Comparing heat stress effects on male-fertile and male-sterile tomatoes

Plant, Cell and Environment (1998) 21, 225 231 ORIGINAL ARTICLE OA 220 EN Comparing heat stress effects on male-fertile and male-sterile tomatoes M. M. PEET, S. SATO & R. G. GARDNER Department of Horticultural Science, Box 7609, North Carolina State University, Raleigh, NC 27695 7609, USA ABSTRACT To separate the effects of heat stress on male and female reproductive tissues, male-sterile (MSs) and male-fertile tomatoes (MFs) were placed in growth chambers at 12 h day/12 h night temperatures of 28/22, 30/24 or 32/26 C from flower appearance to seed maturation (daily mean temperatures of 25, 27 or 29 C). Pollen from MFs was applied individually to MS flowers. As MFs were self-pollinated, heat stress was experienced by both male and female tissues. At growth temperatures of 29 C fruit number, fruit weight per plant, and seed number per fruit were only 10%, 6 4% and 16 4%, respectively, compared with those at 25 C. Heat stress also adversely affected fruitset in MSs, especially when experienced by donor pollen. No fruit at all developed on MSs receiving pollen produced at 29 C, even when ovule development, pollen germination and subsequent embryo development all took place at 25 C. Effects on fruitset in MSs were reduced if donor pollen had not experienced heat stress. MSs grown at 29 C but receiving pollen developing at 25 C produced 73% as much fruit (both on number and weight basis), had 40% as high fruitset and produced 87% of the seed per fruit as MSs grown at 25 C. This use of male-sterile and male-fertile lines of tomato provides new evidence that impairment of pollen and anther development by elevated temperature will be an important contributing factor to decreased fruit set in tomato, and possibly other crops, with global warming. Key-words: Lycopersicon esculentum L. Mill., fruitset; hightemperature stress; male-steriles; high temperature; ovule development; pollen production; seed formation. INTRODUCTION Recent thermotolerance work has focused on physiological responses to short but severe heat shock treatments. While the ability of a plant to survive acute thermal stress is one way to define stress resistance, a more useful definition agronomically is the maintenance of yield when exposed to stress (Mahan, McMichael & Wanjura 1995). Tomato (Lycopersicon esculentum Mill.) is an example of a species with well documented but not well understood Correspondence: Dr Mary M. Peet. Fax: 919 515 2505; e-mail: mary_peet@ncsu.edu 1998 Blackwell Science Ltd sensitivity to high temperatures (Picken 1984). Processes reported to be adversely affected by high temperature (Kinet & Peet 1997) include: (1) meiosis in the pollen and ovule mother cells; (2) stigma position; (3) development of the endothecium in the anther (resulting in reduced dehiscence and pollen shed); (4) the number of pollen grains retained by the stigma; (5) pollen germination; (6) pollen tube growth; (7) ovule viability; (8) fertilization and postfertilization processes; and (9) growth of the endosperm, proembryo and fertilized embryo. In addition to developmental abnormalities in male and female reproductive tissues, reduced supply of photosynthates and poor production of growth regulators in sink tissues have been cited as explanations for poor fruitset in tomatoes at high temperatures (Kinet & Peet 1997). Most researchers have concluded that poor fruitset at high temperature in tomato could not be attributed to a single factor (e.g. Kuo et al. 1979; Rudich, Zamski & Regev 1977). In most of these studies, however, plants were exposed to extreme levels of heat stress. The most detailed investigations of developmental responses were conducted on plants grown at 20 C and exposed to 3 h periods of 40 C temperatures during various developmental stages (e.g. Iwahori 1965, 1966; Sugiyama, Iwahori & Takahashi 1966). In other experiments, daytime temperatures were as high as 37 38 C. Although it is clear that temperatures in the range of 35 40 C injure many components of the fruitset process, it is not clear whether all these injuries are equally likely at summer temperatures typical of warmtemperate regions (25 30 C). Peet, Willits & Gardner (1997) demonstrated that in hand-pollinated male-sterile tomatoes, relative seediness, as well as percentage fruitset, and total number and weight of fruit per plant decreased linearly as mean daily temperature rose from 25 to 29 C, even though pollen developed at low temperatures (26/22 C). The purpose of the present experiment was to compare the effects of heat stress applied during pollen development and release with effects of heat stress applied to developing female gametes and to reproductive processes taking place after pollen release. MATERIALS AND METHODS Procedures for plant growth are described in more detail in Peet et al. (1997). Seeds of NC8288 (cultivar attributes described in Gardner (1990)) were germinated at 22 C 225

226 M. M. Peet et al. starting 29 September 1995 then transplanted 10 d after planting (DAP) into a 26/22 C day/night temperature greenhouse in the NC State University Phytotron. Seedlings of NC8288 carrying the male-sterile allele that confers shortened anthers (MSs) were identified using the green-stem (aa gene) characteristic. The green-stem marker (aa) is located 10 crossover units away from the MS-10 36 gene conferring male sterility. Stems of young NC8288 seedlings lacking this allele (male-fertiles, MFs) have a distinct purplish tinge and develop normal anthers. At 31 DAP both MSs and MFs were transplanted into 9dm 3 containers and distributed among six growth chambers (Fig. 1). Three chambers contained only MSs and the other three contained half MSs and half MFs (mixed chambers), for a total of 72 sterile and 24 fertile plants. This design prevented accidental contamination of MSs by pollen drift from MFs other than those in the assigned temperature treatment group. Once buds were visible (48 DAP), one all-mss and one mixed chamber was assigned to each of three temperature day/night treatments (28/22, 30/24 and 32/26 C). As daylength was 12 h, mean daily temperatures were 25, 27 and 29 C, respectively. Chamber temperatures were continuously measured with periodically calibrated Type T thermocouples placed in Figure 1. Experimental treatments in six growth chambers. Three chambers contain half male-fertile (MF) and half male-sterile (MS) tomato plants. In these chambers, the MFs are vibrated, and the pollen released is collected on a slide to be applied to MSs in the same chamber. Additional pollen collected from the MFs is applied to half the MSs in the two all-ms chambers maintained at different temperatures. This is indicated by the outward arrows from the MFs/MSs chambers. Thus pollen produced by MFs at all three temperatures was applied to MSs grown at all three temperatures. shielded aspirated boxes at plant height. Photon flux at the beginning of each experiment was 550 µmol m 2 s 1. Daytime vapour pressure deficits (VPDs) ranged from 0 38 kpa at 28 C to 0 57 kpa at 32 C. Starting 54 DAP and continuing at approximately 3 d intervals (56, 59, 61, 64 and 68 DAP), pollen from MFs in each of the three mixed chambers was collected on glass slides by vibrating flowers with a commercial pollinating rod. The entire cluster was vibrated as soon as the first flower reached peak pollen production (full anthesis, indicated visually by completely reflexed petals). Pollen was applied to MSs by gently dragging stigmas through deposits on the slide. As crossover restores fertility to 10% of the population with the green-stem characteristic, MSs were inspected periodically to ensure against production of fertile flowers. Any unpollinated MSs found to be setting fruit or producing normal-appearing rather than shortened anthers were taken from the chamber to remove potential pollen sources. Data from these plants were treated as missing values in the analysis. These precautions and the physical separation of MSs and MFs assigned to different temperature treatments (Fig. 1) minimized potential pollen drift between treatments. From 69 to 96 DAP, plants were left in the chamber, but no additional flowers were pollinated. At this time the number of trusses pollinated varied from one or two in the low-temperature-grown MSs receiving pollen from hightemperature-grown MFs to four to seven in high-temperature-grown MSs receiving pollen from the low-temperature-grown MFs. The reasons for this variability were a difference in growth rates in the MSs between temperature treatments and a difference in the MFs in the amount of pollen they produced that was available to be applied to the MSs. Final harvest took place 96 DAP and included immature (pre-breaker) and mature (postbreaker) fruit in approximately equal numbers (132 versus 106). Each fruit was weighed individually and the date of pollination indicated on the tag was recorded. Pollination dates were also recorded for flowers that had abscissed or had not developed into fruit. Seeds were extracted from each fruit by fermentation, washed, and counted. Data were analysed using the JMP Statistical package (SAS Institute, Cary, NC) procedures for linear regression, one-and two way analysis of variance (ANOVA) and Student s t-test. Regression results are presented in the tables, but standard errors derived from ANOVA were used to construct error bars on the figures. In some cases, ANOVA and Student s t-test results are also referenced in the discussion to determine if differences between particular means were significant. For all analyses, individual fruit and flower data for each plant were grouped by cluster. Percentage fruitset was then calculated by dividing the number of fruit present per cluster by the fruit potential (total number of fruit plus any aborted or non-developed flowers). In MSs, fruitset was averaged over all clusters on the plant that had been pollinated, including those without fruitset. Fruitset was not calculated in MFs, because no

Heat stress effects on tomatoes 227 flowers were tagged. Fruit number and fruit weight from all clusters were totalled by plant for analysis. Average fruit weight and seed number per fruit were calculated using only non-zero values. That is, where the flower did not develop into a fruit, the resulting value for average fruit weight and seed number was considered to be missing rather than zero. Thus, average fruit weight and average seed per fruit represent only fruit that weighed at least 40 g. Results are expressed as mean ± SEM. RESULTS Effects of heat stress when experienced by both male and female tissues Negative linear regressions (Table 1) of fruiting characteristics on growth temperature were significant for MFs. They were also significant for MSs grown at the same temperature as donor pollen (Table 2). Fruit number (Fig. 2a), total fruit weight per plant (Fig. 2b), and seed number per fruit (Fig. 2c) declined sharply with increasing temperature in both groups of plants. In MFs grown at 29 C, fruit numbers, fruit weight per plant, and seed number per fruit were only 10%, 6 4% and 16 4%, respectively, that at 25 C. No fruit at all were produced by MSs grown at 29 C and receiving 29 C donor pollen. The decline in fruit weight per plant with increasing temperature was caused more by the decrease in fruit number than by a decrease in weight of individual fruit, although average fruit weight also declined somewhat in both populations (Fig. 2d). The regression of average weight/fruit and seed content on growth temperature was significant for the MFs (Table 1). Regression statistics could not be calculated for the MSs because no fruit were produced in the 29 C treatment, leaving only two treatments with non-missing values for seed content and average fruit weight. Differences in average fruit weight and seed content between the 25 and 27 C treatments were not significant for MSs. Comparison of MFs and MSs Fruit weight and number and seed content of MFs exceeded that of MSs growing at the same temperatures in the same growth chamber (Fig. 2a d). Averaged over all temperature treatments, MFs produced significantly (P = 0 005) more fruit per plant (4 67 ± 0 78) than MSs (2 48 ± 0 35). The total weight per plant of fruit produced was significantly (P = 0 0003) greater in MFs (535 1 g ± 109) compared with the MSs (259 23 ± 37 98). The average number of seeds per fruit was significantly (P = 0 0007) higher in the MFs than MSs (86 6 ± 12 5 versus 45 16 ± 4 9). There was no significant difference between MSs and MFs in average fruit weight, however. Flowering and fruitset in MSs MSs were tagged and pollinated at flower opening, allowing comparison of number of flowers and percentage fruitset among temperature treatments. The effect of growth temperature on flower production was significant, with fewer flowers per plant (12 4) at 25 C compared with 15 0 and 14 8 in the 27 and 29 C treatments, respectively. Growth temperature decreased fruit number significantly in MSs receiving 25 C pollen, but not in plants receiving 27 or 29 C pollen. Because high temperature grown plants Total fruit weight plant 1 294 2 10 78 0 0001 27 29 No. fruit plant 1 1 969 8 10 0 0001 0 2431 Average weight fruit 1 (g) 13 10 3 37 0.0034 3 884 Average no. seed fruit 1 30 38 6 26 0 0001 4 828 Table 1. Single-parameter regressions of the response surfaces for all dependent variables against daily mean temperature for male-fertile tomato plants (MFs) Total fruit weight plant 1 163 6 7 38 0 0001 22 19 No. of fruit plant 1 1 518 7 27 0 0001 0 2086 Average weight fruit 1 (g)* Average no. seeds fruit 1 No. of flowers 0 968 0 98 0 3375 0 9880 Table 2. Single parameter regressions of all dependent variables in male-sterile tomato plants (MSs) against daily mean temperature growth for plants when pollen was grown at the same temperature as the MS plant *No fruit were produced in MSs assigned to 29 C pollen treatment. These were considered to be missing values rather than zero in calculating average fruit weight. Hence, only two points would be available to plot a regression equation. No fruit were produced in MSs assigned to 29 C pollen treatment. These were considered to be missing values rather than zero in calculating average number of seeds per fruit. Hence, only two points would be available to plot a regression equation

228 M. M. Peet et al. Figure 2. Effect of average daily temperature during growth on (a) number of tomato fruit produced per plant; (b) total weight of tomato fruit produced per plant; (c) number of seed per tomato fruit; (d) average tomato fruit weight (g). Values are means ± SE. (Average weight was calculated only for fruit that developed. Where flowers aborted, the fruit weight was analysed as a missing value, rather than zero.) For male-fertile (MF) plants, daily mean growth temperatures of 25, 27, and 29 shown on the figure represent daytime temperatures of 28, 30 and 32 C and night-time temperatures of 22, 24, and 26 C, respectively, experienced from the start of flowering. For male-sterile (MS) plants, daily mean growth temperatures shown on the figure represent growth conditions for the plant from the start of flowering, but flowers were hand-pollinated using pollen produced by MFs exposed to the same temperature treatments. No fruit developed on MSs grown at 29 C and receiving pollen produced at 29 C. in this pollen treatment produced more flowers, but fewer fruit, percentage fruitset was reduced (Fig. 3a). In plants receiving 27 C pollen, however, there was no significant effect of growth temperature of the MSs on fruitset or fruit number (Fig. 3b). No fruit were set in MSs receiving 29 C pollen. Comparative sensitivity in MSs of pollen production and other reproductive processes to heat stress Regardless of the temperature at which they were grown, MSs produced no fruit when donor pollen was produced at 29 C (Fig. 3b). On the other hand, MSs grown at 29 C from flower appearance to seed maturation but receiving donor pollen produced at 25 C, produced 73% as much fruit (on number and weight basis), had 40% of the fruitset and produced 87% of the seed per fruit as MSs kept at 25 C. Overall, heat stress applied to the pollen parent before and during pollen release decreased seed number and fruitfulness much more than heat stress applied to the ovule (MS) parent during its development and after pollen application to the style (Figs 3a d). Most reproductive characteristics were negatively correlated with heat stress experienced by the MF pollen donor (Table 3). Heat stress imposed during development of the ovules and after pollen application did not affect any reproductive characteristics except flower number, which increased, and fruitset, which decreased (Table 4). Lower fruitset in MSs grown at high temperature reflects both increased flower production and decreased fruit number, but was only significant for plants receiving 25 C grown pollen (Fig. 3a). Even in MSs receiving 25 C donor pollen, differences in number of fruit and total fruit weight per plant were only significant between plants grown at 25 C and 29 C. There was no effect of any type of heat stress on average weight per fruit (data not shown) or seed number per fruit (Fig. 3d). Thus, most of the deleterious effect on yield of heat stress applied to either male or female gametes was attributable to reduced fruit number. DISCUSSION Effects of heat stress when experienced by both male and female tissues As mean daily temperature rose from 25 to 29 C, yieldrelated characteristics, including seeds per fruit, declined

Heat stress effects on tomatoes 229 Figure 3. Effect of average daily temperature during growth and release of the pollen produced by MFs and of the temperature during flower development of the MSs and after pollen application. Values are means ± SE. (a) Fruitset in MSs. (b) Number of fruit in MSs. (c) Total weight of fruit produced per plant in MSs. (d) Number of seeds per fruit in MSs. Daily mean growth temperatures of 25, 27, and 29 shown on the figure represent daytime temperatures of 28, 30 and 32 C and night-time temperatures of 22, 24, and 26 C, respectively, experienced from the start of flowering until harvest. Average weight and number of seed per fruit was calculated only for fruit that developed. Where flowers aborted or did not develop, fruit weight and seed number were analysed as a missing value, rather than zero. Hence, no data are presented for a pollen growth temperature of 29 C. Total fruit weight plant 1 141 8 10 61 0 0001 13 36 No. fruit plant 1 1 321 11 01 0 0001 0 120 Average weight fruit 1 (g) 9 523 1 75 0 0893 5 439 Average no. seed fruit 1 8 966 1 89 0 0678 4 748 Percentage fruitset 7 095 8 52 0 0001 0 8324 Table 3. Single-parameter regressions of all dependent variables in male-sterile tomato plants (MSs) against daily mean temperature for pollen applied Total fruit weight plant 1 15 38 0 61 0 5449 25 22 No. of fruit plant 1 0 1595 0 69 0 4957 0 2323 Average weight fruit 1 (g) 3 029 0 83 0 413 3 654 Average no. seed fruit 1 1 116 0 34 0 7326 3 239 No. of flowers 0 5272 0 67 0 5038 0 7826 Percentage fruitset 2 967 2 29 0 0267 1 298 Table 4. Single parameter regressions of all dependent variables in male-sterile tomato plants (MSs) against daily mean growth temperature

230 M. M. Peet et al. sharply, as shown in Fig. 2a d. This confirms that temperatures above 25 C can reduce fruit production in tomatoes (Charles & Harris 1972) and corresponds to the day/night limits of 30/21 C reported by Moore & Thomas (1952). The optimum temperature for vegetative growth of tomatoes is reported to be 18 25 C (Hurd & Cooper 1970). Decreases in total yield in both MFs and MSs were caused by reduced fruit number rather than by reduced weight per fruit, which declined only slightly with increased temperature. In the MFs there was a drastic decline in seeds/fruit as temperatures increased, which was not paralleled by a decrease in average fruit weight. This suggests that in MFs there is an increased tendency for parthenocarpic fruitset at high temperatures. This trend was less clear in the MSs, mainly because no fruit were set in the 29 C pollen treatment. Comparison of MFs and MSs Production of more fruit in MFs may have partially reflected experimental limitations. First, hand pollination and vibration were discontinued 68 DAP, but fruit set by MFs through unassisted pollen shed were allowed to remain on the plant. Secondly, in the 29 C treatment, MFs did not produce enough pollen for all the flowers assigned to that pollen treatment on all possible dates. A much greater ratio of MFs to MSs than 1:3 would be required at a pollen growth temperature of 29 C to ensure adequate pollen supply. The fact that hand-pollinated fruit contained fewer seed compared with MFs suggests that hand pollination (at least with our methods) was not as effective as vibration-assisted self-pollination. Overall, however, MSs and MFs responded similarly to heat stress. Use of a malesterile line to construct a response surface, as in Peet et al. (1997), is therefore probably valid. Hand-pollination, however, may mask some heat stress effects because it precludes consideration of pollen transfer limitations such as exsertion of the style and splitting of the antheridial cone, considered by Levy et al. (1978) and Rudich et al. (1977) to be important factors in cultivar sensitivity to high temperatures. Relative sensitivity of pollen production and other reproductive processes to heat stress There was some heat stress sensitivity of both male and female gametes, and some heat sensitivity both before and after pollen release. For MSs experiencing heat stress but receiving 25 C pollen, there was a progressive decline in fruit number and total fruit weight with increasing growth temperature, confirming observations of Peet et al. (1997). However, the most striking finding of the present study was the much greater adverse effect of heat stress during pollen development compared with heat stress during development of the female reproductive tissues and postpollen application. Even when ovule development, pollen germination, pollen tube growth, fertilization and fruit growth all took place at 29 C, effects were modest compared with MSs receiving 29 C pollen. No MSs receiving pollen from MFs grown at 29 C set fruit. All indices of reproductive success were greatly reduced in MSs when the pollen source was MFs grown at 27 C. Based on visual observation of the glass slide used for collection, pollen amount decreased at high temperature. After several weeks of heat stress, it was necessary to accumulate pollen from the 29 C treatments for several days before application. Minimal pollen was released when MFs were vibrated and this was in clumps rather than being evenly dispersed and powdery, making application to MSs difficult. Low VPD was not the cause of this clumping, as VPDs were slightly higher in the 29 C growth chamber than in the two cooler chambers. A similar clumping of tomato pollen has been reported under low light (Picken 1984). Bean pollen exposed to high temperatures (32/27 C) two days before anthesis was clustered in clumps that did not remain attached to the stigma, possibly because stigmatic secretions or pollen/stigma interactions were impaired by high temperature (Gross & Kigel 1994). Many, but not all studies show reduced pollen viability at high temperatures. In beans, Farlow, Byth & Druger (1979) found that high temperatures (44 48 C) rendered pollen inviable. Dane et al. (1991) reported a decrease in tomato pollen fertility during prolonged periods of heat stress. They also found that heat-tolerant lines maintained a higher level of pollen fertility throughout the season than heat-sensitive lines. Decreases in pollen fertility in tomatoes have been reported by El Ahmadi & Stevens (1979) and Kuo et al. (1979), but Charles & Harris (1972) and Rudich et al. (1977) concluded that tomato cultivar variability in pollen germination was not correlated with their heat setting characteristics. Relatively few other studies have imposed stress differentially on male and female reproductive tissues. In a cross-pollination study using emasculation rather than male-steriles, Levy, Rabinowitch & Kedar (1978) found significant effects when either the male or female parent in the cross experienced temperature stress, but also concluded that overall, microspores were more affected than macrospores. Studies in which heat stress was imposed at various developmental stages also point to the sensitivity of pollen formation to heat stress. Rudich et al. (1977) reported the most serious injury in terms of fruit set in tomatoes occurring when plants were exposed to 40 C for 4 h 9 d before anthesis. Iwahori (1965) and Sugiyama et al. (1966) concluded that pollen damage from heat stress (40 C temperature for 30 h) was more serious than ovule damage 5 7 d pre-anthesis. At other times, this was evidently not the case because application of control pollen did not improve fruit set. Gross & Kigel (1994) reported postfertilization stages and early pod development in beans were less sensitive to high temperature than pre-fertilization stages. In bean, exposure to high temperature at sporogenesis resulted in male sterility because of non-viable pollen and failure of anther dehiscence whereas gynoecium

Heat stress effects on tomatoes 231 function was unaffected. Agtunong et al. (1992) compared heat stress treatments (34/29 C) before and after flowering in heat-sensitive and tolerant beans. No cultivar set seed when stress was applied both before and after flowering. Pre-flowering stress had more effect than post-flowering stress on pod set and seed number. In conclusion, for the present experimental system, heat stress effects on pollen during development and release were highly deleterious to development of fruit and seed. Similar stress treatments when imposed during development of the female gametes and after pollen application were less deleterious. This use of male-sterile and malefertile lines of tomato provides new evidence that impairment of pollen development and release by elevated temperature will be an important contributing factor to decreased fruit set in tomato, and possibly other crops, with global warming. 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