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Genome Wide Identification of Respiratory Burst Oxidase Homolog (Rboh) Genes in Citrus sinensis and Functional Analysis of CsRbohD in Cold Tolerance.

ABSTRACT: Respiratory burst oxidase homologs (Rbohs) are critical enzymes involved in the generation of reactive oxygen species (ROS) that play an important role in plant growth and development as well as various biotic and abiotic stresses in plants. Thus far, there have been few reports on the characterization of the Rboh gene family in Citrus. In this study, seven Rboh genes (CsRbohA~CsRbohG) were identified in the Citrus sinensis genome. The CsRboh proteins were predicted to localize to the cell membrane. Most CsRbohs contained four conserved domains, an EF-hand domain, and a transmembrane region. Phylogenetic analysis demonstrated that the CsRbohs were divided into five groups, suggesting potential distinct functions and evolution. The expression profiles revealed that these seven CsRboh genes displayed tissue-specific expression patterns, and five CsRboh genes were responsive to cold stress. Fourteen putative cis-acting elements related to stress response, hormone response, and development regulation were present within the promoters of CsRboh genes. The in-silico microRNA target transcript analyses indicated that CsRbohE might be targeted by csi-miR164. Further functional and physiological analyses showed that the knockdown of CsRbohD in trifoliate orange impaired resistance to cold stress. As a whole, our results provide valuable information for further functional studies of the CsRboh genes in response to cold stress.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC8776138 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:: The accumulation of lignin in fruit has a significant negative impact on the quality of fruit-producing trees, and in particular the lignin formation stimulates the development of stone cells in pear fruit. Reactive oxygen species (ROS) are essential for lignin polymerization. However, knowledge of the RBOH family, a key enzyme in ROS metabolism, remains unknown in most fruit trees. In this study, a total of 40 RBOHs were identified from five fruit-producing trees (Pyrusbretschneideri, Prunuspersica, Citrussinensis, Vitisvinifera, and Prunusmume), and 10 of these sequences came from Pyrusbretschneideri. Multiple sequence alignments revealed that all 10 PbRBOHs contained the NADPH_Ox domain and the six alpha-helical transmembrane domains (TM-I to TM-VI). Chromosome localization and interspecies phylogenetic tree analysis showed that 10 PbRBOHs irregularly distributed on 8 chromosomes and 3 PbRBOHs (PbRBOHA, PbRBOHB, and PbRBOHD) are closely related to known lignification-related RBOHs. Furthermore, hormone response pattern analysis showed that the transcription of PbRBOHs is regulated by SA, ABA and MeJA. Reverse transcription-quantitative real-time polymerase chain reaction (qRT-PCR) and transcriptome sequencing analysis showed that PbRBOHA, PbRBOHB, and PbRBOHD accumulated high transcript abundance in pear fruit, and the transcriptional trends of PbRBOHA and PbRBOHD was consistent with the change of stone cell content during fruit development. In addition, subcellular localization revealed that PbRBOHA and PbRBOHD are distributed on the plasma membrane. Combining the changes of apoplastic superoxide (O2.-) content and spatio-temporal expression analysis, these results indicate that PbRBOHA and PbRBOHD, which are candidate genes, may play an important role in ROS metabolism during the lignification of pear stone cells. This study not only provided insight into the molecular characteristics of the RBOH family in fruit-producing trees, but also lays the foundation for studying the role of ROS in plant lignification.

Project description:Potassium (K+) is a critical determinant of salinity tolerance, and H2O2 has been recognized as an important signaling molecule that mediates many physiological responses. However, the details of how H2O2 signaling regulates K+ uptake in the root under salt stress remain elusive. In this study, salt-sensitive cucumber and salt-tolerant pumpkin which belong to the same family, Cucurbitaceae, were used to answer the above question. We show that higher salt tolerance in pumpkin was related to its superior ability for K+ uptake and higher H2O2 accumulation in the root apex. Transcriptome analysis showed that salinity induced 5816 (3005 up- and 2811 down-) and 4679 (3965 up- and 714 down-) differentially expressed genes (DEGs) in cucumber and pumpkin, respectively. DEGs encoding NADPH oxidase (respiratory burst oxidase homolog D; RBOHD), 14-3-3 protein (GRF12), plasma membrane H+-ATPase (AHA1), and potassium transporter (HAK5) showed higher expression in pumpkin than in cucumber under salinity stress. Treatment with the NADPH oxidase inhibitor diphenylene iodonium resulted in lower RBOHD, GRF12, AHA1, and HAK5 expression, reduced plasma membrane H+-ATPase activity, and lower K+ uptake, leading to a loss of the salinity tolerance trait in pumpkin. The opposite results were obtained when the plants were pre-treated with exogenous H2O2. Knocking out of RBOHD in pumpkin by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9] editing of coding sequences resulted in lower root apex H2O2 and K+ content and GRF12, AHA1, and HAK5 expression, ultimately resulting in a salt-sensitive phenotype. However, ectopic expression of pumpkin RBOHD in Arabidopsis led to the opposite effect. Taken together, this study shows that RBOHD-dependent H2O2 signaling in the root apex is important for pumpkin salt tolerance and suggests a novel mechanism that confers this trait, namely RBOHD-mediated transcriptional and post-translational activation of plasma membrane H+-ATPase operating upstream of HAK5 K+ uptake transporters.

Project description:Verticillium wilt, mainly caused by a soil-inhabiting fungus Verticillium dahliae, can seriously reduce the yield and quality of cotton. The complex mechanism underlying cotton resistance to Verticillium wilt remains largely unknown. In plants, reactive oxygen species (ROS) mediated by Rbohs is one of the earliest responses of plants to biotic and abiotic stresses. In our previous study, we performed a time-course phospho-proteomic analysis of roots of resistant and susceptible cotton varieties in response to V. dahliae, and found early differentially expressed protein burst oxidase homolog protein D (GhRbohD). However, the role of GhRbohD-mediated ROS in cotton defense against V. dahliae needs further investigation. In this study, we analyzed the function of GhRbohD-mediated resistance of cotton against V. dahliae in vitro and in vivo. Bioinformatics analysis showed that GhRbohD possessed the conservative structural attributes of Rbohs family, 12 members of RbohD out of 57 Rbohs in cotton. The expression of GhRbohD was significantly upregulated after V. dahliae inoculation, peaking at 6 hpi, and the phosphorylation level was also increased. A VIGS test demonstrated that ROS production, NO, H2O2 and Ca2+ contents of GhRbohD-silenced cotton plants were significantly reduced, and lignin synthesis and callose accumulation were damaged, important reasons for the impairment of GhRbohD-silenced cotton's defense against V. dahliae. The expression levels of resistance-related genes were downregulated in GhRbohD-silenced cotton by qRT-PCR, mainly involving the lignin metabolism pathway and the jasmonic acid signaling pathway. However, overexpression of GhRbohD enhanced resistance of transgenic Arabidopsis to V. dahliae challenge. Furthermore, Y2H assays were applied to find that GhPBL9 and GhRPL12C may interact with GhRbohD. These results strongly support that GhRbohD activates ROS production to positively regulate the resistance of plants against V. dahliae.

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Genome Wide Identification of Respiratory Burst Oxidase Homolog (Rboh) Genes in Citrus sinensis and Functional Analysis of CsRbohD in Cold Tolerance.

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