Low Salicylic Acid Level Improves Pollen Development Under Long-Term Mild Heat Conditions in Tomato

Exposure to high temperatures leads to failure in pollen development, which may have significant implications for food security with ongoing climate change. We hypothesized that the stress response-associated hormone salicylic acid (SA) affects pollen tolerance to long-term mild heat (LTMH) (≥14 days exposure to day-/nighttime temperature of 30–34/24–28°C, depending on the genotype), either positively, by inducing acclimation, or negatively, by reducing investment in reproductive development. Here, we investigated these hypotheses assessing the pollen thermotolerance of a 35S:nahG tomato line, which has low SA levels. We found that reducing the SA level resulted in increased pollen viability of plants grown in LTMH and further characterized this line by transcriptome, carbohydrate, and hormone analyses. Low expression of JAZ genes in 35S:nahG and LTMH hypersensitivity of low-jasmonic acid (JA) genotypes together suggest that the increased pollen thermotolerance in the low-SA line involves enhanced JA signal in developing anthers in LTMH. These findings have potential application in the development of more thermotolerant crops

Long-Term Mild Heat Causes Post-Mitotic Pollen Abortion Through a Local Effect on Flowers

Crop reproductive success is significantly challenged by heatwaves, which are increasing in frequency and severity globally. Heat-induced male sterility is mainly due to aborted pollen development, but it is not clear whether this is through direct or systemic effects. Here, long-term mild heat (LTMH) treatment, mimicking a heatwave, was applied locally to tomato flowers or whole plants and followed up by cytological, transcriptomic, and biochemical analyses. By analyzing pollen viability, LTMH was shown to act directly on the flowers and not via effects on other plant tissue. The meiosis to early microspore stage of pollen development was the most sensitive to LTMH and 3 days of exposure around this period was sufficient to significantly reduce pollen viability at the flower anthesis stage. Extensive cytological analysis showed that abnormalities in pollen development could first be observed after pollen mitosis I, while no deviations in tapetum development were observed. Transcriptomic and biochemical analyses suggested that pollen development suffered from tapetal ER stress and that there was a limited role for oxidative stress. Our results provide the first evidence that heat acts directly on flowers to induce pollen sterility, and that the molecular-physiological responses of developing anthers to the LTMH are different from those to severe heat shock

Relative expression of B- and C-class genes under control and continuous mild heat conditions (CMH32) in anthers of the tomato cultivar Red setter.

<p>A, expression of the B-class genes <i>TAP3</i>, <i>TM6</i>, <i>TPI</i> and <i>LePI</i> and the C-class genes <i>TAG1</i> and <i>TAGL1</i> in young anthers of 2–3 mm. B, gene expression in mature anthers. Values represent the mean ± SE, with the mean expression in the control condition set to 1. *, significantly different from the control treatment, P<0.05; **, P<0.01; ***, P<0.001.</p

List of primers used in this study.

<p>List of primers used in this study.</p

Presence of anther deformations, pollen germination rate and pollen number of wild-type Microtom and the <i>TM6</i>–RNAi line under control and CMH30 conditions.

<p>Presence of anther deformations, pollen germination rate and pollen number of wild-type Microtom and the <i>TM6</i>–RNAi line under control and CMH30 conditions.</p

Continous mild heat conditions affect pollen viability and flower deformation simultaneously.

<p>A, Frequency of anther deformations and pollen germination rate of tomato plants (cv. Red Setter) grown under different temperature regimes (CT, CMH32, CMH34). Values represent the mean ± SD, different letters indicate statistically significant differences (per trait, P<0.01). B, Mature flowers of wild-type tomato (cv. Red Setter) from control (CT) and CMH32 conditions. Insets show the adaxial side of the indicated boxed regions. Scale bars: 1 mm and 0,5 mm (insets). a, anther; ovl, ovule-like structures; p, petal; pi, pistil; s, sepal. C, Frequency of anther deformations and pollen germination rate of wild type (WT) tomato plants (cv. Rubicon) and transgenic lines <i>AtGRXS17-3</i> and <i>AtGRXS17-9</i> grown under CMH32. Under control conditions, no anther deformations were observed, and percentage pollen viability was 60 ± 12 (SD), 60 ± 13 and 56 ± 9 for WT, <i>AtGRXS17-3</i> and <i>AtGRXS17-9</i>, respectively (no significant differences between genotypes). Values represent the mean ± SD, different letters indicate statistically significant differences (per trait, P<0.05).</p

Cross sections of anthers and pistils from mature flowers of the tomato cultivar Red Setter grown under control and continuous mild heat conditions.

<p>A, overview of anthers grown under control conditions (CT). Scale bar: 300 μm. B, overview of anthers and pistil from CHM32. Scale bar 300 μm. C, transmitting-tissue-like cells from an anther grown under CMH32 (close up of B) on the left, and true stylar transmitting tissue from control conditions on the right. Scale bar 20 μm. D, ovule-like deformation from an anther grown under CMH32 (scale bar 30 μm). Upper inset shows overview of CMH32 anther with ovule-like deformation (scale bar 300 μm), lower inset shows a true ovule from control conditions (scale bar 50 μm). a, anther; l, locule; ov, ovule; ovl, ovule-like structure; st, style; tt, transmitting tissue; ttl, transmitting tissue-like cells; v, vascular bundle.</p

Relative expression of pistil marker genes in anthers and pistils of Red Setter grown under control conditions and continuous mild heat (CMH32).

<p>A, B, Relative expression of the <i>SlTTS</i> (A) and <i>TAGL11</i> (B) in anthers and pistils from 2-3mm flower buds and mature flowers (grown under control conditions). C, D, relative expression of <i>SlTTS</i> (C) and <i>TAGL11</i> (D) in anthers grown under control conditions (CT) and CMH32. Values represent the mean ± SE, with the mean expression in anthers (A, B) or under control conditions (C, D) at each developmental stage set to 1. *, significantly different from anther (A, B) or the control treatment (C, D), P<0.05; **, P<0.01; ***, P<0.001.</p