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.

Cyclic guanosine monophosphate improves salt tolerance in Solanum lycopersicum.

The cyclic nucleotide cyclic guanosine monophosphate (cGMP) is a powerful cell signaling molecule involved in biotic and abiotic stress perception and signal transduction. In the model plant Arabidopsis thaliana, salt and osmotic stress rapidly induce increase in cGMP which plays role by modulating the activity of monovalent cation transporters, possibly by direct binding to these proteins and by altering the expression of many abiotic stress responsive genes. In a recent study, a membrane permeable analogue of cGMP (8-bromo-cGMP) was found to have a promotive effect on soluble sugar, flavonoids and lignin content, and membrane integrity in Solanum lycopersicum seedlings under salt stress. However, it remains to be elucidated how salt stress affects the endogenous cGMP level in S. lycopersicum and if Br-cGMP-induced improvement in salt tolerance in S. lycopersicum involves altered cation fluxes. The current study was conducted to answer these questions. A rapid increase (within 30 s) in endogenous cGMP level was determined in S. lycopersicum roots after treatment with 100 mM NaCl. Addition of membrane permeable Br-cGMP in growth medium remarkably ameliorated the inhibitory effects of NaCl on seedlings' growth parameters, chlorophyll content and net photosynthesis rate. In salt stressed plants, Br-cGMP significantly decreased Na+ content by reducing its influx and increasing efflux while it improved plants K+ content by reducing its efflux and enhancing influx. Furthermore, supplementation with Br-cGMP improved plant's proline content and total antioxidant capacity, resulting in markedly decreased electrolyte leakage under salt stress. Br-cGMP increased the expression of Na+/H+ antiporter genes in roots and shoots of S. lycopersicum growing under salt stress, potentially enhancing plant's ability to sequester Na+ into the vacuole. The findings of this study provide insights into the mechanism of cGMP-induced salt stress tolerance in S. lycopersicum.

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.

Decolorization of azo dyes by a novel aerobic bacterial strain Bacillus cereus strain ROC.

Synthetic dyes are widely used as colorant compounds in various industries for different purposes. Among all the dyestuffs, azo dyes constitute the largest and the most used class of dyes. These dyes and their intermediate products are common contaminants of ground water and soil in developing countries. Biological methods have been found to be promising for the treatment and degradation of these compounds. In the present study, we focused on the biological removal of azo dyes (Reactive orange 16 and Reactive black 5) under aerobic conditions using an indigenous bacterial strain isolated from contaminated industrial areas. The bacterial isolate was identified as Bacillus cereus strain ROC. Degradation experiments under agitation with both free and immobilized cells indicates that this strain degrades both azo- dyes in 5 days. The immobilized cells were more proficient than their free cell counterparts. The toxicity of the biotransformation products formed after decolorization were assessed by conducting bacteriotoxic and phytotoxic assays. All the toxicity assays indicate that the dyes' degraded products were non-toxic in nature, as compared to the dyes themselves. The kinetics of the azo dyes' degradation was also studied at various initial concentration ranges from 50 mg/L to 250 mg/L by growth independent kinetic models. Zero-order kinetics were fit to the experimental data, producing values of least squares regression (R2) greater than 0.98, which indicates that the bacterial strain degrades both dyes by co-metabolism rather than utilizing them as sole energy source. These results indicate that the Bacillus cereus ROC strain has great potential to degrade dye-contaminated water and soil.

Graphene quantum dot and iron co-doped TiO2 photocatalysts: Synthesis, performance evaluation and phytotoxicity studies.

The present study reports the synthesis, photocatalytic decolorization of reactive black 5 dye and phytotoxicity of graphene quantum dots (GQDs) and iron co-doped TiO2 photocatalysts via modified sol gel method. GQDs were synthesized by direct pyrolysis of citric acid (CA). Scanning electron microscopy (SEM) and energy dispersion spectroscopy (EDS), Raman spectroscopy, atomic force microscopy (AFM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), diffuse reflectance spectroscopy (DRS), Brunauer-Emmett-Teller (BET) and photoluminescence spectroscopy (PL) were used to determine the physicochemical properties of the best performing photocatalysts. The results indicated improved physicochemical properties of GQD-0.1Fe-TiO2-300 with root mean square roughness (Rz) (33.82 nm), higher surface area (170.79 m2 g-1), pore volume (0.08 cm3 g-1), and bandgap (2.94 eV). Moreover, GQD-0.1Fe co-doping of TiO2 greatly improved the photocatalytic decolorization efficiency for RB5 dye. The photocatalytic reaction followed the pseudo first order reaction with gradual decrease in Kapp values for increment in RB5 concentration. The KC value was obtained as 2.45 mg L-1 min-1 while the KLH value was 0.45 L mg-1 indicating the heterogeneous reaction system followed the Langmuir-Hinshelwood isotherm and simultaneously occurring adsorption and photocatalytic processes. Photocatalytic reaction mechanism studies exhibited the holes and OH radicals as the main active species in the GQD-0.1Fe-TiO2-300 responsible for the decolorization of RB5. The proposed reaction pathway showed that both Fe-TiO2 and GQDs play important role in generation of electrons and holes. Additionally, GQD-0.1Fe-TiO2-300 were durable up to four cycles. Phytotoxicity assay displayed that treated water and best performing photocatalysts had no effect on Lycopersicon esculentum seed germination. Therefore, the proposed system can pave a viable solution for safe usage of dye loaded wastewater and effluent for irrigation after treatment.

The expression of constitutively active CPK3 impairs potassium uptake and transport in Arabidopsis under low K+ stress.

Potassium (K+) is a vital cation and is involved in multiple physiological functions in plants. K+ uptake from outer medium by roots is a tightly regulated process and is mainly carried out by two high affinity K+ transport proteins AKT1 and HAK5. It has been shown that calcium (Ca2+) signaling plays important roles in the regulation of K+ transport in plants. Ca2+-dependent protein kinases (CPKs) are involved in regulation of multiple K+ channels in different tissues. However, it remains to be studied whether CPKs are involved in the regulation of AKT1 and, thereby, K+ transport. Here, we have shown that constitutively active version of CPK3 (CPK3CA) is involved in K+ transport in Arabidopsis via regulating AKT1 under low K+ conditions. The constitutively active version of CPK3 (CPK3CA), as well as CPK21 (CPK21CA), inhibited K+ currents of AKT1 in Xenopus oocytes. CPK3CA inhibited only channel conductance but had no effect on channel open probability. Further, CPK3 in vivo interacted with AKT1. Under low K+ conditions, cpk3 knock-out mutants had no distinct phenotype, while the seedlings of 35S-CPK3CA overexpressing lines died even at normal K+ concentration. Further, the transgenic lines expressing CPK3CA under AKT1 promoter (ProAKT1-CPK3CA) exhibited the same phenotype as akt1 mutant with a defective root growth and leaf chlorosis. Moreover, ProAKT1-CPK3CA transgenic lines had lower root and shoot K+ contents than Col. Overall, the data reported here demonstrate that the expression of constitutively active of CPK3 impairs potassium uptake and transports in Arabidopsis under low K+ stress by inhibiting the activity of AKT1.

Expression of cyanobacterial genes enhanced CO2 assimilation and biomass production in transgenic Arabidopsis thaliana.

BACKGROUND: Photosynthesis is a key process in plants that is compromised by the oxygenase activity of Rubisco, which leads to the production of toxic compound phosphoglycolate that is catabolized by photorespiratory pathway. Transformation of plants with photorespiratory bypasses have been shown to reduce photorespiration and enhance plant biomass. Interestingly, engineering of a single gene from such photorespiratory bypasses has also improved photosynthesis and plant productivity. Although single gene transformations may not completely reduce photorespiration, increases in plant biomass accumulation have still been observed indicating an alternative role in regulating different metabolic processes. Therefore, the current study was aimed at evaluating the underlying mechanism (s) associated with the effects of introducing a single cyanobacterial glycolate decarboxylation pathway gene on photosynthesis and plant performance. METHODS: Transgenic Arabidopsis thaliana plants (GD, HD, OX) expressing independently cyanobacterial decarboxylation pathway genes i.e., glycolate dehydrogenase, hydroxyacid dehydrogenase, and oxalate decarboxylase, respectively, were utilized. Photosynthetic, fluorescence related, and growth parameters were analyzed. Additionally, transcriptomic analysis of GD transgenic plants was also performed. RESULTS: The GD plants exhibited a significant increase (16%) in net photosynthesis rate while both HD and OX plants showed a non-significant (11%) increase as compared to wild type plants (WT). The stomatal conductance was significantly higher (24%) in GD and HD plants than the WT plants. The quantum efficiencies of photosystem II, carbon dioxide assimilation and the chlorophyll fluorescence-based photosynthetic electron transport rate were also higher than WT plants. The OX plants displayed significant reductions in the rate of photorespiration relative to gross photosynthesis and increase in the ratio of the photosynthetic electron flow attributable to carboxylation reactions over that attributable to oxygenation reactions. GD, HD and OX plants accumulated significantly higher biomass and seed weight. Soluble sugars were significantly increased in GD and HD plants, while the starch levels were higher in all transgenic plants. The transcriptomic analysis of GD plants revealed 650 up-regulated genes mainly related to photosynthesis, photorespiratory pathway, sucrose metabolism, chlorophyll biosynthesis and glutathione metabolism. CONCLUSION: This study revealed the potential of introduced cyanobacterial pathway genes to enhance photosynthetic and growth-related parameters. The upregulation of genes related to different pathways provided evidence of the underlying mechanisms involved particularly in GD plants. However, transcriptomic profiling of HD and OX plants can further help to identify other potential mechanisms involved in improved plant productivity.

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.

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.

Yellow nutsedge WRI4-like gene improves drought tolerance in Arabidopsis thaliana by promoting cuticular wax biosynthesis.

BACKGROUND: Cuticular wax plays important role in protecting plants from drought stress. In Arabidopsis WRI4 improves drought tolerance by regulating the biosynthesis of fatty acids and cuticular wax. Cyperus esculentus (yellow nutsedge) is a tough weed found in tropical and temperate zones as well as in cooler regions. In the current study, we report the molecular cloning of a WRI4-like gene from Cyperus esculentus and its functional characterization in Arabidopsis. RESULTS: Using RACE PCR, full-length WRI-like gene was amplified from yellow nutsedge. Phylogenetic analyses and amino acid comparison suggested it to be a WRI4-like gene. According to the tissue-specific expression data, the highest expression of WRI4-like gene was found in leaves, followed by roots and tuber. Transgenic Arabidopsis plants expressing nutsedge WRI4-like gene manifested improved drought stress tolerance. Transgenic lines showed significantly reduced stomatal conductance, transpiration rate, chlorophyll leaching, water loss and improved water use efficiency (WUE). In the absence of drought stress, expression of key genes for fatty acid biosynthesis was not significantly different between transgenic lines and WT while that of cuticular wax biosynthesis genes was significantly higher in transgenic lines than WT. The PEG-simulated drought stress significantly increased expression of key genes for fatty acid as well as wax biosynthesis in transgenic Arabidopsis lines but not in WT plants. Consistent with the gene expression data, cuticular wax load and deposition was significantly higher in stem and leaves of transgenic lines compared with WT under control as well as drought stress conditions. CONCLUSIONS: WRI4-like gene from Cyperus esculentus improves drought tolerance in Arabidopsis probably by promoting cuticular wax biosynthesis and deposition. This in turn lowers chlorophyll leaching, stomatal conductance, transpiration rate, water loss and improves water use efficiency under drought stress conditions. Therefore, CeWRI4-like gene could be a good candidate for improving drought tolerance in crops.

Immobilized cells of a novel bacterium increased the degradation of N-methylated carbamates under low temperature conditions.

Carbamates are synthetic pesticides, extensively used throughout the world due to their broad specificity against various insect pests. However, their enormous and inadequate use have made them a potential threat to the environment. At low temperature, degradation of carbamates becomes difficult mainly because of low biological activity. In the present study, we isolated a bacterial strain from a low temperature climate where the N-methylated carbamates are used for crop protection. The bacterium, was identified as Pseudomonas plecoglossicida strain (TA3) by 16S rRNA analysis. Degradation experiments with both free and immobilized cells in minimal salt medium indicated that the strain TA3 utilized carbaryl, carbofuran and aldicarb as both carbon and nitrogen source. TA3 can grow well at 4 °C and demonstrated the ability to degrade three carbamates (50 µgml-1) at low temperature. The immobilized cells were found more efficient than their free cells counter parts. Immobilized cells has ability to degrade 100% of carbamates at 30 °C while 80% at 4 °C but incase of their free cells counter parts the efficiency to degrade carbamates was less which was 60% at 4 °C and 80% at 30 °C. TA3 free cellsextract also depicted high activity against all the three carbamates even at 4 °C indicating a possible enzymatic mechanism of degradation.

Constitutive cyclic GMP accumulation in Arabidopsis thaliana compromises systemic acquired resistance induced by an avirulent pathogen by modulating local signals.

The infection of Arabidopsis thaliana plants with avirulent pathogens causes the accumulation of cGMP with a biphasic profile downstream of nitric oxide signalling. However, plant enzymes that modulate cGMP levels have yet to be identified, so we generated transgenic A. thaliana plants expressing the rat soluble guanylate cyclase (GC) to increase genetically the level of cGMP and to study the function of cGMP in plant defence responses. Once confirmed that cGMP levels were higher in the GC transgenic lines than in wild-type controls, the GC transgenic plants were then challenged with bacterial pathogens and their defence responses were characterized. Although local resistance was similar in the GC transgenic and wild-type lines, differences in the redox state suggested potential cross-talk between cGMP and the glutathione redox system. Furthermore, large-scale transcriptomic and proteomic analysis highlighted the significant modulation of both gene expression and protein abundance at the infection site, inhibiting the establishment of systemic acquired resistance. Our data indicate that cGMP plays a key role in local responses controlling the induction of systemic acquired resistance in plants challenged with avirulent pathogens.

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.

Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis.

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca(2+) gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca(2+) channels in pollen tube tips are core components that regulate Ca(2+) gradients by mediating and regulating external Ca(2+) influx. Therefore, Ca(2+) channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca(2+) channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca(2+) gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/-) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18's transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca(2+) channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca(2+) channel for pollen tube guidance 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.