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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. 

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 Age-dependent changes in reactive oxygen species (ROS) levels are critical in leaf senescence. While H2O2-reducing enzymes such as catalases and cytosolic ASCORBATE PEROXIDASE1 (APX1) tightly control the oxidative load during senescence, their regulation and function are not specific to senescence. Previously, we identified the role of ASCORBATE PEROXIDASE6 (APX6) during seed maturation in Arabidopsis (Arabidopsis thaliana). Here, we show that APX6 is a bona fide senescence-associated gene. APX6 expression is specifically induced in aging leaves and in response to senescence-promoting stimuli such as abscisic acid (ABA), extended darkness, and osmotic stress. apx6 mutants showed early developmental senescence and increased sensitivity to dark stress. Reduced APX activity, increased H2O2 level, and altered redox state of the ascorbate pool in mature pre-senescing green leaves of the apx6 mutants correlated with the early onset of senescence. Using transient expression assays in Nicotiana benthamiana leaves, we unraveled the age-dependent post-transcriptional regulation of APX6. We then identified the coding sequence of APX6 as a potential target of miR398, which is a key regulator of copper redistribution. Furthermore, we showed that mutants of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), the master regulator of copper homeostasis and miR398 expression, have a higher APX6 level compared with the wild type, which further increased under copper deficiency. Our study suggests that APX6 is a modulator of ROS/redox homeostasis and signaling in aging leaves that plays an important role in developmental- and stress-induced senescence programs. 

Abstract Key message Arabidopsis pollen transcriptome analysis revealed new intergenic transcripts of unknown function, many of which are long non-coding RNAs, that may function in pollen-specific processes, including the heat stress response. Abstract The male gametophyte is the most heat sensitive of all plant tissues. In recent years, long noncoding RNAs (lncRNAs) have emerged as important components of cellular regulatory networks involved in most biological processes, including response to stress. While examining RNAseq datasets of developing and germinating Arabidopsis thaliana pollen exposed to heat stress (HS), we identified 66 novel and 246 recently annotated intergenic expressed loci (XLOCs) of unknown function, with the majority encoding lncRNAs. Comparison with HS in cauline leaves and other RNAseq experiments indicated that 74% of the 312 XLOCs are pollen-specific, and at least 42% are HS-responsive. Phylogenetic analysis revealed that 96% of the genes evolved recently in Brassicaceae . We found that 50 genes are putative targets of microRNAs and that 30% of the XLOCs contain small open reading frames (ORFs) with homology to protein sequences. Finally, RNAseq of ribosome-protected RNA fragments together with predictions of periodic footprint of the ribosome P-sites indicated that 23 of these ORFs are likely to be translated. Our findings indicate that many of the 312 unknown genes might be functional and play a significant role in pollen biology, including the HS response. 

<bold>Summary</bold> Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROSpollen germinated with a frequency that was 35‐fold higher than the low‐ROSpollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantifyROSdynamics within a large pollen population was shown by dose‐dependent alterations inDCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increasedROSlevels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen. 

Summary Plants show a rapid systemic response to a wide range of environmental stresses, where the signals from the site of stimulus perception are transmitted to distal organs to elicit plant‐wide responses. A wide range of signaling molecules are trafficked through the plant, but a trio of potentially interacting messengers, reactive oxygen species (ROS), Ca²⁺and electrical signaling (‘trio signaling’) appear to form a network supporting rapid signal transmission. The molecular components underlying this rapid communication are beginning to be identified, such as theROSproducingNAPDHoxidaseRBOHD, the ion channel two pore channel 1 (TPC1), and glutamate receptor‐like channelsGLR3.3 andGLR3.6. The plant cell wall presents a plant‐specific route for possible propagation of signals from cell to cell. However, the degree to which the cell wall limits information exchange between cells via transfer of small molecules through an extracellular route, or whether it provides an environment to facilitate transmission of regulators such asROSor H⁺remains to be determined. Similarly, the role of plasmodesmata as both conduits and gatekeepers for the propagation of rapid cell‐to‐cell signaling remains a key open question. Regardless of how signals move from cell to cell, they help prepare distant parts of the plant for impending challenges from specific biotic or abiotic stresses.