The Heat Is On: Maize Pollen Development after a Heat Wave

The yield of key food crops (i.e. wheat [Triticum aestivum], rice [Oryza sativa], maize [Zea mays], and sorghum [Sorghum biocolor]) relies on their production of seeds, while the propagation of almost all crops is seed-dependent. Seed set in turn relies on the production of flowers containing the male and female gametophytes, successful fertilization, and the subsequent growth and development of the embryo and endosperm. Temperature fluctuations often have a particularly detrimental effect during reproduction, with extremes leading to a reduction in fertility in many plant species. Thermosensitivity in response to heat results from developmental or physiological effects on meiosis during male and female gametogenesis, pollen tube germination and growth, fertilization, and subsequent development of the seed (Giorno et al., 2013; Saini et al., 1983). Cell-to-cell interactions between the gametophytic tissues may also be disrupted (Giorno et al., 2013). With climate change set to increase the frequency of extreme weather events, it is imperative to understand how heat and cold affects each of the steps from flower initiation to seed development, and how these effects can be mitigated.

Anther and pollen development are more sensitive to temperature fluctuations than are other steps in gametophyte development, fertilization, and seed set (e.g. Warrag and Hall, 1984; Erickson and Markhart, 2002; Kim et al., 2001). As an example of the impact of a heatwave, under higher temperatures, tomato (Solanum lycopersicum) pollen grains accumulate less starch prior to anthesis and less soluble sugars at anthesis (Pressman et al., 2002). Starch accumulation during late stages of pollen grain maturation is important, as it provides an energy source for the male gametophyte during germination and pollen tube growth. Heat stress also affects mono- and disaccharide levels, leading to decreased Glc and Fru and increased Suc levels in tomato pollen (Sato et al., 2006).

In this issue of Plant Physiology, Begcy et al. (2019) from the University of Regensberg, Helmholtz Center Munich, and University of Vienna present a study on the longer-term effects of transient heat stress on subsequent pollen development and fertilization in maize. Plants with pollen grains at the tetrad stage were subjected to 2 d of heat stress followed by physiological and morphological analysis. Two weeks later, the mature pollen was examined by developmental and morphological assays, metabolomics, and transcriptomics.

Begcy et al. (2019) validated the immediate effects of their heat stress treatment through the presence of tetrad microspores with irregular morphology. Interestingly, staining with the DNA and tubulin-binding compound 4′,6-diamidino-2-phenylindole (DAPI; Bonne et al., 1985) led to less intense coloration of heat-stressed microspores, indicating that DNA may be damaged or decondensed, the cytoskeleton influenced by increased temperature, or the cell wall affected in such a manner that DAPI uptake is impacted. Whether reduced DAPI staining is transient or continues throughout pollen development once maize plants are returned to more conducive temperatures remains to be tested.

Having confirmed that 2 d of heat stress at an early stage of gametogenesis is sufficient to affect short-term maize pollen development, the authors turned their focus to the longer-term effects of this transient increase in temperature. Histological analysis demonstrated a delay in some aspects of pollen development that is evident as the grains mature. Loss of pollen adherence to the anthers was accelerated, which may be linked to the low germination and fertilization rates of pollen from heat-stressed plants (Fig. 1).

Figure 1.

Transiently heat stressed maize pollen has reduced fertility. When non-heat-stressed female parents are fertilized with pollen from heat-stressed plants, seed set is severely reduced (right) compared to fertilization with control non-heat-stressed pollen (left). Reprint of figure 3F in Begcy et al. (2019).

At a metabolic level, starch granules and total starch were reduced in mature pollen grains from heat-stressed plants, as has previously been observed in tomato (Pressman et al., 2002). To test whether this decrease in starch was caused by enzymatic limitations, mature pollen grains were stained with fluorescein diacetate, a vital stain that is hydrolyzed nonspecifically by esterases, lipases, and proteases. The authors speculate that the reduction in photoassimilates could have affected enzymatic activity, influencing starch/sugar levels and lipid composition and hence pollen grain function. To more directly link changes in photoassimilates with pollen viability and alterations in metabolites, transient heat stress will need to be applied to only nonreproductive or reproductive tissues and the assays focused on the activity of specific enzymes.

A detailed transcriptomic and metabolomic analysis of mature pollen grains followed. RNA sequencing identified a subset of genes with differential expression between transiently stressed and control pollen grains, including those encoding heat shock proteins, a potential regulator of apoptosis, and a JAZ/TIFY protein that may repress expression of transcription factors involved in plant defense along with other upregulated genes linked to stress responses. Further analysis of the RNA levels of transcription factors showed induction of some families that are usually expressed during early pollen development rather than in mature pollen (MYB and MYB related), and downregulation of other families that are more usually expressed later in pollen development (Golden-like 2; GLK). Genes linked to translation and growth were downregulated, while a correlation analysis revealed misregulation of a number of kinases. In some instances, changes in gene expression could result from differences in the development of the mature pollen grains after transient heat stress. Further analysis of specific signaling and cell wall-associated protein families did not identify global changes in gene expression. Auxin responses were also not altered in mature maize pollen despite a decrease in auxin levels in anthers of other plant species upon heat stress (Sakata et al., 2010).

One outcome of the RNA-sequencing correlation analysis was identification of misregulated genes involved in carbohydrate metabolism, with downregulation of ADP-Glc pyrophosphate, phosphate dehydrogenase, and enolase and upregulation of an invertase and a phosphofructokinase. Gas chromatography confirmed that transcriptional changes were accompanied by increased Fru, Suc, and Glc levels, while pyruvate was decreased, with the increase in Suc echoing previous results from tomato (Sato et al., 2006). The decrease in pyruvate could conceivably be linked to lipid biosynthesis and basic metabolism, as pyruvate is used in the generation of acetyl-coenzyme A, which in turn is used in the synthesis of lipids and ATP. Metabolic analyses indicated a decrease in long-chain saturated fatty acids and an increase in long-chain unsaturated fatty acids that could limit the germination of pollen and growth of pollen tubes due to their reliance on lipids for cell membrane formation. Therefore, even 2 weeks after a transient heat stress, the effects on the transcriptome and metabolome are likely to result in reduced male gametophyte viability due to changes in lipid composition and energy biosynthesis via pyruvate.

Whether this knowledge can be used to make grains and seeds more thermotolerant remains to be explored. The applicability of these results to further crop species will also need to be confirmed. Conceivably, crop varieties could be bred or developed that retain higher starch levels and pyruvate levels, lower Suc, Glc, and Fru levels, and normal lipid profiles under transient heat stress. The expectation is that pollen viability may be retained at a higher level in such plants upon transient heat stress and thus that thermotolerance could be increased in the face of more frequent heatwaves.