Rice kinase OsMRLK63 contributes to drought tolerance by regulating reactive oxygen species production.

Drought is a major adverse environmental factor that plants face in nature but the molecular mechanism by which plants transduce stress signals and further endow themselves with tolerance remains unclear. Malectin/malectin-like domains containing receptor-like kinases (MRLKs) have been proposed to act as receptors in multiple biological signaling pathways, but limited studies show their roles in drought-stress signaling and tolerance. In this study, we demonstrate OsMRLK63 in rice (Oryza sativa L.) functions in drought tolerance by acting as the receptor of 2 rapid alkalization factors, OsRALF45 and OsRALF46. We show OsMRLK63 is a typical receptor-like kinase that positively regulates drought tolerance and reactive oxygen species (ROS) production. OsMRLK63 interacts with and phosphorylates several nicotinamide adenine dinucleotide phosphate (NADPH) oxidases with the primarily phosphorylated site at Ser26 in the N-terminal of RESPIRATORY BURST OXIDASE HOMOLOGUE A (OsRbohA). The application of the 2 small signal peptides (OsRALF45/46) on rice can greatly alleviate the dehydration of plants induced by mimic drought. This function depends on the existence of OsMRLK63 and the NADPH oxidase-dependent ROS production. The 2 RALFs interact with OsMRLK63 by binding to its extracellular domain, suggesting they may act as drought/dehydration signal sensors for the OsMRLK63-mediated process. Our study reveals a OsRALF45/46-OsMRLK63-OsRbohs module which contributes to drought-stress signaling and tolerance in rice.

OsBBX19-OsBTB97/OsBBX11 module regulates spikelet development and yield production in rice.

Spikelet and floral-related organs are important agronomic traits for rice grain yield. BTB (broad-complex, tram track, and bric-abrac) proteins control various developmental functions in plants; however, the molecular mechanism of BTB proteins underlying grain development and yield production is still unknown. Here, we evaluated the molecular mechanism of a previously unrecognized functional gene, namely OsBTB97 that regulates the floral and spikelet-related organs which greatly affect the final grain yield. We found that the knockdown of the OsBTB97 gene had significant impacts on the development of spikelet-related organs and grain size, resulting in a decrease in yield, by altering the transcript levels of various spikelet- and grain-related genes. Furthermore, we found that the knockout mutants of two BBX genes, OsBBX11 and OsBBX19, which interact with the OsBTB97 protein at translation and transcriptional level, respectively, displayed lower OsBTB97 expression, suggesting the genetic relationship between the BTB protein and the BBX transcription factors in rice. Taken together, our study dissects the function of the novel OsBTB97 by interacting with two BBX proteins and an OsBBX19-OsBTB97/OsBBX11 module might function in the spikelet development and seed production in rice. The outcome of the present study provides promising knowledge about BTB proteins in the improvement of crop production in plants.

Rice Carbohydrate-Binding Malectin-Like Protein, OsCBM1, Contributes to Drought-Stress Tolerance by Participating in NADPH Oxidase-Mediated ROS Production.

Carbohydrate-binding malectin/malectin-like domain-containing proteins (CBMs) are a recently identified protein subfamily of lectins that participates various functional bioprocesses in the animal, bacterial, and plant kingdoms. However, little is known the roles of CBMs in rice development and stress response. In this study, OsCBM1, which encodes a protein containing only one malectin-like domain, was cloned and characterized. OsCBM1 is localized in both the endoplasmic reticulum and plasma membrane. Its transcripts are dominantly expressed in leaves and could be significantly stimulated by a number of phytohormone applications and abiotic stress treatments. Overexpression of OsCBM1 increased drought tolerance and reactive oxygen species production in rice, whereas the knockdown of the gene decreased them. OsCBM1 physically interacts with OsRbohA, a NADPH oxidase, and the expression of OsCBM1 in osrbohA, an OsRbohA-knockout mutant, is significantly downregulated under both normal growth and drought stress conditions. Meanwhile, OsCBM1 can also physically interacts with OsRacGEF1, a specific guanine nucleotide exchange factor for the Rop/Rac GTPase OsRac1, and transient coexpression of OsCBM1 with OaRacGEF1 significantly enhanced ROS production. Further transcriptome analysis showed that multiple signaling regulatory mechanisms are involved in the OsCBM1-mediated processes. All these results suggest that OsCBM1 participates in NADPH oxidase-mediated ROS production by interacting with OsRbohA and OsRacGEF1, contributing to drought stress tolerance of rice. Multiple signaling pathways are likely involved in the OsCBM1-mediated stress tolerance in rice.

The highly interactive BTB domain targeting other functional domains to diversify the function of BTB proteins in rice growth and development.

The BTB (broad-complex, tram track, and bric-abrac) proteins are involved in developmental processes, biotic, and abiotic stress responses in various plants, but the molecular basis of protein interactions is yet to be investiagted in rice. In this study, the identified BTB proteins were divided into BTB-TAZ, MATH-BTB, BTB-NPH, BTB-ANK, BTB-Skp, BTB-DUF, and BTB-TPR subfamilies based on the additional functional domains found together with the BTB domain at N- and C-terminal as well. This suggesting that the extension region at both terminal sites could play a vital role in the BTB gene family expansion in plants. The yeast two-hybrid system, firefly luciferase complementation imaging (LCI) assay and bimolecular fluorescence complementation (BiFC) assay further confirmed that BTB proteins interact with several other proteins to perform a certain developmental process in plants. The overexpression of BTB genes of each subfamily in Arabidopsis revealed that BTB genes including OsBTB4, OsBTB8, OsBTB64, OsBTB62, OsBTB138, and OsBTB147, containing certain additional functional domains, could play a potential role in the early flowering, branching, leaf, and silique development. Thus we concluded that the presence of other functional domains such as TAZ, SKP, DUF, ANK, NPH, BACK, PQQ, and MATH could be the factor driving the diverse functions of BTB proteins in plant biology.

Zinc Finger-Homeodomain Genes: Evolution, Functional Differentiation, and Expression Profiling Under Flowering-Related Treatments and Abiotic Stresses in Plants.

Zinc finger-homeodomain (ZHD) proteins constitute a plant-specific transcription factor family that play important roles in plant growth, development, and stress responses. In this study, we investigated a total of 10, 17, and 31 ZHD gene members in the peach, Arabidopsis, and apple genome, respectively. The phylogenetic tree divided the identified ZHD genes into 4 subfamilies based on their domain organization, gene structure, and motif distribution with minor variations. The ZHD gene family members were unevenly distributed throughout in apple, peach, and Arabidopsis genomes. Segmental duplication was observed for 14 pairs of genes in apple. Transcript analysis found that ZHD genes mostly expressed in various tissues, particularly in leaves and flowers. Moreover, the transcript of most ZHD genes was significantly affected at different time points in response to various flowering-related exogenous hormones (sugar, gibberellin [GA], and 6-benzylaminopurine [6-BA]), signifying their possible role in the flowering induction in apple. Furthermore, the transcripts of CaZHD6, CaZHD7, CaZHD3, and CaZHD8 have induced in response to abiotic stresses including heat, drought, salt, and cold, indicating their possible involvement in response to abiotic stresses. Our research work systemically presents the different roles of ZHD genes. We believe that this study will provide a platform for future functional characterization of ZHD genes and to deeply unfold their roles in the regulation of flowering induction in plants.

A systematic in silico prediction of gibberellic acid stimulated GASA family members: A novel small peptide contributes to floral architecture and transcriptomic changes induced by external stimuli in rice.

The GASA (GA-stimulated Arabidopsis) gene family is highly specific to plants, signifying a crucial role in plant growth and development. Herein, we retrieved 119 GASA genes in 10 different plant species in two major lineages (monocots and eudicots). Further, in the phylogenetic tree we classified these genes into four well-conserved subgroups. All the proteins contain a conserved GASA domain with similar characteristics and a highly specific 12-cysteine residue of the C-terminus position. According to the global microarray data and qRT-PCR based analysis, the OsGASA gene family was dominantly expressed in the seedling and transition phase of floral stages. Despite this, OsGASA genes profoundly contribute to rice grain size and length, whereas the highest abundance of transcript level was noticed in stage-2 (Inf 6, 3.0-cm-long spikelet) and stage-3 (Inf 7, 5.0-cm-long spikelet) under GA treatment during panicle formation. Additionally, the maximum expression level of these genes was recorded in response to GA and ABA in young seedlings. Further, in response to abiotic stresses, OsGASA1/8/10 was up- regulated by salt, OsGASA2/5/7 by drought, OsGASA3/6 by cold, and OsGASA4/9 by heat stress. With the exception of OsGASA4, the higher transcription levels of all the other GASA genes were induced by Cd and Cr metal stresses (8-10 fold changes) at various time points. Finally, the GO ontology analysis of GASAs revealed the biological involvement in the GA-mediated signaling pathway and abiotic stresses. Prominently, most of these proteins are localized in cellular components such as the cell wall and extracellular region, where the molecular functions such as ATP binding and protein binding were observed. These results imply that GASAs are significantly involved in rice panicle developmental stages, responses to external stimuli, and hormones.

Characterization of B-BOX gene family and their expression profiles under hormonal, abiotic and metal stresses in Poaceae plants.

BACKGROUND: B-box (BBX) proteins play important roles in plant growth regulation and development including photomorphogenesis, photoperiodic regulation of flowering, and responses to biotic and abiotic stresses. RESULTS: In the present study we retrieved total 131 BBX members from five Poaceae species including 36 from maize, 30 from rice, 24 from sorghum, 22 from stiff brome, and 19 from Millet. All the BBX genes were grouped into five subfamilies on the basis of their phylogenetic relationships and structural features. The expression profiles of 12 OsBBX genes in different tissues were evaluated through qRT-PCR, and we found that most rice BBX members showed high expression level in the heading stage compared to seedling and booting stages. The expression of OsBBX1, OsBBX2, OsBBX8, OsBBX19, and OsBBX24 was strongly induced by abiotic stresses such as drought, cold and salt stresses. Furthermore, the expression of OsBBX2, OsBBX7, OsBBX17, OsBBX19, and OsBBX24 genes was up-regulated under GA, SA and MeJA hormones at different time points. Similarly, the transcripts level of OsBBX1, OsBBX7, OsBBX8, OsBBX17, and OsBBX19 genes were significantly affected by heavy metals such as Fe, Ni, Cr and Cd. CONCLUSION: Change in the expression pattern of BBX members in response to abiotic, hormone and heavy metal stresses signifies their potential roles in plant growth and development and in response to multivariate stresses. The findings suggest that BBX genes could be used as potential genetic markers for the plants, particularly in functional analysis and determining their roles under multivariate stresses.

Comparative in Silico Analysis of Ferric Reduction Oxidase (FRO) Genes Expression Patterns in Response to Abiotic Stresses, Metal and Hormone Applications.

The ferric reduction oxidase (FRO) gene family is involved in various biological processes widely found in plants and may play an essential role in metal homeostasis, tolerance and intricate signaling networks in response to a number of abiotic stresses. Our study describes the identification, characterization and evolutionary relationships of FRO genes families. Here, total 50 FRO genes in Plantae and 15 'FRO like' genes in non-Plantae were retrieved from 16 different species. The entire FRO genes have been divided into seven clades according to close similarity in biological and functional behavior. Three conserved domains were common in FRO genes while in two FROs sub genome have an extra NADPH-Ox domain, separating the function of plant FROs. OsFRO1 and OsFRO7 genes were expressed constitutively in rice plant. Real-time RT-PCR analysis demonstrated that the expression of OsFRO1 was high in flag leaf, and OsFRO7 gene expression was maximum in leaf blade and flag leaf. Both genes showed vigorous expressions level in response to different abiotic and hormones treatments. Moreover, the expression of both genes was also substantial under heavy metal stresses. OsFRO1 gene expression was triggered following 6 h under Zn, Pb, Co and Ni treatments, whereas OsFRO7 gene expression under Fe, Pb and Ni after 12 h, Zn and Cr after 6 h, and Mn and Co after 3 h treatments. These findings suggest the possible involvement of both the genes under abiotic and metal stress and the regulation of phytohormones. Therefore, our current work may provide the foundation for further functional characterization of rice FRO genes family.

The Fundamental Role of NOX Family Proteins in Plant Immunity and Their Regulation.

NADPH oxidases (NOXs), also known as respiratory burst oxidase homologs (RBOHs), are the major source of reactive oxygen species (ROS), and are involved in many important processes in plants such as regulation of acclimatory signaling and programmed cell death (PCD). Increasing evidence shows that NOXs play crucial roles in plant immunity and their functions in plant immune responses are not as separate individuals but with other signal molecules such as kinases, Rac/Rop small GTPases and hormones, mediating a series of signal transmissions. In a similar way, NOX-mediated signaling also participates in abiotic stress response of plants. We summarized here the complex role and regulation mechanism of NOXs in mediating plant immune response, and the viewpoint that abiotic stress response of plants may be a kind of special plant immunity is also proposed.