Resting cytosol Ca2+ level maintained by Ca2+ pumps affects environmental responses in Arabidopsis.

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.

Overexpressing Vitamin C Defective 2 reduces fertility and alters Ca2+ signals in Arabidopsis pollen.

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.

A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16).

BACKGROUND: In flowering plants, the male gametophyte (pollen) is one of the most vulnerable cells to temperature stress. In Arabidopsis thaliana, a pollen-specific Cyclic Nucleotide-Gated cation Channel 16 (cngc16), is required for plant reproduction under temperature-stress conditions. Plants harboring a cncg16 knockout are nearly sterile under conditions of hot days and cold nights. To understand the underlying cause, RNA-Seq was used to compare the pollen transcriptomes of wild type (WT) and cngc16 under normal and heat stress (HS) conditions. RESULTS: Here we show that a heat-stress response (HSR) in WT pollen resulted in 2102 statistically significant transcriptome changes (≥ 2-fold changes with adjusted p-value ≤0.01), representing approximately 15% of 14,226 quantified transcripts. Of these changes, 89 corresponded to transcription factors, with 27 showing a preferential expression in pollen over seedling tissues. In contrast to WT, cngc16 pollen showed 1.9-fold more HS-dependent changes (3936 total, with 2776 differences between WT and cngc16). In a quantitative direct comparison between WT and cngc16 transcriptomes, the number of statistically significant differences increased from 21 pre-existing differences under normal conditions to 192 differences under HS. Of the 20 HS-dependent changes in WT that were most different in cngc16, half corresponded to genes encoding proteins predicted to impact cell wall features or membrane dynamics. CONCLUSIONS: Results here define an extensive HS-dependent reprogramming of approximately 15% of the WT pollen transcriptome, and identify at least 27 transcription factor changes that could provide unique contributions to a pollen HSR. The number of statistically significant transcriptome differences between WT and cngc16 increased by more than 9-fold under HS, with most of the largest magnitude changes having the potential to specifically impact cell walls or membrane dynamics, and thereby potentiate cngc16 pollen to be hypersensitive to HS. However, HS-hypersensitivity could also be caused by the extensive number of differences throughout the transcriptome having a cumulative effect on multiple cellular pathways required for tip growth and fertilization. Regardless, results here support a model in which a functional HS-dependent reprogramming of the pollen transcriptome requires a specific calcium-permeable Cyclic Nucleotide-Gated cation Channel, CNGC16.

Disruption of pollen tube homogalacturonan synthesis relieves pollen tube penetration defects in the Arabidopsis O-FUCOSYLTRANSFERASE1 mutant.

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.

A Ratiometric Calcium Reporter CGf Reveals Calcium Dynamics Both in the Single Cell and Whole Plant Levels Under Heat Stress.

Land plants evolved to quickly sense and adapt to temperature changes, such as hot days and cold nights. Given that calcium (Ca2+) signaling networks are implicated in most abiotic stress responses, heat-triggered changes in cytosolic Ca2+ were investigated in Arabidopsis leaves and pollen. Plants were engineered with a reporter called CGf, a ratiometric, genetically encoded Ca2+ reporter with an mCherry reference domain fused to an intensiometric Ca2+ reporter GCaMP6f. Relative changes in [Ca2+]cyt were estimated based on CGf's apparent K D around 220 nM. The ratiometric output provided an opportunity to compare Ca2+ dynamics between different tissues, cell types, or subcellular locations. In leaves, CGf detected heat-triggered cytosolic Ca2+ signals, comprised of three different signatures showing similarly rapid rates of Ca2+ influx followed by differing rates of efflux (50% durations ranging from 5 to 19 min). These heat-triggered Ca2+ 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 [Ca2+]cyt oscillations to shift to a pattern characteristic of a growth arrest (22%), or an almost undetectable [Ca2+]cyt (78%). Together, these contrasting examples of heat-triggered Ca2+ responses in leaves and pollen highlight the diversity of Ca2+ signals in plants, inviting speculations about their differing kinetic features and biological functions.

Decapitation Crosses to Test Pollen Fertility Mutations for Defects in Stigma-Style Penetration.

During sexual reproduction in flowering plants, pollen grains germinate on the stigma surface and grow through the stigma-style tissue to reach the ovary and deliver sperm cells for fertilization. Here, we outline a method to test whether a pollen fertility mutation specifically disrupts pollen penetration through the stigma-style barrier. This method surgically removes the stigma-style (stigma decapitation) to test whether transferring pollen directly onto an exposed ovary surface significantly improves the transmission efficiency (TE) of a mutant allele. To illustrate this technique, we applied stigma decapitation to investigate a loss-of-function mutation in Arabidopsis OFT1, a gene encoding a putative o-fucosyl transferase functioning in the secretory pathway. oft1-3 mutant pollen showed a significant decrease in transmission efficiency compared to wild type. Decapitation crosses (described here) indicated that the removal of the stigma-style barrier alleviated the transmission deficiency from 858-fold to a 2.6-fold, providing evidence that most, but not all, oft1 pollen deficiencies can be attributed to a reduced ability to penetrate through the stigma-style barrier. This method outlines a genetic strategy to quantify a mutation's impact on the ability of pollen to navigate through the stigma-style barrier on its journey to the ovule.