowpea, male sterility occurs if high temperatures coincide with floral development, due to premature degradation of the tapetum and a lack of endothecial development (Ahmed et al., 1992). In Arabidopsis, heat shock disrupts pollen development in a stage specific manner with critical effects at the meiotic stage of pollen mother cell development (Kim et al., 2001). In rice night temperatures above 35 ◦ C result in a significant reduction in pollen tube length and viability (Das et al., 2014). In the present study, similar effects were observed in tomato genotypes CA4 and CLN2498E which despite producing similar percentages of viable pollen grains under non stress conditions produced no pollen under heat stressed conditions. Tomato lines CLN1621L and CLN5915-93D produced a mixture of very few viable pollen grains and large number of non-viable pollen grains despite having morphologically sound flowers (data not shown). The observed phenomenon could partly be explained by the up-regulation of the three compatible solute genes SPS for sucrose synthesis, SuSy for sucrose cleavage and P5CS for proline synthesis. The observed increase in the number of mRNA transcripts in heat tolerant and not in heat sensitive cultivars suggests the potential for proline synthesis and sucrose re-synthesis in non-photosynthetic tissues. Im (2004), reported in maize, the possibility of sucrose re-synthesis in non-photosynthetic tissues such as anther, subsequent to the failure of the normal functioning of the hexose transporter gene (Datta et al., 2002). The failure of pollen formation and abnormal flower development could potentially be explained by the disruption of sugar metabolism and proline transport (Sato et al., 2006) which affected not only the pollens but also injured the anther cells and tissues (Sakata and Higashitani, 2008). The tolerant tomato cultivars (CL5915-93D and CLN1621L) showed an increased expression of compatible solute genes (SPS, SuSy, and P5CS) in at least in one stage of pollen development, and non-significant expression in either the previous or subsequent stages. These expression patterns suggest that heat tolerant lines have developed a unique metabolic switch
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which activates synthesis of compatible solutes just after the interference of sugar and proline transporter metabolism. Conclusions The data presented in this study emphasize the importance of maintaining appropriate levels of soluble sugars during pollen development under heat stress as exemplified by the increased expression of SPS, P5CS and SuSy genes in heat tolerant lines as opposed to heat sensitive lines. The ability of tomato genotype to achieve soluble sugar homeostasis during heat stress depends upon its ability to switch from metabolic sugar translocation in anther tissues to soluble sugar biosynthesis. However, it was not clear how this expression is regulated under heat stress. Acknowledgements This work was funded by GTZ project No: 07.7860.5-001.00 AVRDC project. The authors would like to thank the molecular biotechnology unit at AVRDC Headquarters, Taiwan and the Botany Department University of Dar es Salaam, Tanzania. References Abdul-Baki AA, Stommel JR. Pollen viability and fruit set of tomato genotypes under optimum and high-temperature regimes. Hortscience 1995;30:115–7. Ahmed FE, Hall AE, DeMason DA. Heat injury during floral development in Cowpea (Vigna Unguiculata, Fabaceae). Am J Bot 1992;79:784–91. Ainsworth EA, Ort DR. How do we improve crop production in a warming world. Plant Physiol 2010;154:526–30. Aloni B, Karni L, Zaidman Z, Schaffer AA. Changes of carbohydrates in pepper (Capsicum annuum L.) flowers in relation to their abscission under different shading regimes. Ann Bot 1996;78:163–8. Barnaba B, Jager K, Feher A. The effect of drought and heat stress on reproductive processes in cereals. Plant Cell Environ 2008;31:11–38. Battisti DS, Naylor RL. Historical warnings of future food insecurity with unprecedented seasonal heat. Science 2009;323:240–4. Bruinsma J. The quantitative analysis of chlorophyll a and b in plant extracts. Photochem Photobiol 1963;2:241–9. Camejo D, Rodriguez P, Morales MA, Dell’Amico JM. High temperature effects on photosynthetic activity of two tomato cultivars with different heat susceptibility. J Plant Physiol 2005;162:281–9. Comlekcioglu N, Kemal Soylu M. Determination of high temperature tolerance via screening of flower and fruit formation in tomato. J Agric Sci 2010;20:123–30. Dane F, Hunter AG, Chambliss OL. Fruit set, pollen fertility, and combining ability of selected tomato genotypes under high-temperature field conditions. J Am Soc Hortic Sci 1991;116:906–10. D’Aoust MA, Yelle S, Nguyen-Quoc B. Antisense inhibition of tomato fruit synthase decreases fruit setting and the sucrose unloading capacity of young fruit. Plant Cell 1999;11:2407–18. Das S, Krishnan P, Nayak M, Ramakrishnan B. High temperature stress effects on pollens of rice (Oryza sativa L.) genotypes. Environ Exp Bot 2014;101:36–46. Datta R, Chamusco KC, Chourey PS. Starch biosynthesis during pollen maturation is associated with altered patterns of gene expression in Maize. J Plant Physiol 2002;130:1645–56. Demnitz-King A, Ho LC, Baker DA. Activity of sucrose hydrolysing enzymes and sugar accumulation during tomato fruit development. Plant Growth Regul 1997;22:193–201. Erickson AN, Markhart AH. Flower developmental stage and organ sensitivity of bell pepper (Capsicum annuum L.) to elevated temperature. Plant Cell Environ 2002;25:123–30. Firon N, Shaked R, Peet MM, Pharr DM, Zamski E, Rosenfeld K, et al. Pollen Grains of heat tolerant tomato cultivars retain higher carbohydrate concentration under heat stress conditions. Sci Hort 2006;109:212–7. Fischer RA, Edmeades GO. Breeding and cereal yield progress. Crop Sci 2010;50:85–98. FAO. Food and Agriculture Organisation of United Nations report; April, 2005, Website database: http://www.fao.org. Giorno F, Wolters-Arts M, Mariani C, Rieu I. Ensuring reproduction at high temperatures: the heat stress response during anther and pollen development. Plants 2013;2:489–506.
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