Calcineurin B-like Research Articles

A novel role of the cotton calcium sensor CBL3 was involved in Verticillium wilt resistance in cotton.

Verticillium wilt, causes mainly by the soilborne pathogen Verticillium dahliae, is a devastated vascular disease resulting in huge financial losses in cotton, so research on improving V. dahliae stress tolerance in cotton is the utmost importance. Calcium as the second messenger acts as a crucial role in plant innate immunity. Cytosolic Ca2+during the pathogen infection is a significant increase in plant immune responses. Calcineurin B-like (CBL) proteins are widely known calcium sensors that regulate abiotic stress responses. However, the role of cotton CBLs in response to V. dahliae stress remains unclear. To discover and utilize the gene to Verticillium wilt resistance and defense response mechanism of cotton. Through screening the gene to Verticillium wilt resistance in cotton, four GhCBL3 copies were obtained from the current common cotton genome sequences. The protein domain and phylogenetic analyses of GhCBL3 were performed using NCBI Blast, DNAMAN, and MotifScan programs. Real-time RT-PCR was used to detect the expression of GhCBL3 gene in cotton seedlings under various stress treatments. The expression construct including GhCBL3 cDNA was transduced into Agrobacterium tumefaciens (GV3101) by heat shock method and transformed into cotton plants by Virus-Induced Gene Silencing (VIGS) method. The results of silencing of GhCBl3 on ROS accumulation and plant disease resistance in cotton plants were assessed. A member of calcineurin B-like proteins (defined as GhCBL3) in cotton was obtained. The expression of GhCBL3 was significantly induced and raised by various stressors, including dahliae, jasmonic acid (JA) and H2O2 stresses. Knockdown GhCBL3 in cotton by Virus-Induced Gene Silencing analysis enhanced Verticillium wilt tolerance and changed the occurrence of reactive oxygen species. Some disease-resistant genes were increased in GhCBL3-silencing cotton lines. GhCBL3 may function on regulating the Verticillium dahliae stress response of plants.

Genome-wide profiling of CBL interacting protein kinases (CIPKs) in banana unveils their role in abiotic stress signaling and stress tolerance enhancement

Calcineurin B-like (CBL)-interacting protein kinases (CIPKs) play a crucial role in the complex molecular systems of plants, acting as coordinators in different plant development processes, manage hormone signaling, and respond to environmental stress. Despite their well-established importance in various physiological process, the CIPK gene family in banana has remained an unexplored, creating a knowledge gap that this research aims to address. Through comprehensive analysis, we identified and characterized 34 CIPK genes in the banana genome. Structural diversity analysis revealed distinct clades characterized by homologs with varying intron-rich and intron-poor members. Particularly, evolutionary conservation was observed in intron-less members. Exploring conserved motifs and physicochemical properties provided insights into the detailed structure of specific homologs. Subcellular localization predictions indicated that most CIPK proteins primarily reside in the cytoplasm and nucleus, indicating their involvement in essential cellular activities. The predicted chromosomal distribution pattern suggests that the CIPK gene family largely expanded through segmental duplications in the banana genome. The phylogenetic analysis has uncovered two monophyletic clades for intron-less and intron-containing homologs, providing insights into two separate evolutionary history and expansion of the CIPK gene family. The clustering pattern in the phylogeny suggest that sequence of CIPKs members in the different genomes are largely conserved during course of evolution after evolution of higher plants. However, a few members having diverse sequences that these homologs are evolving with higher rate of evolution. Diverse physiological roles of banana CIPKs members are further supported by the presence of numerous cis-acting elements associated with growth, development, hormone responsiveness, and stress, with certain MaCIPK promoters exhibiting multiple hormone-sensitive elements. The protein-protein interaction network sheds light on the functional associations between MaCIPKs and MaCBLs, highlighting specific clusters of interactions. 3D protein structure predictions provide additional insights, revealing variations among the MaCIPK proteins. Real-time expression analysis across various tissues and developmental stages emphasizes the significant roles played by CIPKs in plant development. Under stress conditions, 16 in leaves and 22 in root MaCIPK gene transcript levels were up –regulated in response to drought and cold stress. These insights into the dynamic expression and functional roles of CIPK genes provide valuable information for understanding the mechanisms underlying banana's resilience to stress, with direct implications for the development of stress-resistant banana varieties. Overall this research offers promising prospects for enhancing global food security by ensuring the sustainability of banana production in the face of changing environmental conditions.

Mechanisms of calcium homeostasis orchestrate plant growth and immunity.

Calcium (Ca2+) is an essential nutrient for plants and a cellular signal, but excessive levels can be toxic and inhibit growth1,2. To thrive in dynamic environments, plants must monitor and maintain cytosolic Ca2+ homeostasis by regulating numerous Ca2+ transporters3. Here we report two signalling pathways in Arabidopsis thaliana that converge on the activation of vacuolar Ca2+/H+ exchangers (CAXs) to scavenge excess cytosolic Ca2+ in plants. One mechanism, activated in response to an elevated external Ca2+ level, entails calcineurin B-like (CBL) Ca2+ sensors and CBL-interacting protein kinases (CIPKs), which activate CAXs by phosphorylating a serine (S) cluster in the auto-inhibitory domain. The second pathway, triggered by molecular patterns associated with microorganisms, engages the immune receptor complex FLS2-BAK1 and the associated cytoplasmic kinases BIK1 and PBL1, which phosphorylate the same S-cluster in CAXs to modulate Ca2+ signals in immunity. These Ca2+-dependent (CBL-CIPK) and Ca2+-independent (FLS2-BAK1-BIK1/PBL1) mechanisms combine to balance plant growth and immunity by regulating cytosolic Ca2+ homeostasis.

Genome‐wide characterization of trihelix genes reveals Cqtrihelix23 enhances the salt tolerance in quinoa (Chenopodium quinoa)

AbstractSoil salinity poses an escalating threat to global environmental sustainability, agricultural productivity, and food safety. Quinoa (Chenopodium quinoa), recognized as a halophyte, has emerged as a promising crop due to its high nutritional value and stress resistance. Nevertheless, the current understanding of salt tolerance genes in quinoa remains incomplete. This comprehensive study aimed to identify the quinoa trihelix family and families associated with salt stress, including Na+/H+ antiporter (NHX) and calcineurin B‐like (CBL). Through expression analysis, Cqtrihelix23 was identified as responsive to salt stress. Subsequent transient transformation experiments revealed that Cqtrihelix23 enhanced quinoa's tolerance to salt stress by promoting root development, maintaining the antioxidant system, and reducing the Na+/K+ ratio. Additionally, it was discovered that Cqtrihelix23 upregulated the expression of CqCBL10 and CqNHX4. Further protein interaction experiments confirmed the interaction between Cqtrihelix23, CqCBL10, and CqNHX4. Notably, CqCBL10 and CqNHX4 also contributed to salt stress resistance, and in combination with Cqtrihelix23, they synergistically enhanced salt stress tolerance. In conclusion, this study highlights the significance of Cqtrihelix23, CqCBL10, and CqNHX4 as key contributors within the regulatory network associated with quinoa's salt tolerance. These findings lay a solid groundwork for the development of salt‐tolerant quinoa varieties.

The Roles of Calcineurin B-like Proteins in Plants under Salt Stress

Salinity stands as a significant environmental stressor, severely impacting crop productivity. Plants exposed to salt stress undergo physiological alterations that influence their growth and development. Meanwhile, plants have also evolved mechanisms to endure the detrimental effects of salinity-induced salt stress. Within plants, Calcineurin B-like (CBL) proteins act as vital Ca2+ sensors, binding to Ca2+ and subsequently transmitting signals to downstream response pathways. CBLs engage with CBL-interacting protein kinases (CIPKs), forming complexes that regulate a multitude of plant growth and developmental processes, notably ion homeostasis in response to salinity conditions. This review introduces the repercussions of salt stress, including osmotic stress, diminished photosynthesis, and oxidative damage. It also explores how CBLs modulate the response to salt stress in plants, outlining the functions of the CBL-CIPK modules involved. Comprehending the mechanisms through which CBL proteins mediate salt tolerance can accelerate the development of cultivars resistant to salinity.

A novel function of the tomato CALCINEURIN-B LIKE 10 gene as a root-located negative regulator of salt stress.

Climate change exacerbates abiotic stresses like salinization, negatively impacting crop yield, so development of strategies, like using salt-tolerant rootstocks, is crucial. The CALCINEURIN B-LIKE 10 (SlCBL10) gene has been previously identified as a positive regulator of salt tolerance in the tomato shoot. Here, we report a different function of SlCBL10 in tomato shoot and root, as disruption of SlCBL10 only induced salt sensitivity when it was used in the scion but not in the rootstock. The use of SlCBL10 silencing rootstocks (Slcbl10 mutant and RNAi line) improved salt tolerance on the basis of fruit yield. These changes were associated with improved Na+ and K+ homoeostasis, as SlCBL10 silencing reduced the Na+ content and increased the K+ content under salinity, not only in the rootstock but also in the shoot. Improvement of Na+ homoeostasis in Slcbl10 rootstock seems to be mainly due to induction of SlSOS1 expression, while the higher K+ accumulation in roots seems to be mainly determined by expression of LKT1 transporter and SlSKOR channel. These findings demonstrate that SlCBL10 is a negative regulator of salt tolerance in the root, so the use of downregulated SlCBL10 rootstocks may provide a suitable strategy to increase tomato fruit production under salinity.

Understanding the salt overly sensitive pathway in Prunus: Identification and characterization of NHX, CIPK, and CBL genes.

Salinity is a major abiotic stress factor that can significantly impact crop growth, and productivity. In response to salt stress, the plant Salt Overly Sensitive (SOS) signaling pathway regulates the homeostasis of intracellular sodium ion concentration. The SOS1, SOS2, and SOS3 genes play critical roles in the SOS pathway, which belongs to the members of Na+/H+ exchanger (NHX), CBL-interacting protein kinase (CIPK), and calcineurin B-like (CBL) gene families, respectively. In this study, we performed genome-wide identifications and phylogenetic analyses of NHX, CIPK, and CBL genes in six Rosaceae species: Prunus persica, Prunus dulcis, Prunus mume, Prunus armeniaca, Pyrus ussuriensis × Pyrus communis, and Rosa chinensis. NHX, CIPK, and CBL genes of Arabidopsis thaliana were used as controls for phylogenetic analyses. Our analysis revealed the lineage-specific and adaptive evolutions of Rosaceae genes. Our observations indicated the existence of two primary classes of CIPK genes: those that are intron-rich and those that are intron-less. Intron-rich CIPKs in Rosaceae and Arabidopsis can be traced back to algae CIPKs and CIPKs found in early plants, suggesting that intron-less CIPKs evolved from their intron-rich counterparts. This study identified one gene for each member of the SOS signaling pathway in P. persica: PpSOS1, PpSOS2, and PpSOS3. Gene expression analyses indicated that all three genes of P. persica were expressed in roots and leaves. Yeast two-hybrid-based protein-protein interaction analyses revealed a direct interaction between PpSOS3 and PpSOS2; and between PpSOS2 and PpSOS1C-terminus region. Our findings indicate that the SOS signaling pathway is highly conserved in P. persica.

Functional Characterization of a New Salt Stress Response Gene, PeCBL4, in Populus euphratica Oliv

Populus euphratica is a typical stress-resistant tree species that provides valuable natural genetic resources for breeding salt-tolerant plants. The calcineurin B-like (CBL)-interacting protein kinase (CIPK) network plays an important role in regulating plant responses to abiotic stresses. The aim of this study was to characterize the function of a new CBL member, PeCBL4, in response to abiotic stresses. PeCBL4 was cloned, and sequence analysis was performed. The subcellular localization of PeCBL4 was determined using the fusion expression vector of GFP. Yeast two-hybrid assays and bimolecular fluorescence complementation were performed to identify PeCIPK members that interacted with PeCBL4. PeCBL4 was then transformed into the corresponding Arabidopsis thaliana mutants. Na+ and K+ content as well as their net fluxes were determined under high salt stress and low K+ stress. Phylogenetic tree analysis showed that PeCBL4 was clustered together with PtCBL4 and belonged to the same subgroup as AtCBL4. Subcellular localization indicated that PeCBL4 was expressed on the plasma membrane. Yeast two-hybrid assays and bimolecular fluorescence complementation showed that PeCBL4 interacted with PeCIPK24 and PeCIPK26. In addition, under high salt stress, the Na+ efflux capacities of seedlings decreased in sos3 mutants, and transgenic plants of PeCBL4 enhanced efflux capacities. In addition, the overexpression of PeCBL4 negatively influenced the influx capacity of K+. PeCBL4 interacts with PeCIPK24 and PeCIPK26 and regulates Na+/K+ balance under low K+ and high salt stress.

A salt stress-activated GSO1-SOS2-SOS1 module protects the Arabidopsis root stem cell niche by enhancing sodium ion extrusion.

Soil salinity impairs plant growth reducing crop productivity. Toxic accumulation of sodium ions is counteracted by the Salt Overly Sensitive (SOS) pathway for Na+ extrusion, comprising the Na+ transporter SOS1, the kinase SOS2, and SOS3 as one of several Calcineurin-B-like (CBL) Ca2 + sensors. Here, we report that the receptor-like kinase GSO1/SGN3 activates SOS2, independently of SOS3 binding, by physical interaction and phosphorylation at Thr16. Loss of GSO1 function renders plants salt sensitive and GSO1 is both sufficient and required for activating the SOS2-SOS1 module in yeast and in planta. Salt stress causes the accumulation of GSO1 in two specific and spatially defined areas of the root tip: in the endodermis section undergoing Casparian strip (CS) formation, where it reinforces the CIF-GSO1-SGN1 axis for CS barrier formation; and in the meristem, where it creates the GSO1-SOS2-SOS1 axis for Na+ detoxification. Thus, GSO1 simultaneously prevents Na+ both from diffusing into the vasculature, and from poisoning unprotected stem cells in the meristem. By protecting the meristem, receptor-like kinase-conferred activation of the SOS2-SOS1 module allows root growth to be maintained in adverse environments.

Nitrate supply regulates tissue calcium abundance and transcript level of Calcineurin B-like (CBL) gene family in wheat

Calcium ion (Ca2+) is the most ubiquitous signalling molecule and is sensed by different classes of Ca2+ sensor proteins. Recent evidences underscore the role of calcium signalling in plant response to nitrogen/nitrate supply. Recently we found that under nitrate deficiency, a short-term supply of calcium could improve the plant biomass, nitrate assimilation, anthocyanin accumulation and expression of nitrate uptake and signalling genes. Long-term calcium supply, on the other hand, was not beneficial. Calcineurin B-like (CBL) proteins are one of the vital plant Ca2+ sensory protein family which is essential for stress perception and signaling. To understand the dynamics of CBL-mediated stress signalling in bread wheat, we identified CBL genes in bread wheat (Triticum aestivum) and its progenitors, namely Triticum dicoccoides, Triticum urartu and Aegilops tauschii with the aid of newly available whole-genome sequence. The expression of different CBLs and the changes in root Ca2+ localization in response to nitrate provision or deficiency were analysed. Expression of the CBLs were studied in two bread wheat genotypes with comparatively higher (B.T. Schomburgk, BTS) and lower (Gluyas early, GE) nitrate responsiveness and nitrogen use efficiency. High N promoted the expression of CBLs in seedling leaves while in roots the expression was promoted by N deficiency. At the 5 days after anthesis stage, nitrate starvation downregulated the expression of CBLs while nitrate supply enhanced the expression. At anthesis stage, expression of CBL6 was significantly promoted by HN in panicles of both the genotypes, the highest expression was recorded in BTS. Expression of CBL6 was significantly upregulated by short term nitrate treatment also suggesting its role in Primary nitrate response (PNR) in wheat. There was a significant down regulation of CBL6 expression post nitrate starvation, making it a probable regulator of nitrogen starvation response (NSR) as well. In seedling roots, the tissue localization of Ca2+ was increased both by high and low nitrate treatments, albeit at different magnitudes. Our results suggest that calcium signalling might be a major signalling pathway governing nitrogen responsiveness and CBL6 might be playing pivotal role in NSR and PNR in wheat.

Genome-wide identification of CBL family genes in Nicotiana tabacum and the functional analysis of NtCBL4A-1 under salt stress

The CALCINEURIN B-LIKE PROTEIN family genes (CBLs) encode a group of plant-specific calcium sensor proteins that play critical roles in plant development and stress response. Although genome-wide analysis of CBL family genes has been conducted in several diploid plant species, less is known about their functions in allopolyploid plant species. This study conducted a detailed analysis of tobacco CBL family genes. Of the 24 NtCBL genes identified, 14 were derived from the maternal genome donor N. sylvestris, and 10 were from the paternal genome donor N. tomentosiformis. Phylogenetic analysis revealed that NtCBLs could be clustered into three different groups with distinct N-terminal characteristics. All NtCBLs contain EF-hand motifs, but amino acid substitutions and configuration changes may be responsible for variations in their Ca2+-binding abilities. Alternative splicing is common for NtCBL gene transcripts, which may contribute to variations in translation efficiency or protein interaction preferences. Several NtCBL genes exhibited shoot- or root-predominant expression patterns, and some were responsive to salt and/or drought stress. NtCBL4A-1, an ortholog of Arabidopsis thaliana AtCBL4 (SOS3), was predominantly expressed in roots and exhibited a lower gene expression level under salt stress. Further functional analysis showed that overexpression of NtCBL4A-1 increased the sensitivity of transgenic tobacco to Na+-induced salt stress, which is inconsistent with the role of AtCBL4 in contributing to salt tolerance. This study provides data for identifying CBL gene functions and selecting candidate stress-tolerant genes in N. tabacum. Additionally, the results may aid gene function studies in other allopolyploid plant species.

Characterization of Dendrobium catenatum CBL-CIPK signaling networks and their response to abiotic stress

Dendrobium catenatum is a traditional Chinese medicine listing as rare and endangered due to environmental impacts. But little is known about its stress resistance mechanism. The CBL-CIPK signaling pathway played vital roles in various stress responses. In this study, we identified 9 calcineurin B-like (CBL) genes and 28 CBL-interacting protein kinase (CIPK) genes from D. catenatum. Phylogenetic analysis showed that DcCBL and DcCIPK families could be divided into four and six subgroups, respectively. Members in each subgroup had similar gene structures. Cis-acting element analyses showed that these genes were involved in stress responses and hormone signaling. Spatial expression profiles showed that they were tissue-specific, and expressed lower in vegetative organs than reproductive organs. Gene expression analyses revealed that these genes were involved in drought, heat, cold, and salt responses and depended on abscisic acid (ABA) and salicylic acid (SA) signaling pathways. Furthermore, we cloned 19 DcCIPK genes and 9 DcCBL genes and detected ten interacting CBL-CIPK combinations using yeast two-hybrid system. Finally, we constructed 20 CBL-CIPK signaling pathways based on their expression patterns and interaction relationships. These results established CBL-CIPK signaling pathway responding to abiotic stress and provided a molecular basis for improving D. catenatum stress resistance in the future.

CIPK-B is essential for salt stress signalling in Marchantia polymorpha.

Calcium signalling is central to many plant processes, with families of calcium decoder proteins having expanded across the green lineage and redundancy existing between decoders. The liverwort Marchantia polymorpha has fast become a new model plant, but the calcium decoders that exist in this species remain unclear. We performed phylogenetic analyses to identify the calcineurin B-like (CBL) and CBL-interacting protein kinase (CIPK) network of M.polymorpha. We analysed CBL-CIPK expression during salt stress, and determined protein-protein interactions using yeast two-hybrid and bimolecular fluorescence complementation. We also created genetic knockouts using CRISPR/Cas9. We confirm that M.polymorpha has two CIPKs and three CBLs. Both CIPKs and one CBL show pronounced salt-responsive transcriptional changes. All M.polymorpha CBL-CIPKs interact with each other in planta. Knocking out CIPK-B causes increased sensitivity to salt, suggesting that this CIPK is involved in salt signalling. We have identified CBL-CIPKs that form part of a salt tolerance pathway in M.polymorpha. Phylogeny and interaction studies imply that these CBL-CIPKs form an evolutionarily conserved salt overly sensitive pathway. Hence, salt responses may be some of the early functions of CBL-CIPK networks and increased abiotic stress tolerance required for land plant emergence.

Genome-wide discovery of CBL genes in Nitraria tangutorum Bobr. and functional analysis of NtCBL1-1 under drought and salt stress.

Calcineurin B-like (CBL) proteins are a class of important Ca2+ receptors that play key roles in plant stress response. CBLs have been shown to participate in responses to abiotic stresses such as drought, salt, and cold in many plant species, including Arabidopsis and rice. However, little is known about their potential functions in the desert halophyte Nitraria tangutorum. Here, we have identified 11 CBL genes distributed across six chromosomes of N. tangutorum and categorized them into four groups through phylogenetic analysis. Synteny analysis showed that they have strong collinear relationships and have undergone purifying selection during their evolution. NtCBL promoter regions contain a variety of cis-acting elements related to hormone and environmental stress responses. Real-time quantitative PCR showed that the expression of NtCBLs differed significantly among various tissues and was upregulated by salt and drought stress. We chose NtCBL1-1 for an in-depth functional characterization and observed that transgenic Arabidopsis plants expressing NtCBL1-1 exhibited increased tolerance to both drought and salt stress. Compared to wild-type Arabidopsis, transgenic lines showed higher germination rates, slower chlorophyll degradation, more soluble proteins, and reduced accumulation of the oxidative stress marker malondialdehyde. These findings indicate that NtCBL1-1 plays a significant role in responding to drought and salt stress, laying the foundation for further investigations into the functional mechanisms of NtCBL genes in N. tangutorum.

Halophyte Nitraria billardieri CIPK25 promotes photosynthesis in Arabidopsis under salt stress.

The calcineurin B-like (CBL)-interacting protein kinases (CIPKs), a type of plant-specific genes in the calcium signaling pathway, function in response to adverse environments. However, few halophyte derived CIPKs have been studied for their role in plant physiological and developmental adaptation during abiotic stresses, which inhibits the potential application of these genes to improve environmental adaptability of glycophytes. In this study, we constructed Nitraria billardieri CIPK25 overexpressing Arabidopsis and analyzed the seedling development under salt treatment. Our results show that Arabidopsis with NbCIPK25 expression exhibits more vigorous growth than wild type plants under salt condition. To gain insight into the molecular mechanisms underlying salt tolerance, we profiled the transcriptome of WT and transgenic plants via RNA-seq. GO and KEGG analyses revealed that upregulated genes in NbCIPK25 overexpressing seedlings under salt stress are enriched in photosynthesis related terms; Calvin-cycle genes including glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) are significantly upregulated in transgenic plants, which is consistent with a decreased level of NADPH (GAPDH substrate) and increased level of NADP+. Accordingly, NbCIPK25 overexpressing plants exhibited more efficient photosynthesis; soluble sugar and proteins, as photosynthesis products, showed a higher accumulation in transgenic plants. These results provide molecular insight into how NbCIPK25 promotes the expression of genes involved in photosynthesis, thereby maintaining plant growth under salt stress. Our finding supports the potential application of halophyte-derived NbCIPK25 in genetic modification for better salt adaptation.

Kinase CIPK9 integrates glucose and abscisic acid signaling to regulate seed oil metabolism in rapeseed.

Rapeseed (Brassica napus), an important oil crop worldwide, provides large amounts of lipids for human requirements. Calcineurin B-like (CBL)-interacting protein kinase 9 (CIPK9) was reported to regulate seed oil content in the plant. Here, we generated gene-silenced lines through RNA interference biotechnology and loss-of-function mutant bnacipk9 using CRISPR/Cas9 to further study BnaCIPK9 functions in the seed oil metabolism of rapeseeds. We discovered that compared with wild-type (WT) lines, gene-silenced and bnacipk9 lines had substantially different oil contents and fatty acid compositions: seed oil content was improved by 3%-5% and 1%-6% in bnacipk9 lines and gene-silenced lines, respectively; both lines were with increased levels of monounsaturated fatty acids and decreased levels of polyunsaturated fatty acids. Additionally, hormone and glucose content analyses revealed that compared with WT lines the bnacipk9 lines showed significant differences: in bnacipk9 seeds, indoleacetic acid and abscisic acid (ABA) levels were higher; glucose and sucrose contents were higher with a higher hexose-to-sucrose ratio in bnacipk9 mid-to-late maturation development seeds. Furthermore, the bnacipk9 was less sensitive to glucose and ABA than the WT according to stomatal aperture regulation assays and the expression levels of genes involved in glucose and ABA regulating pathways in rapeseeds. Notably, in Arabidopsis (Arabidopsis thaliana), exogenous ABA and glucose imposed on developing seeds revealed the effects of ABA and glucose signaling on seed oil accumulation. Altogether, our results strongly suggest a role of CIPK9 in mediating the interaction between glucose flux and ABA hormone signaling to regulate seed oil metabolism in rapeseed.

HPCA1 is required for systemic reactive oxygen species and calcium cell-to-cell signaling and plant acclimation to stress.

Reactive oxygen species (ROS), produced by respiratory burst oxidase homologs (RBOHs) at the apoplast, play a key role in local and systemic cell-to-cell signaling, required for plant acclimation to stress. Here we reveal that the Arabidopsis thaliana leucine-rich-repeat receptor-like kinase H2O2-INDUCED CA2+ INCREASES 1 (HPCA1) acts as a central ROS receptor required for the propagation of cell-to-cell ROS signals, systemic signaling in response to different biotic and abiotic stresses, stress responses at the local and systemic tissues, and plant acclimation to stress, following a local treatment of high light (HL) stress. We further report that HPCA1 is required for systemic calcium signals, but not systemic membrane depolarization responses, and identify the calcium-permeable channel MECHANOSENSITIVE ION CHANNEL LIKE 3, CALCINEURIN B-LIKE CALCIUM SENSOR 4 (CBL4), CBL4-INTERACTING PROTEIN KINASE 26 and Sucrose-non-fermenting-1-related Protein Kinase 2.6/OPEN STOMATA 1 (OST1) as required for the propagation of cell-to-cell ROS signals. In addition, we identify serine residues S343 and S347 of RBOHD (the putative targets of OST1) as playing a key role in cell-to-cell ROS signaling in response to a local application of HL stress. Our findings reveal that HPCA1 plays a key role in mediating and coordinating systemic cell-to-cell ROS and calcium signals required for plant acclimation to stress.

EF-hand domain-containing proteins in Triticum aestivum: Insight into their roles in stress response and signalling

In plants, Ca2+ is an integral element in several stresses related signallings. During environmental stresses, the cytosolic Ca2+ concentration tends to rise rapidly, which is further co-ordinate with EF-hand domain-containing proteins. In our study, a total of 586 EF-hand domain-containing proteins were identified in Triticum aestivum as calmodulin (CaM)/Calmodulin-like (CML), calcineurin B-like (CBL), protein kinase (CDPK, CRK, CCaMK), respiratory burst oxidase homologs (RBOHs), miscellaneous proteins and un-categorized EF-hand proteins (UEPs). These proteins either carried the EF-hand domain singly or along with other domains. Phylogenetically, the proteins were clustered into eight groups, each with diverse types of proteins. The genes were found on all the chromosomes and sub-genomes of T. aestivum. The gene structure analysis revealed their conserved architecture. The diverse expression patterns of genes indicated their involvement in plant growth and development. The modulated expression of various genes to heat, drought, combined heat drought, salt and two fungal stresses unveiled their functioning in defence responses. Moreover, the altered expression under CaCl2 treatment suggested their role in stress signalling. Furthermore, the miRNA interaction analysis revealed their interaction with various plant development and stress resistance associated miRNAs, which also showed their participation in these phenomenon. Additionally, protein-protein and protein-chemical analyses revealed the interaction of EF-hand proteins with various Ca2+ binding/sensing proteins which regulate Ca2+- mediated signallings.This study will be useful in the functional characterization of potential candidate genes in future studies.

Differential Gene Expression Analysis of SoCBL Family Calcineurin B-like Proteins: Potential Involvement in Sugarcane Cold Stress.

Sugarcan e is a major crop for sugar and biofuel production and is cultivated in tropical and subtropical areas worldwide. Sugarcane growth is constrained because of winter’s low-temperature stress, and cold resistance is an important limitation in sugarcane growth enhancement. Therefore, in this study, we identified a gene involved in the low-temperature stress response of sugarcane. Calcineurin B-like (CBL) protein is a calcium signal receptor involved in the cold stress response. Five sugarcane CBL genes were cloned, sequenced, and named SoCBL1, SoCBL3, SoCBL5, SoCBL6, and SoCBL9. The protein sequences of these genes were analyzed. The calculated molecular weight of these proteins was 24.5, 25.9, 25.2, 25.6, and 26.3 kD, respectively. Subcellular localization analysis revealed that SoCBL1, SoCBL3, SoCBL6, and SoCBL9 were situated in the cytoplasm, while SoCBL5 was present in mitochondria. Secondary structure analysis showed that these five CBL proteins had similar secondary structures. Conserved domain analysis displayed that each sugarcane CBL protein contained three conserved EF domains. According to the self-expanding values of the phylogenetic tree, the CBL gene family was divided into four groups. The CBL1 and CBL9 genes were classified into one group, illustrating that these two genes might possess a similar function. The expression analysis of the SoCBL gene under low temperatures showed that SoCBL3 and SoCBL5 were affected significantly, while SoCBL1 and SoCBL9 were less affected. These results demonstrate that the CBL genes in sugarcane have similar characteristics and present differences in genetic diversity and gene expression response to low temperatures. Therefore, these genes might be novel candidates for fighting cold stress in sugarcane.

Comprehensive genomic insight deciphers significance of EF-hand gene family in foxtail millet [Setaria italica (L.) P. Beauv.]

As a signaling molecule, calcium plays a vital role in the intracellular signaling in plants in response to various physiological and environmental factors. Signaling processes start just after stimulus-receptor interaction, and these signals are relayed and decoded by a range of calcium-binding proteins. The majority of calcium-binding proteins comprise the EF-hand motif with a helix-loop-helix structure. This study reports genome-wide analysis of EF-hand (SiEF-hand) gene family in foxtail millet [Setaria italica (L.) P. Beauv.]. The analysis resulted in the identification of 180 SiEF-hand genes. Further, we carried out phylogenetic, gene structure and motif analysis that resulted in clustering of the SiEF-hand genes in 13 clades broadly as per different subfamilies - calmodulins (CaMs), calmodulins-like proteins (CMLs), calcium-dependent proteins (CDPKs), calcineurin B-like (CBLs), respiratory burst oxidase homologs (Rboh) and others. The domain architecture analysis highlighted 24 different domain compositions. Further, SiEF-hand genes are mapped to all nine chromosomes with uneven distribution. Cis-regulatory element analysis showed the presence of multiple stress and hormone-responsive elements in the promoter region of SiEF-hand genes. The most abundant cis-regulatory elements in SiEF-hand genes are ABRE, TGACG-motif, G-box, MYC, and MYB. Expression profiling of SiEF-hand genes in different tissues leaf, root, stem, and tassel inflorescence revealed differential expression patterns. Further, 8 SiEF-hand genes exhibited tissue-specific expression - four are expressed in roots and four in tassel inflorescences. In a nutshell, the present study provides a detailed insight into the SiEF-hand gene family and may serve as a base for further characterization of Ca2+ sensors.