Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.

Genome-wide identification of WRKY transcription factors in Casuarina equisetifolia and the function analysis of CeqWRKY11 in response to NaCl/NaHCO3 stresses.

BACKGROUND: Casuarina equisetifolia (C. equisetifolia) is a woody species with many excellent features. It has natural resistance against drought, salt and saline-alkali stresses. WRKY transcription factors (TFs) play significant roles in plant response to abiotic stresses, therefore, molecular characterization of WRKY gene family under abiotic stresses holds great significance for improvement of forest trees through molecular biological tools. At present, WRKY TFs from C. equisetifolia have not been thoroughly studied with respect to their role in salt and saline-alkali stresses response. The current study was conducted to bridge the same knowledge gap. RESULTS: A total of 64 WRKYs were identified in C. equisetifolia and divided into three major groups i.e. group I, II and III, consisting of 10, 42 and 12 WRKY members, respectively. The WRKY members in group II were further divided into 5 subgroups according to their homology with Arabidopsis counterparts. WRKYs belonging to the same group exhibited higher similarities in gene structure and the presence of conserved motifs. Promoter analysis data showed the presence of various response elements, especially those related to hormone signaling and abiotic stresses, such as ABRE (ABA), TGACG (MeJA), W-box ((C/T) TGAC (T/C)) and TC-rich motif. Tissue specific expression data showed that CeqWRKYs were mainly expressed in root under normal growth conditions. Furthermore, most of the CeqWRKYs were up-regulated by NaCl and NaHCO3 stresses with few of WRKYs showing early responsiveness to both stresses while few others exhibiting late response. Although the expressions of CeqWRKYs were also induced by cold stress, the response was delayed compared with other stresses. Transgenic C. equisetifolia plants overexpressing CeqWRKY11 displayed lower electrolyte leakage, higher chlorophyll content, and enhanced tolerance to both stresses. The higher expression of abiotic stress related genes, especially CeqHKT1 and CeqPOD7, in overexpression lines points to the maintenance of optimum Na+/K+ ratio, and ROS scavenging as possible key molecular mechanisms underlying salt stress tolerance. CONCLUSIONS: Our results show that CeqWRKYs might be key regulators of NaCl and NaHCO3 stresses response in C. equisetifolia. In addition, positive correlation of CeqWRKY11 expression with increased stress tolerance in C. equisetifolia encourages further research on other WRKY family members through functional genomic tools. The best candidates could be incorporated in other woody plant species for improving stress tolerance.

Genome-wide identification and expression analysis of the HAK/KUP/KT gene family in Moso bamboo.

The K+ uptake permease/high-affinity K+/K+ transporter (KUP/HAK/KT) family is the most prominent group of potassium (K+) transporters, playing a key role in K+ uptake, transport, plant growth and development, and stress tolerance. However, the presence and functions of the KUP/HAK/KT family in Moso bamboo (Phyllostachys edulis (Carriere) J. Houzeau), the fastest-growing plant, have not been studied. In this study, we identified 41 KUP/HAK/KT genes (PeHAKs) distributed across 18 chromosomal scaffolds of the Moso bamboo genome. PeHAK is a typical membrane protein with a conserved structural domain and motifs. Phylogenetic tree analysis classified PeHAKs into four distinct clusters, while collinearity analysis revealed gene duplications resulting from purifying selection, including both tandem and segmental duplications. Enrichment analysis of promoter cis-acting elements suggested their plausible role in abiotic stress response and hormone induction. Transcriptomic data and STEM analyses indicated that PeHAKs were involved in tissue and organ development, rapid growth, and responded to different abiotic stress conditions. Subcellular localization analysis demonstrated that PeHAKs are predominantly expressed at the cell membrane. In-situ PCR experiments confirmed that PeHAK was mainly expressed in the lateral root primordia. Furthermore, the involvement of PeHAKs in potassium ion transport was confirmed by studying the potassium ion transport properties of a yeast mutant. Additionally, through homology modeling, we revealed the structural properties of HAK as a transmembrane protein associated with potassium ion transport. This research provides a solid basis for understanding the classification, characterization, and functional analysis of the PeHAK family in Moso bamboo.

Calcium signaling-mediated transcriptional reprogramming during abiotic stress response in plants.

Calcium (Ca2+) is a second messenger in plants growth and development, as well as in stress responses. The transient elevation in cytosolic Ca2+ concentration have been reported to be involved in plants response to abiotic and biotic stresses. In plants, Ca2+-induced transcriptional changes trigger molecular mechanisms by which plants adapt and respond to environment stresses. The mechanism for transcription regulation by Ca2+ could be either rapid in which Ca2+ signals directly cause the related response through the gene transcript and protein activities, or involved amplification of Ca2+ signals by up-regulation the expression of Ca2+ responsive genes, and then increase the transmission of Ca2+ signals. Ca2+ regulates the expression of genes by directly binding to the transcription factors (TFs), or indirectly through its sensors like calmodulin, calcium-dependent protein kinases (CDPK) and calcineurin B-like protein (CBL). In recent years, significant progress has been made in understanding the role of Ca2+-mediated transcriptional regulation in different processes in plants. In this review, we have provided a comprehensive overview of Ca2+-mediated transcriptional regulation in plants in response to abiotic stresses including nutrition deficiency, temperature stresses (like heat and cold), dehydration stress, osmotic stress, hypoxic, salt stress, acid rain, and heavy metal stress.

Transcriptome profiling of Arabidopsis slac1-3 mutant reveals compensatory alterations in gene expression underlying defective stomatal closure.

Plants adjust their stomatal aperture for regulating CO2 uptake and transpiration. S-type anion channel SLAC1 (slow anion channel-associated 1) is required for stomatal closure in response to various stimuli such as abscisic acid, CO2, and light/dark transitions etc. Arabidopsis slac1 mutants exhibited defects in stimulus-induced stomatal closure, reduced sensitivity to darkness, and faster water loss from detached leaves. The global transcriptomic response of a plant with defective stimuli-induced stomatal closure (particularly because of defects in SLAC1) remains to be explored. In the current research we attempted to address the same biological question by comparing the global transcriptomic changes in Arabidopsis slac1-3 mutant and wild-type (WT) under dark, and dehydration stress, using RNA-sequencing. Abscisic acid (ABA)- and dark-induced stomatal closure was defective in Arabidopsis slac1-3 mutants, consequently the mutants had cooler leaf temperature than WT. Next, we determined the transcriptomic response of the slac1-3 mutant and WT under dark and dehydration stress. Under dehydration stress, the molecular response of slac1-3 mutant was clearly distinct from WT; the number of differentially expressed genes (DEGs) was significantly higher in mutant than WT. Dehydration induced DEGs in mutant were related to hormone signaling pathways, and biotic and abiotic stress response. Although, overall number of DEGs in both genotypes was not different under dark, however, the expression pattern was very much distinct; whereas majority of DEGs in WT were found to be downregulated, in slac1-3 majority were upregulated under dark. Further, a set 262 DEGs was identified with opposite expression pattern between WT and mutant under light-darkness transition. Amongst these, DEGs belonging to stress hormone pathways, and biotic and abiotic stress response were over-represented. To sum up, we have reported gene expression reprogramming underlying slac1-3 mutation and resultantly defective stomatal closure in Arabidopsis. Moreover, the induction of biotic and abiotic response in mutant under dehydration and darkness could be suggestive of the role of stomata as a switch in triggering these responses. To summarize, the data presented here provides useful insights into the gene expression reprogramming underlying slac1-3 mutation and resultant defects in stomatal closure.

Calcium Signaling in Plant Programmed Cell Death.

Programmed cell death (PCD) is a process intended for the maintenance of cellular homeostasis by eliminating old, damaged, or unwanted cells. In plants, PCD takes place during developmental processes and in response to biotic and abiotic stresses. In contrast to the field of animal studies, PCD is not well understood in plants. Calcium (Ca2+) is a universal cell signaling entity and regulates numerous physiological activities across all the kingdoms of life. The cytosolic increase in Ca2+ is a prerequisite for the induction of PCD in plants. Although over the past years, we have witnessed significant progress in understanding the role of Ca2+ in the regulation of PCD, it is still unclear how the upstream stress perception leads to the Ca2+ elevation and how the signal is further propagated to result in the onset of PCD. In this review article, we discuss recent advancements in the field, and compare the role of Ca2+ signaling in PCD in biotic and abiotic stresses. Moreover, we discuss the upstream and downstream components of Ca2+ signaling and its crosstalk with other signaling pathways in PCD. The review is expected to provide new insights into the role of Ca2+ signaling in PCD and to identify gaps for future research efforts.

Slow anion channel GhSLAC1 is essential for stomatal closure in response to drought stress in cotton.

Drought is one of the abiotic stresses which affects the growth and development of plants, including cotton. The role of stomatal anion channel SLAC1 has been well established in regulating stomatal closure in response to drought stress in several plant species. However, the gene encoding for the main S-type anion channel SLAC1 in cotton has not been identified hence its role in drought stress response remains uncharacterized. In this study, we identified Gh_A08G1582 as the gene encoding for GhSLAC1 in cotton. The gene exhibited abundant expression in leaves and was localized in cell membrane. Furthermore, the expression of GhSLAC1 in Arabidopsis slac1-3 mutants rescued the defective stomatal movement phenotypes of the mutants, pointing to its role in stomata regulation. GhSLAC1 channel was activated by AtOST1 in Xenopus laevis oocytes and showed greater permeability for nitrate than chloride. Further data demonstrated that transgenic cotton lines with silenced GhSLAC1 exhibited obvious leaf wilting phenotype and strong stomatal closure insensitivity under drought stress. Taken together, these results demonstrate that GhSLAC1 is an essential element for stomatal closure in response to drought in cotton.

Genome-Wide Identification of JRL Genes in Moso Bamboo and Their Expression Profiles in Response to Multiple Hormones and Abiotic Stresses.

Jacalin-related lectins (JRLs) are a new subfamily of plant lectins that has recently been recognized and plays an important role in plant growth, development, and abiotic stress response. Although moso bamboo (Phyllostachys edulis) is an economically and industrially important bamboo worldwide, there has been no systematic identification of JRLs in this species. Here, we identified 25 JRL genes in moso bamboo, and these genes are unequally distributed among 10 genome scaffolds. Phylogenetic analysis showed that the moso bamboo JRLs were clustered into four JRL subgroups: I, II, V, and VII. Numerous stress-responsive and hormone-regulated cis-elements were detected in the upstream promoter regions of the JRLs. Genome collinearity analyses showed that the JRL genes of moso bamboo are more closely related to those of Brachypodium distachyon than to those of Oryza sativa and Zea mays. Sixty-four percent of the PeJRL genes are present as segmental and tandem duplicates. qRT-PCR expression analysis showed that JRL genes in the same subgroup were significantly downregulated in response to salicylic acid (SA), abscisic acid (ABA), and methyl jasmonate (MeJA) treatments and significantly upregulated under low temperature, drought, and salt stress; they also exhibited tissue-specific expression patterns. Subcellular localization experiments revealed that PeJRL04 and PeJRL13 were localized to the cell membrane, nucleus, and cytoplasm. Three dimensional structure prediction and yeast two-hybrid assays were used to verify that PeJRL13 exists as a self-interacting homodimer in vivo. These findings provide an important reference for understanding the functions of specific moso bamboo JRL genes and for the effective selection of stress-related genes.

Optimization of Tissue Culturing and Genetic Transformation Protocol for Casuarina equisetifolia.

Casuarina equisetifolia is widely used in agroforestry plantations for soil stabilization, ecosystem rehabilitation, reclamation, and coastal protection. Moreover, C. equisetifolia has remarkable resistance to typhoons, desert, low soil fertility, drought, and salinity, but not cold. Therefore, it is significant to breed high-quality Casuarina varieties to improve the tolerance and adaptability to cold weather by molecular techniques. The establishment of a rapid and efficient callus induction and regeneration system via tissue culture is pre-requisite for the genetic transformation of C. equisetifolia, which is so far lacking. In this study, we reported an efficient and rapid regeneration system using stem segment explants, in which callus induction was found to be optimal in a basal medium supplemented with 0.1 mg⋅L-1 TDZ and 0.1 mg⋅L-1 NAA, and proliferation in a basal medium containing 0.1 mg⋅L-1 TDZ and 0.5 mg⋅L-1 6-BA. For bud regeneration and rooting, the preferred plant growth regulator (PGR) in basal medium was 0.5 mg⋅L-1 6-BA, and a combination of 0.02 mg⋅L-1 IBA and 0.4 mg⋅L-1 IAA, respectively. We also optimized genetic a transformation protocol using Agrobacterium tumefaciens harboring the binary vector pCAMBIA1301 with ß-glucuronidase (GUS) as a reporter gene. Consequently, 5 mg L-1 hygromycin, 20 mg L-1 acetosyringone (As), and 2 days of co-cultivation duration were optimized to improve the transformation efficiency. With these optimized parameters, transgenic plants were obtained in about 4 months. Besides that, Agrobacterium rhizogenes-mediated transformation involving adventitious root induction was also optimized. Our findings will not only increase the transformation efficiency but also shorten the time for developing transgenic C. equisetifolia plants. Taken together, this pioneer study on tissue culturing and genetic transformation of C. equisetifolia will pave the way for further genetic manipulation and functional genomics of C. equisetifolia.

An S-ribonuclease binding protein EBS1 and brassinolide signaling are specifically required for Arabidopsis tolerance to bicarbonate.

Bicarbonate (NaHCO3) present in soils is usually considered to be a mixed stress for plants, with salts and high pH. NaHCO3-specific signaling in plants has rarely been reported. In this study, transcriptome analyses were conducted in order to identify NaHCO3-specific signaling in Arabidopsis. Weighted correlation network analysis was performed to isolate NaHCO3-specific modules in comparison with acetate treatment. The genes in the NaHCO3-root-specific module, which exhibited opposite expression to that in sodium acetate treatments, were further examined with their corresponding knock-out mutants. The gene Exclusively Bicarbonate Sensitive 1 (EBS1) encoding an S-ribonuclease binding protein, was identified to be specifically involved in plant tolerance to NaHCO3, but not to the other two alkaline salts, acetate and phosphate. We also identified the genes that are commonly regulated by bicarbonate, acetate and phosphate. Multiple brassinosteroid-associated gene ontology terms were enriched in these genes. Genetic assays showed that brassinosteroid signaling positively regulated plant tolerance to NaHCO3 stress, but negatively regulated tolerance to acetate and phosphate. Overall, our data identified bicarbonate-specific genes, and confirmed that alkaline stress is mainly dependent on the specificities of the weak acid ions, rather than high pH.

Brassicaceae transcriptomes reveal convergent evolution of super-accumulation of sinigrin.

Wasabi, horseradish and mustard are popular pungent crops in which the characteristic bioactive hydrolysis of specialized glucosinolates (GSLs) occurs. Although the metabolic pathways of GSLs are well elucidated, how plants have evolved convergent mechanisms to accumulate identical GSL components remains largely unknown. In this study, we discovered that sinigrin is predominantly synthesized in wasabi, horseradish and mustard in Brassicaceae. We de novo assembled the transcriptomes of the three species, revealing the expression patterns of gene clusters associated with chain elongation, side chain modification and transport. Our analysis further revealed that several gene clusters were convergently selected during evolution, exhibiting convergent shifts in amino acid preferences in mustard, wasabi and horseradish. Collectively, our findings provide insights into how unrelated crop species evolve the capacity for sinigrin super-accumulation and thus promise a potent strategy for engineering metabolic pathways at multiple checkpoints to fortify bioactive compounds for condiment or pharmaceutical purposes.

The S-Type Anion Channel ZmSLAC1 Plays Essential Roles in Stomatal Closure by Mediating Nitrate Efflux in Maize.

Diverse stimuli induce stomatal closure by triggering the efflux of osmotic anions, which is mainly mediated by the main anion channel SLAC1 in plants, and the anion permeability and selectivity of SLAC1 channels from several plant species have been reported to be variable. However, the genetic identity as well as the anion permeability and selectivity of the main S-type anion channel ZmSLAC1 in maize are still unknown. In this study, we identified GRMZM2G106921 as the gene encoding ZmSLAC1 in maize, and the maize mutants zmslac1-1 and zmslac1-2 harboring a mutator (Mu) transposon in ZmSLAC1 exhibited strong insensitive phenotypes of stomatal closure in response to diverse stimuli. We further found that ZmSLAC1 functions as a nitrate-selective anion channel without obvious permeability to chloride, sulfate and malate, clearly different from SLAC1 channels of Arabidopsis thaliana, Brassica rapa ssp. chinensis and Solanum lycopersicum L. Further experimental data show that the expression of ZmSLAC1 successfully rescued the stomatal movement phenotypes of the Arabidopsis double mutant atslac1-3atslah3-2 by mainly restoring nitrate-carried anion channel currents of guard cells. Together, these findings demonstrate that ZmSLAC1 is involved in stomatal closure mainly by mediating the efflux of nitrate in maize.

S-type Anion Channels SLAC1 and SLAH3 Function as Essential Negative Regulators of Inward K+ Channels and Stomatal Opening in Arabidopsis.

Drought stress induces stomatal closure and inhibits stomatal opening simultaneously. However, the underlying molecular mechanism is still largely unknown. Here we show that S-type anion channels SLAC1 and SLAH3 mainly inhibit inward K+ (K+in) channel KAT1 by protein-protein interaction, and consequently prevent stomatal opening in Arabidopsis. Voltage-clamp results demonstrated that SLAC1 inhibited KAT1 dramatically, but did not inhibit KAT2. SLAH3 inhibited KAT1 to a weaker degree relative to SLAC1. Both the N terminus and the C terminuses of SLAC1 inhibited KAT1, but the inhibition by the N terminus was stronger. The C terminus was essential for the inhibition of KAT1 by SLAC1. Furthermore, drought stress strongly up-regulated the expression of SLAC1 and SLAH3 in Arabidopsis guard cells, and the over-expression of wild type and truncated SLAC1 dramatically impaired K+in currents of guard cells and light-induced stomatal opening. Additionally, the inhibition of KAT1 by SLAC1 and KC1 only partially overlapped, suggesting that SLAC1 and KC1 inhibited K+in channels using different molecular mechanisms. Taken together, we discovered a novel regulatory mechanism for stomatal movement, in which singling pathways for stomatal closure and opening are directly coupled together by protein-protein interaction between SLAC1/SLAH3 and KAT1 in Arabidopsis.

Protein kinase OsSAPK8 functions as an essential activator of S-type anion channel OsSLAC1, which is nitrate-selective in rice.

MAIN CONCLUSION: OsSAPK8 is an essential activator of OsSLAC1 by phosphorylation, and OsSLAC1 is a nitrate-selective anion channel. S-type anion channel AtSLAC1 and protein kinase AtOST1 have been well-characterized as two core components of ABA signaling cascade in Arabidopsis guard cells, and AtOST1 functions as a main upstream activator of AtSLAC1 for drought stress- and ABA-induced stomata closure. However, the identity of the ortholog of AtOST1 in rice, the main activator of OsSLAC1, is still unknown. Here, we report that protein kinase OsSAPK8 interacts with and activates OsSLAC1 mainly by phosphorylating serine 129 (S129) of OsSLAC1, and this phosphorylating site corresponds to the specific phosphorylating site serine 120 (S120) of AtSLAC1 for AtOST1. Additionally, we found that OsSLAC1 is a nitrate-selective anion channel without obvious permeability to chloride, malate, and sulfate, and the expression of OsSLAC1 in Arabidopsis slac1-3 (atslac1-3) mutant successfully rescued the hypersensitive phenotype of this mutant to drought stress. Together, this research suggests that OsSAPK8 is a counterpart of AtOST1 for the activation of OsSLAC1, which is a nitrate-selective anion channel.

The Os-AKT1 channel is critical for K+ uptake in rice roots and is modulated by the rice CBL1-CIPK23 complex.

Potassium (K(+)) is one of the essential nutrient elements for plant growth and development. Plants absorb K(+) ions from the environment via root cell K(+) channels and/or transporters. In this study, the Shaker K(+) channel Os-AKT1 was characterized for its function in K(+) uptake in rice (Oryza sativa) roots, and its regulation by Os-CBL1 (Calcineurin B-Like protein1) and Os-CIPK23 (CBL-Interacting Protein Kinase23) was investigated. As an inward K(+) channel, Os-AKT1 could carry out K(+) uptake and rescue the low-K(+)-sensitive phenotype of Arabidopsis thaliana akt1 mutant plants. Rice Os-akt1 mutant plants showed decreased K(+) uptake and displayed an obvious low-K(+)-sensitive phenotype. Disruption of Os-AKT1 significantly reduced the K(+) content, which resulted in inhibition of plant growth and development. Similar to the AKT1 regulation in Arabidopsis, Os-CBL1 and Os-CIPK23 were identified as the upstream regulators of Os-AKT1 in rice. The Os-CBL1-Os-CIPK23 complex could enhance Os-AKT1-mediated K(+) uptake. A phenotype test confirmed that Os-CIPK23 RNAi lines exhibited similar K(+)-deficient symptoms as the Os-akt1 mutant under low K(+) conditions. These findings demonstrate that Os-AKT1-mediated K(+) uptake in rice roots is modulated by the Os-CBL1-Os-CIPK23 complex.

Calcineurin B-like protein CBL10 directly interacts with AKT1 and modulates K+ homeostasis in Arabidopsis.

Potassium transporters and channels play crucial roles in K+ uptake and translocation in plant cells. These roles are essential for plant growth and development. AKT1 is an important K+ channel in Arabidopsis roots that is involved in K+ uptake. It is known that AKT1 is activated by a protein kinase CIPK23 interacting with two calcineurin B-like proteins CBL1/CBL9. The present study showed that another calcineurin B-like protein (CBL10) may also regulate AKT1 activity. The CBL10-over-expressing lines showed a phenotype as sensitive as that of the akt1 mutant under low-K+ conditions. In addition, the K+ content of both CBL10-over-expressing lines and akt1 mutant plants were significantly reduced compared with wild-type plants. Moreover, CBL10 directly interacted with AKT1, as verified in yeast two-hybrid, BiFC and co-immunoprecipitation experiments. The results of electrophysiological analysis in both Xenopus oocytes and Arabidopsis root cell protoplasts demonstrated that CBL10 impairs AKT1-mediated inward K+ currents. Furthermore, the results from the yeast two-hybrid competition assay indicated that CBL10 may compete with CIPK23 for binding to AKT1 and negatively modulate AKT1 activity. The present study revealed a CBL-interacting protein kinase-independent regulatory mechanism of calcineurin B-like proteins in which CBL10 directly regulates AKT1 activity and affects ion homeostasis in plant cells.