Evolutionary Analysis of Respiratory Burst Oxidase Homolog (RBOH) Genes in Plants and Characterization of ZmRBOHs.

The respiratory burst oxidase homolog (RBOH), as the key producer of reactive oxygen species (ROS), plays an essential role in plant development. In this study, a bioinformatic analysis was performed on 22 plant species, and 181 RBOH homologues were identified. A typical RBOH family was identified only in terrestrial plants, and the number of RBOHs increased from non-angiosperms to angiosperms. Whole genome duplication (WGD)/segmental duplication played a key role in RBOH gene family expansion. Amino acid numbers of 181 RBOHs ranged from 98 to 1461, and the encoded proteins had molecular weights from 11.1 to 163.6 kDa, respectively. All plant RBOHs contained a conserved NADPH_Ox domain, while some of them lacked the FAD_binding_8 domain. Plant RBOHs were classified into five main subgroups by phylogenetic analysis. Most RBOH members in the same subgroup showed conservation in both motif distribution and gene structure composition. Fifteen ZmRBOHs were identified in maize genome and were positioned in eight maize chromosomes. A total of three pairs of orthologous genes were found in maize, including ZmRBOH6/ZmRBOH8, ZmRBOH4/ZmRBOH10 and ZmRBOH15/ZmRBOH2. A Ka/Ks calculation confirmed that purifying selection was the main driving force in their evolution. ZmRBOHs had typical conserved domains and similar protein structures. cis-element analyses together with the expression profiles of the ZmRBOH genes in various tissues and stages of development suggested that ZmRBOH was involved in distinct biological processes and stress responses. Based on the RNA-Seq data and qRT-PCR analysis, the transcriptional response of ZmRBOH genes was examined under various abiotic stresses, and most of ZmRBOH genes were up-regulated by cold stress. These findings provide valuable information for further revealing the biological roles of ZmRBOH genes in plant development and abiotic stress responses.

Nitrate inhibits primary root growth by reducing accumulation of reactive oxygen species in the root tip in Medicago truncatula.

In Medicago truncatula, nitrate, acting as a signal perceived by NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER FAMILY 6.8 (MtNPF6.8), inhibits primary root growth through a reduction of root cell elongation. Since reactive oxygen species (ROS) produced and converted in root tip (O2•- → H2O2 → •OH) have been reported to control cell elongation, the impact of nitrate on the distribution of these ROS in the primary root of M. truncatula was analyzed. We found that nitrate reduced the content of O2•-, H2O2 and •OH in the root tip of three wild type genotypes sensitive to nitrate (R108, DZA, A17), inhibition of root growth and O2•- accumulation being highly correlated. Nitrate also modified the capacity of R108 root tip to produce or remove ROS. The ROS content decrease observed in R108 in response to nitrate is linked to changes in peroxidase activity (EC1.11.1.7) with an increase in peroxidative activity that scavenge H2O2 and a decrease in hydroxylic activity that converts H2O2 into •OH. These changes impair the accumulation of H2O2 and then the accumulation of •OH, the species responsible for cell wall loosening and cell elongation. Accordingly, nitrate inhibitory effect was abolished by externally added H2O2 or mimicked by KI, an H2O2 scavenger. In contrast, nitrate has no effect on ROS production or removal capacities in npf6.8-2, a knockdown line insensitive to nitrate, affected in the nitrate transporter MtNPF6.8 (in R108 background) by RNAi. Altogether, our data show that ROS are mediators acting downstream of MtNPF6.8 in the nitrate signaling pathway.

H2O2 and NADPH oxidases involve in regulation of 2-(2-phenylethyl)chromones accumulation during salt stress in Aquilaria sinensis calli.

2-(2-Phenylethyl)chromones are the main compounds responsible for the quality of agarwood, which is widely used in traditional medicines, incenses and perfumes. H2O2 and NADPH oxidases (also known as respiratory burst oxidase homologs, Rbohs) mediate diverse physiological and biochemical processes in environmental stress responses. However, little is known about the function of H2O2 and NADPH oxidases in 2-(2-phenylethyl)chromones accumulation. In this study, we found that salt stress induced a transient increase in content of H2O2 and 2-(2-phenylethyl)chromones accumulation in Aquilaria sinensis calli. Exogenous H2O2 remarkably decreased the production of 2-(2-phenylethyl)chromones, while dimethylthiourea (DMTU), a scavenger of H2O2, significantly increased 2-(2-phenylethyl)chromones accumulation in salt treated calli. Three new H2O2-generating genes, named AsRbohA-C, were isolated and characterized from A. sinensis. Salt stress also induced a transient increase in AsRbohA-C expression and NADPH oxidase activity. Furthermore, exogenous H2O2 increased AsRbohA-C expression and NADPH oxidase activity, while DMTU inhibited AsRbohA-C expression and NADPH oxidase activity under salt stress. Moreover, diphenylene iodonium (DPI), the inhibitor of NADPH oxidases, reduced AsRbohA-C expression and NADPH oxidase activity, but significantly induced 2-(2-phenylethyl)chromones accumulation during salt stress. These results clearly demonstrated the central role of H2O2 and NADPH oxidases in regulation of salt-induced 2-(2-phenylethyl)chromones accumulation in A. sinensis calli.