Involvement of NADPH oxidase isoforms in the production of O_2⁻ manipulated by ABA in the senescing leaves of early-senescence-leaf (<i>esl</i>) mutant rice (<i>Oryza sativa</i>)

Abstract

<div><p>In this study, the differences in reactive oxygen species (ROS) generation and abscisic acid (ABA) accumulation in senescing leaves were investigated by early-senescence-leaf (<i>esl</i>) mutant and its wild type, to clarify the relationship among ABA levels, ROS generation, and NADPH oxidase (Nox) in senescing leaves of rice (<i>Oryza sativa</i>). The temporal expression levels of <i>OsNox</i> isoforms in senescing leaves and their expression patterns in response to ABA treatment were determined through quantitative real-time reverse transcription PCR (qRT-PCR). Results showed that the flag leaf of the <i>esl</i> mutant generated more O_2^- concentrations and accumulated higher ABA levels than the wild-type cultivar did in the grain-filling stage. Exogenous ABA treatment induced O_2^- generation; however, it was depressed by diphenyleneiodonium chloride (DPI) pretreatment in the detached leaf segments. This finding suggested the involvement of NADPH oxidase in ABA-induced O_2^- generation. The <i>esl</i> mutant exhibited significantly higher expression of <i>OsNox2</i>, <i>OsNox5</i>, <i>OsNox6</i>, and <i>OsNox7</i> in the initial of grain-filling stage, followed by sharply decrease. The transcriptional levels of <i>OsNox1</i>, <i>OsNox3</i>, and <i>OsFR07</i> in the flag leaf of the <i>esl</i> mutant were significantly lower than those in the wild-type cultivar. The expression levels of <i>OsNox2</i>, <i>OsNox5</i>, <i>OsNox6</i>, and <i>OsNox7</i> were significantly enhanced by exogenous ABA treatments. The enhanced expression levels of <i>OsNox2</i> and <i>OsNox6</i> were dependent on the duration of ABA treatment. The inducible expression levels of <i>OsNox5</i> and <i>OsNox7</i> were dependent on ABA concentrations. By contrast, exogenous ABA treatment severely repressed the transcripts of <i>OsNox1</i>, <i>OsNox3</i>, and <i>OsFR07</i> in the detached leaf segments. Therefore, <i>OsNox2</i>, <i>OsNox5</i>, <i>OsNox6</i>, and <i>OsNox7</i> were probably involved in the ABA-induced O_2^- generation in the initial stage of leaf senescence. Subsequently, other oxidases activated in deteriorating cells were associated with ROS generation and accumulation in the senescing leaves of the <i>esl</i> mutant. Conversely, <i>OsNox1</i>, <i>OsNox3</i>, and <i>OsFR07</i> were not associated with ABA-induced O_2^- generation during leaf senescence.</p></div