Contrasting anther glucose-6-phosphate dehydrogenase activities between two bean varieties suggest an important role in reproductive heat tolerance

Abstract

Common beans (Phaseolus vulgaris) are highly sensitive to elevated temperatures, and rising global temperatures threaten bean production. Plants at the reproductive stage are especially susceptible to heat stress due to damage to male (anthers) and female (ovary) reproductive tissues, with anthers being more sensitive to heat. Heat damage promotes early tapetal cell degradation, and in beans this was shown to cause male infertility. In this study, we focus on understanding how changes in leaf carbon export in response to elevated temperature stress contribute to heat-induced infertility. We hypothesize that anther glucose-6-phosphate dehydrogenase (G6PDH) activity plays an important role at elevated temperature and promotes thermotolerance. To test this hypothesis, we compared heat-tolerant and susceptible common bean genotypes using a combination of phenotypic, biochemical, and physiological approaches. Our results identified changes in leaf sucrose export, anther sugar accumulation and G6PDH activity and anther H₂ O₂ levels and antioxidant-related enzymes between genotypes at elevated temperature. Further, anther respiration rate was found to be lower at high temperature in both bean varieties. Overall, our results support the hypothesis that enhanced male reproductive heat tolerance involves changes in the anther oxidative pentose phosphate pathway, which supplies reductants to critical H₂ O₂ scavenging enzymes.

Keywords: ROS quenching; anther metabolism; antioxidant enzymes; heat stress; pollen development; reactive oxygen species; sucrose phloem loading.

FIGURE 1

Analysis of male reproductive tissues of Phaseolus vulgaris. (a) Comparative image of anther dehiscence of common bean heat‐tolerant (Sacramento) and heat‐susceptible (Red Hawk) cultivars exposed at control or high temperature treatment. (b) Analysis of pollen grain number formed in common bean anthers under control or high temperature stress (n = 9 individual anthers, each from individual flowers). Data presented as total number of pollen grains in five microscope fields of vision. (c) Percent pollen viability of pollen grains collected from anthers that developed under control or heat treatment at 7–5 days pre‐anthesis (n = 9–10 anthers, each from individual flowers). Data were analysed using two‐way ANOVA with post hoc Tukey tests (letters indicate significant differences between groups at p < .05). Error bars depict SEM. Thick line inside the boxes depict the mean and thin lines depict the median [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Analysis of (a) hydrogen peroxide content (n = 3–6 pools of anthers) and (b) catalase enzyme activity (n = 4–5 pools of anthers) in heat‐tolerant and heat‐sensitive common bean anthers at control and high temperature treatment. (c) Diagram of the glutathione‐ascorbate mechanism for H₂O₂ scavenging. APX, ascorbate peroxidase; DHAR, dehydroascorbate reductase; GPX, glutathione peroxidase; GR, glutathione reductase. Pools of anthers each consist of anthers collected from eight flowers. Enzyme activity is expressed per μg of anther total protein. Data were analysed using two‐way ANOVA with post hoc Tukey tests (letters indicate significant differences between groups at p < .05). Error bars depict SEM. Thick line inside the boxes depict the mean and thin lines depict the median [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Analysis of (a) anther ascorbate peroxidase (APX; n = 5 pools of anthers) and (b) dehydroascorbate reductase (DHAR; n = 4–5 pools of anthers) activities. Data were analysed using two‐way ANOVAs with post hoc Tukey tests (letters indicate significant differences between groups at p < .05). Error bars depict SEM. Thick line inside the boxes depict the mean and thin lines depict the median [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Analysis of anther enzyme activities and leaf to anthers transport of sucrose. (a) Anther G6PDH activity (n = 4–5 pools of anthers). (b) Anther glutathione reductase (GR) activity (n = 4–5 pools of anthers). (c) Anther glutathione peroxidase (GPX) activity (n = 4–5 pools of anthers). (d) qRT‐PCR expression analysis of PvSUT1.1 transporter gene (n = 4–5). (e) Measurement of phloem exudate sucrose levels collected from common bean plants 30 days after sowing (n = 8–9). (f) Source leaf feeding of ¹⁴CO₂ and detection of radioactivity in isolated anthers of Sacramento and Red Hawk under control and high temperature stress (n = 5–6 plants, each plant a pool of 30 anthers). Pools of anthers each consist of anthers collected from eight flowers. Enzyme activity is expressed per μg of anther total protein. Data were analysed using two‐way ANOVA with post hoc Tukey tests (letters indicate significant differences between groups at p < .05). Error bars depict SEM. Thick line inside the boxes depict the mean and thin line depict the median [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

Analysis of soluble carbohydrates in flowers and anthers of two common bean cultivars. Flowers and anthers that developed after onset of heat treatment were collected at 7–5 days before anthesis. (a) Flower sucrose levels in Sacramento and Red Hawk subjected to control or heat stress conditions (n = 7). (b) Anther sucrose levels from common beans (n = 5–7 pools of anthers). (c) Anther glucose levels (n = 5–7 pools of anthers). Data were analysed using two‐way ANOVAs with post hoc Tukey tests (letters indicate significant differences between groups at p < .05). Error bars depict SEM. Thick line inside the boxes depict the mean and thin lines depict the median [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 6

Measurement of anther respiration rate under control and heat stress conditions. A pool of 30 anthers from flowers in each plant that developed under heat stress were dissected and placed in an insect respiration chamber connected to a LI‐COR 6800. Each pool of anthers represents individual biological repeat. Respiration rate was quantified as the amount of CO₂ released by the anthers (n = 6). Data were analysed using two‐way ANOVA with post hoc Tukey test (letters indicate significant differences between groups at p < .05). Error bars depict SEM. Thick line inside the boxes depict the mean and thin line depict the median [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 7

Overall model of carbon flux beginning from phloem unloading of sucrose in anthers and the effect on cellular respiration, tapetum health, and pollen production at elevated temperature. Black arrows depict flux of carbon and arrow thickness indicates increased or decreased flux. Red arrows depict progression of processes and arrow thickness indicate increased or decreased occurrence [Colour figure can be viewed at wileyonlinelibrary.com]