Genetic Analyses Of Calcium Circuits In Arabidopsis Vegetative And Reproductive Development

Calcium homeostasis is maintained by the action of different calcium transporters including channels, antiporters, and calcium pumps. Calcium channels provide an influx pathway whereas calcium antiporters and pumps provide an efflux system. Together, influx and efflux systems create a calcium circuit. One of the calcium circuits operating in plant cells is associated with the endoplasmic reticulum (ER). In chapter II, results are presented on the functional characterization of three calcium pumps in Arabidopsis with ER and ER-like localization. In chapter III and IV, Arabidopsis pollen transcriptome data are presented, which compare heat-stress induced changes in the pollen transcriptomes of wild type and a mutant harboring a knockout of a cyclic nucleotide gated channel (cngc16).Calcium pumps ACA1, 2, and 7 belong to the type IIB subfamily of Ca2+-ATPases. In a comparison of protein sequences for all ten ACA Ca2+-pumps in Arabidopsis, ACA1, 2, and 7 cluster as a distinct subgroup. Members of this subgroup all appeared to localize to the ER or ER-like structures when expressed in stably transformed plants with a YFP tag and imaged with confocal microscopy. A loss-of-function for all three ACAs, ACA1, 2, and 7, resulted in plants showing a lesion-like phenotype in the leaves. This phenotype was suppressed by the expression of a NahG transgene, which encodes an enzyme that degrades salicylic acid, indicating that the lesion phenotype is dependent on salicylic acid. In addition, a triple knockout of aca1/2/7 resulted in a decrease in pollen transmission efficiency when assayed in competition with wild type pollen. This pollen transmission deficiency was exacerbated when crosses were done under temperature stress conditions. Both phenotypes could be rescued by the expression of any of the threeACA1, 2 or 7 genes, suggesting that all three genes encode Ca2+-pumps that are functionally interchangeable. Together, these results establish ACA1, 2, and 7 as a subgroup of ER-localized autoinhibited Ca2+-ATPases that normally function to suppress an SA-dependent programmed cell death pathway in leaves, and are also important for pollen fitness under normal and temperature stress conditions.Under temperature stress conditions, pollen harboring a cngc16 knockout are nearly sterile, and show a greater than 10-fold decrease in pollen transmission efficiency compared to wild-type pollen. To gain further insight into mechanisms underlying this hypersensitivity, an RNA-Seq experiment was performed to compare the stress-dependent changes in the pollen transcriptomes from wild-type and a cngc16 knockout parent. In response to a heat stress, the wild-type pollen transcriptome showed 2,612 changes (≥2-fold changes and adjusted p<0.01). Of these, 641 changes (~ 25%) failed to occur in the cngc16 knockout, identifying a subset of failed responses that might contribute to the poor stress tolerance displayed by cngc16 pollen. At the same time, the mutant displayed 2,733 changes that were not seen in wild-type pollen. Of these differences between wild type and cngc16, 148 were associated with transcription factors, suggesting that a primary defect in the cngc16 mutant lies in its inability to properly reprogram the transcriptome in response to a heat stress. Overall, the RNA-Seq experiment here provides a great resource for research community to explore genes function of potential importance to pollen thermotolerance

Tapping into the plasticity of plant architecture for increased stress resilience [version 1; peer review: 2 approved, 1 approved with reservations]

Plant architecture develops post-embryonically and emerges from a dialogue between the developmental signals and environmental cues. Length and branching of the vegetative and reproductive tissues were the focus of improvement of plant performance from the early days of plant breeding. Current breeding priorities are changing, as we need to prioritize plant productivity under increasingly challenging environmental conditions. While it has been widely recognized that plant architecture changes in response to the environment, its contribution to plant productivity in the changing climate remains to be fully explored. This review will summarize prior discoveries of genetic control of plant architecture traits and their effect on plant performance under environmental stress. We review new tools in phenotyping that will guide future discoveries of genes contributing to plant architecture, its plasticity, and its contributions to stress resilience. Subsequently, we provide a perspective into how integrating the study of new species, modern phenotyping techniques, and modeling can lead to discovering new genetic targets underlying the plasticity of plant architecture and stress resilience. Altogether, this review provides a new perspective on the plasticity of plant architecture and how it can be harnessed for increased performance under environmental stress

A cyclic nucleotide-gated channel (CNGC16) in pollen is critical for stress tolerance in pollen reproductive development

Cyclic nucleotide-gated channels (CNGCs) have been implicated in diverse aspects of plant growth and development, including responses to biotic and abiotic stress, as well as pollen tube growth and fertility. Here, genetic evidence identifies CNGC16 in Arabidopsis (Arabidopsis thaliana) as critical for pollen fertility under conditions of heat stress and drought. Two independent transfer DNA disruptions of cngc16 resulted in a greater than 10-fold stress-dependent reduction in pollen fitness and seed set. This phenotype was fully rescued through pollen expression of a CNGC16 transgene, indicating that cngc16-1 and 16-2 were both loss-of-function null alleles. The most stress-sensitive period for cngc16 pollen was during germination and the initiation of pollen tube tip growth. Pollen viability assays indicate that mutant pollen are also hypersensitive to external calcium chloride, a phenomenon analogous to calcium chloride hypersensitivities observed in other cngc mutants. A heat stress was found to increase concentrations of 3′,5′-cyclic guanyl monophosphate in both pollen and leaves, as detected using an antibody-binding assay. A quantitative PCR analysis indicates that cngc16 mutant pollen have attenuated expression of several heat-stress response genes, including two heat shock transcription factor genes, HsfA2 and HsfB1. Together, these results provide evidence for a heat stress response pathway in pollen that connects a cyclic nucleotide signal, a Ca(2+)-permeable ion channel, and a signaling network that activates a downstream transcriptional heat shock response

Guard cell endomembrane Ca2+-ATPases underpin a ‘carbon memory’ of photosynthetic assimilation that impacts on water-use efficiency

Stomata of most plants close to preserve water when the demand for CO2 by photosynthesis is reduced. Stomatal responses are slow compared with photosynthesis, and this kinetic difference erodes assimilation and water-use efficiency under fluctuating light. Despite a deep knowledge of guard cells that regulate the stoma, efforts to enhance stomatal kinetics are limited by our understanding of its control by foliar CO2. Guided by mechanistic modelling that incorporates foliar CO2 diffusion and mesophyll photosynthesis, here we uncover a central role for endomembrane Ca2+ stores in guard cell responsiveness to fluctuating light and CO2. Modelling predicted and experiments demonstrated a delay in Ca2+ cycling that was enhanced by endomembrane Ca2+-ATPase mutants, altering stomatal conductance and reducing assimilation and water-use efficiency. Our findings illustrate the power of modelling to bridge the gap from the guard cell to whole-plant photosynthesis, and they demonstrate an unforeseen latency, or ‘carbon memory’, of guard cells that affects stomatal dynamics, photosynthesis and water-use efficiency

Additional file 13: of 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)

A comparison of HS-dependent changes in pollen to 67 multi-stress response genes in vegetative tissues. From a list of 67 multi-stress genes curated by Swindell 2006 (PMCID: PMC1698639 [47]; highlighted in purple), 19 genes showed detectable expression in pollen. Among those, only three genes showed significant changes in pollen HS (red font). (XLSX 596 kb

Additional file 5: of 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)

A transcript profile comparison to evaluate purity of pollen samples used for RNA-Seq. A subset of 12 genes was used to compare relative purities of pollen samples in the current pollen transcriptome study to those from a RNA-Seq study from Loraine et al. [22] (yellow highlights) or a microarray experiment from Qin et al. 2009 [23] (purple highlights). Four references genes were chosen to generate normalization factors that could be used to adjust expression values in Loraine et al. [22] and Qin et al. 2009 [23] to allow a relative comparison of the three data sets for WT pollen under control (normal) conditions. For a control group, three CNGC genes were chosen that displayed low to moderate levels of expression (Tunc-Ozdemir et al. 2013 [24] and Frietsch et al. 2007 [25]). As markers for potential contamination from photosynthetic tissues, five different nuclear encoded genes were chosen that are associated with either photosystems I/II, or chlorophyll A-B binding proteins (Umate 2010 [26]). Average relative ratios are shown for each of the four different pollen samples in comparison to both Loraine et al. [22] and Qin et al. [23]. (XLSX 19 kb

Additional file 2: of 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)

Raw expression counts for WT and cngc16 pollen with and without HS. Read counts generated via FeatureCounts, see Methods) for all 12 replicates are shown before normalization and exclusions. (XLSX 4358 kb

Additional file 1: of 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)

Hot/Cold stress cycle. Diagram showing the Hot/Cold stress-cycle used here for growing plants from which pollen samples for RNA-Seq experiment were harvested at the end of HS-peak at 40 °C. See Methods and [20] for more details. (PPTX 50 kb

Additional file 7: of 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)

RNA-Seq validation using real-time Q-PCR. a. Comparison of expression values obtained from Q-PCR and RNA-Seq normalized to WT control (normal). The analysis was performed on two different reference genes separately (CYCP2 (AT3G21870) and UBQ7 (AT2G35635)). b. Primer sequences used for real-time Q-PCR. (XLSX 16 kb