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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. 

Abstract 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. 

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.