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In flowering plants, male gametes are immotile and carried by dry pollen grains to the female organ. Dehydrated pollen is thought to withstand abiotic stress when grains are dispersed from the anther to the pistil, after which sperm cells are delivered via pollen tube growth for fertilization and seed set. Yet, the underlying molecular changes accompanying dehydration and the impact on pollen development are poorly understood. To gain a systems perspective, we analyzed published transcriptomes and proteomes of developing Arabidopsis thaliana pollen. Waves of transcripts are evident as microspores develop to bicellular, tricellular, and mature pollen. Between the “early”- and “late”-pollen-expressed genes, an unrecognized cluster of transcripts accumulated, including those encoding late-embryogenesis abundant (LEA), desiccation-related protein, transporters, lipid-droplet associated proteins, pectin modifiers, cysteine-rich proteins, and mRNA-binding proteins. Results suggest dehydration onset initiates after bicellular pollen is formed. Proteins accumulating in mature pollen like ribosomal proteins, initiation factors, and chaperones are likely components of mRNA-protein condensates resembling “stress” granules. Our analysis has revealed many new transcripts and proteins that accompany dehydration in developing pollen. Together with published functional studies, our results point to multiple processes, including (1) protect developing pollen from hyperosmotic stress, (2) remodel the endomembrane system and walls, (3) maintain energy metabolism, (4) stabilize presynthesized mRNA and proteins in condensates of dry pollen, and (5) equip pollen for compatibility determination at the stigma and for recovery at rehydration. These findings offer novel models and molecular candidates to further determine the mechanistic basis of dehydration and desiccation tolerance in plants.

 

During angiosperm sexual reproduction, pollen tubes must penetrate through multiple cell types in the pistil to mediate successful fertilization. Although this process is highly choreographed and requires complex chemical and mechanical signaling to guide the pollen tube to its destination, aspects of our understanding of pollen tube penetration through the pistil are incomplete. Our previous work demonstrated that disruption of the Arabidopsis thaliana O-FUCOSYLTRANSFERASE1 (OFT1) gene resulted in decreased pollen tube penetration through the stigma-style interface. Here, we demonstrate that second site mutations of Arabidopsis GALACTURONOSYLTRANSFERASE 14 (GAUT14) effectively suppress the phenotype of oft1 mutants, partially restoring silique length, seed set, pollen transmission, and pollen tube penetration deficiencies in navigating the female reproductive tract. These results suggest that disruption of pectic homogalacturonan (HG) synthesis can alleviate the penetrative defects associated with the oft1 mutant and may implicate pectic HG deposition in the process of pollen tube penetration through the stigma-style interface in Arabidopsis. These results also support a model in which OFT1 function directly or indirectly modifies structural features associated with the cell wall, with the loss of oft1 leading to an imbalance in the wall composition that can be compensated for by a reduction in pectic HG deposition. 

Calcium ion transporting systems control cytosol Ca2+ levels ([Ca2+]cyt) and generate transient calcium (Ca2+) signatures that are key to environmental responses. Here, we report an impact of resting [Ca2+]cyt on plants from the functional study of calmodulin-regulated Ca2+ pumps or Ca2+-ATPases in Arabidopsis (Arabidopsis thaliana). The plasma membrane-localized pumps ACA8 (autoinhibited Ca2+-ATPase) and ACA10, as well as the vacuole-localized pumps ACA4 and ACA11, were critical in maintaining low resting [Ca2+]cyt and essential for plant survival under chilling and heat-stress conditions. Their loss-of-function mutants aca8 aca10 and aca4 aca11 had autoimmunity at normal temperatures, and this deregulated immune activation was enhanced by low temperature, leading to chilling lethality. Furthermore, these mutants showed an elevated resting [Ca2+]cyt, and a reduction of external Ca2+ lowered [Ca2+]cyt and repressed their autoimmunity and cold susceptibility. The aca8 aca10 and the aca4 aca11 mutants were also susceptible to heat, likely resulting from more closed stomata and higher leaf surface temperature than the wild type. These observations support a model in which the regulation of resting [Ca2+]cyt is critical to how plants regulate biotic and abiotic responses.

 

Abstract A potential strategy to mitigate oxidative damage in plants is to increase the abundance of antioxidants, such as ascorbate (i.e. vitamin C). In Arabidopsis (A. thaliana), a rate-limiting step in ascorbate biosynthesis is a phosphorylase encoded by Vitamin C Defective 2 (VTC2). To specifically overexpress VTC2 (VTC2 OE) in pollen, the coding region was expressed using a promoter from a gene with ∼150-fold higher expression in pollen, leading to pollen grains with an eight-fold increased VTC2 mRNA. VTC2 OE resulted in a near-sterile phenotype with a 50-fold decrease in pollen transmission efficiency and a five-fold reduction in the number of seeds per silique. In vitro assays revealed pollen grains were more prone to bursting (greater than two-fold) or produced shorter, morphologically abnormal pollen tubes. The inclusion of a genetically encoded Ca2+ reporter, mCherry-GCaMP6fast (CGf), revealed pollen tubes with altered tip-focused Ca2+ dynamics and increased bursting frequency during periods of oscillatory and arrested growth. Despite these phenotypes, VTC2 OE pollen failed to show expected increases in ascorbate or reductions in reactive oxygen species, as measured using a redox-sensitive dye or a roGFP2. However, mRNA expression analyses revealed greater than two-fold reductions in mRNA encoding two enzymes critical to biosynthetic pathways related to cell walls or glyco-modifications of lipids and proteins: GDP-d-mannose pyrophosphorylase (GMP) and GDP-d-mannose 3′,5′ epimerase (GME). These results support a model in which the near-sterile defects resulting from VTC2 OE in pollen are associated with feedback mechanisms that can alter one or more signaling or metabolic pathways critical to pollen tube growth and fertility. 

Abstract Sexual reproduction in flowering plants takes place without an aqueous environment. Sperm are carried by pollen through air to reach the female gametophyte, though the molecular basis underlying the protective strategy of the male gametophyte is poorly understood. Here we compared the published transcriptomes of Arabidopsis thaliana pollen, and of heat-responsive genes, and uncovered insights into how mature pollen (MP) tolerates desiccation, while developing and germinating pollen are vulnerable to heat stress. Germinating pollen expresses molecular chaperones or “heat shock proteins” in the absence of heat stress. Furthermore, pollen tubes that grew through pistils at basal temperature showed induction of the endoplasmic reticulum (ER) stress response, which is a characteristic of stressed vegetative tissues. Recent studies show MP contains mRNA–protein (mRNP) aggregates that resemble “stress” granules triggered by heat or other stresses to protect cells. Based on these observations, we postulate that mRNP particles are formed in maturing pollen in response to developmentally programmed dehydration. Dry pollen can withstand harsh conditions as it is dispersed in air. We propose that, when pollen lands on a compatible pistil and hydrates, mRNAs stored in particles are released, aided by molecular chaperones, to become translationally active. Pollen responds to osmotic, mechanical, oxidative, and peptide cues that promote ER-mediated proteostasis and membrane trafficking for tube growth and sperm discharge. Unlike vegetative tissues, pollen depends on stress-protection strategies for its normal development and function. Thus, heat stress during reproduction likely triggers changes that interfere with the normal pollen responses, thereby compromising male fertility. This holistic perspective provides a framework to understand the basis of heat-tolerant strains in the reproduction of crops. 

Land plants evolved to quickly sense and adapt to temperature changes, such as hot days and cold nights. Given that calcium (Ca 2+ ) signaling networks are implicated in most abiotic stress responses, heat-triggered changes in cytosolic Ca 2+ were investigated in Arabidopsis leaves and pollen. Plants were engineered with a reporter called CGf, a ratiometric, genetically encoded Ca 2+ reporter with an m C herry reference domain fused to an intensiometric Ca 2+ reporter G CaMP6 f . Relative changes in [Ca 2+ ] cyt were estimated based on CGf’s apparent K D around 220 nM. The ratiometric output provided an opportunity to compare Ca 2+ dynamics between different tissues, cell types, or subcellular locations. In leaves, CGf detected heat-triggered cytosolic Ca 2+ signals, comprised of three different signatures showing similarly rapid rates of Ca 2+ influx followed by differing rates of efflux (50% durations ranging from 5 to 19 min). These heat-triggered Ca 2+ signals were approximately 1.5-fold greater in magnitude than blue light-triggered signals in the same leaves. In contrast, growing pollen tubes showed two different heat-triggered responses. Exposure to heat caused tip-focused steady growth [Ca 2+ ] cyt oscillations to shift to a pattern characteristic of a growth arrest (22%), or an almost undetectable [Ca 2+ ] cyt (78%). Together, these contrasting examples of heat-triggered Ca 2+ responses in leaves and pollen highlight the diversity of Ca 2+ signals in plants, inviting speculations about their differing kinetic features and biological functions. 

Climate change has created an environment where heat stress conditions are becoming more frequent as temperatures continue to raise in crop production areas around the world. This situation leads to decreased crop production due to plant sensitivity to heat stress. Reproductive success is critically dependent on plants’ ability to produce functional pollen grains, which are the most thermo-sensitive tissue. Flavonols are plant secondary metabolites known for their potent antioxidative activity, essential for male fertility in several species including tomato, and implicated in heat stress tolerance. Since flavonols are highly abundant in fruits of the tomato high pigment 2 ( hp2 ) mutant, we tested the level of flavonols in pollen of this mutant, under the hypothesis that increased accumulation of flavonols would render pollen more tolerant to heat stress. Indeed, pollen from two alleles of the hp2 mutant was found to have flavonols levels increased by 18 and 280% compared with wild-type (WT) under moderate chronic heat stress (MCHS) conditions. This mutant produced on average 7.8-fold higher levels of viable pollen and displayed better germination competence under heat stress conditions. The percentage of fully seeded fruits and the number of seeds per fruit were maintained in the mutant under heat stress conditions while decreased in wild-type plants. Our results strongly suggest that increased concentrations of pollen flavonols enhance pollen thermotolerance and reproductive success under heat stress conditions. Thus, the high flavonols trait may help frame the model for improving crop resilience to heat stress. 

Abstract The lipid bilayer of biological membranes has a complex composition, including high chemical heterogeneity, the presence of nanodomains of specific lipids, and asymmetry with respect to lipid composition between the two membrane leaflets. In membrane trafficking, membrane vesicles constantly bud off from one membrane compartment and fuse with another, and both budding and fusion events have been proposed to require membrane lipid asymmetry. One mechanism for generating asymmetry in lipid bilayers involves the action of the P4 ATPase family of lipid flippases; these are biological pumps that use ATP as an energy source to flip lipids from one leaflet to the other. The model plant Arabidopsis (Arabidopsis thaliana) contains 12 P4 ATPases (AMINOPHOSPHOLIPID ATPASE1–12; ALA1–12), many of which are functionally redundant. Studies of P4 ATPase mutants have confirmed the essential physiological functions of these pumps and pleiotropic mutant phenotypes have been observed, as expected when genes required for basal cellular functions are disrupted. For instance, phenotypes associated with ala3 (dwarfism, pollen defects, sensitivity to pathogens and cold, and reduced polar cell growth) can be related to membrane trafficking problems. P5 ATPases are evolutionarily related to P4 ATPases, and may be the counterpart of P4 ATPases in the endoplasmic reticulum. The absence of P4 and P5 ATPases from prokaryotes and their ubiquitous presence in eukaryotes make these biological pumps a defining feature of eukaryotic cells. Here, we review recent advances in the field of plant P4 and P5 ATPases. 

Abstract Generating cellular Ca2+ signals requires coordinated transport activities from both Ca2+ influx and efflux pathways. In Arabidopsis (Arabidopsis thaliana), multiple efflux pathways exist, some of which involve Ca2+-pumps belonging to the Autoinhibited Ca2+-ATPase (ACA) family. Here, we show that ACA1, 2, and 7 localize to the endoplasmic reticulum (ER) and are important for plant growth and pollen fertility. While phenotypes for plants harboring single-gene knockouts (KOs) were weak or undetected, a triple KO of aca1/2/7 displayed a 2.6-fold decrease in pollen transmission efficiency, whereas inheritance through female gametes was normal. The triple KO also resulted in smaller rosettes showing a high frequency of lesions. Both vegetative and reproductive phenotypes were rescued by transgenes encoding either ACA1, 2, or 7, suggesting that all three isoforms are biochemically redundant. Lesions were suppressed by expression of a transgene encoding NahG, an enzyme that degrades salicylic acid (SA). Triple KO mutants showed elevated mRNA expression for two SA-inducible marker genes, Pathogenesis-related1 (PR1) and PR2. The aca1/2/7 lesion phenotype was similar but less severe than SA-dependent lesions associated with a double KO of vacuolar pumps aca4 and 11. Imaging of Ca2+ dynamics triggered by blue light or the pathogen elicitor flg22 revealed that aca1/2/7 mutants display Ca2+ transients with increased magnitudes and durations. Together, these results indicate that ER-localized ACAs play important roles in regulating Ca2+ signals, and that the loss of these pumps results in male fertility and vegetative growth deficiencies.