Mutation Of Arabidopsis CATALASE2Results In Hyponastic Leaves By Changes Of Auxin Levels

H2O2, a major reactive oxygen species (ROS), functions as an important signal molecule in various aspects of plant growth. Three catalase genes (CAT1, CAT2and CAT3) have been reported in Arabidopsis；however, photorespiratory CAT2cannot be entirely replaced by either of the other two catalase isoforms or by other antioxidative enzymes. Thus, the cat2-1mutant exhibits the reduced catalase activity and accumulates high levels of H2O2under photorespiratory conditions. The knockout mutant of Arabidopsis CAT2provides a useful tool for investigating the effects of increases in intracellular H2O2, with the advantage that the excess H2O2signal can be switched on and off by transferring plants between photorespiratory and non-photorespiratory conditions, respectively, such as high and low light. Auxin as a growth-promoting hormone is crucial for plant growth and environmental response, and its homeostasis is controlled temporally and spatially with various mechanisms including its biosynthesis. While the possibility for auxin-H2O2interactions has attracted much attention with some parallel data for possible regulation of auxin’s role by ROS including H2O2, further studies must be carried out to elucidate whether and how H2O2modulates auxin.In this paper, we demonstrate for the first time that auxin level is modulated by increased H2O2in the cat2-1mutant and this change in auxin content results in hyponastic leaves with physiological, biochemical and genetic data. The major research results are as follows:1、Cat2-1plants grown under moderate-intensity light (150μmol m-2s-1) and LD shows a hyponastic leaf phenotype. Our results showed that up-curling of leaves were rescued in cat2-1CAT2::CAT2transformants, demonstrating that the altered phenotype resulted from CAT2knockout. We found that the H2O2contents in the leaves of cat2-1was higher than that in the leaves of the complemented and wild-type plants, suggesting that the upward curling of cat2-1leaves could be due to a greater accumulation of H2O2in the leaves.2、DR5::GUS, an auxin-responsive reporter gene line, was crossed with the cat2-1mutant and the resultant progeny were subjected to GUS staining to determine auxin level. Compared to the DR5::GUS line in the wild-type background, cat2-1DR5::GUS showed a remarkable reduction in GUS expression in leaves when grown under moderate intensity light and LD conditions. We then found that IAA content in cat2-1 leaves was lower than that in wild-type leaves when the plants were grown under moderate intensity light and LD conditions, suggesting that the up-curled leaf phenotype could be due to the reduced indole acetic acid (IAA) contents in the cat2-1mutant.3、Elevated auxin levels can rescue the hyponastic leaf phenotype in cat2-1. Our data showed while H2O2centent was elevated with up-curled leaves, auxin level was decreased in the cat2-l mutant under the light of150μmol m-2sec-1. When cat2-1plants were transferred to the light of30μmol m-2sec-1, up-curled leaves were rescued with lower H2O2concentration and higher auxin level, suggesting that H2O2plays its role by modulating auxin level. This conclusion was further enforced by exogenously applying auxin or endogenously manipulating auxin level. When auxin was directly applied to cat2-1leaves, the up-curled leaves turned to be down-curled. If cat2-1was transformed with pCAT2::iaaM for higher auxin level，the transgenic lincs rescued hyponastic leaves.4, To further explore how H2O2affects auxin level in the cat2-1mutant, transcriptional suppression of auxin synthesis-related genes in the mutant was evidenced by qRT-PCR assays. These data suggest that the lower concentration of auxin in the mutant was mainly due to transcriptional suppression of the auxin biosynthetic genes.5^Furthermore, glutathione application can rescue up-curled leaves and increase auxin level, but did not change H2O2content in cat2-1, indicating the involvement of glutathione redox status in H2O2-mediated changes of auxin accumulation in the leaves of cat2-1.6、Furthermore, the curvature regulation genes including TCP3, TCP4, TCP10and HASTY are repressed when cat2-1is incubated under moderate-intensity light.7、In our experiments, the spontaneous lesions observed in the leaves of cat2-1plants grown under moderate light intensity were not restored when the plants were treated with GSH. The finding that the hyponastic leaf phenotype, but not the spontaneous lesions could be rescued by changes in glutathione redox state and auxin level indicated that different downstream pathways governed the formation of H2O2-mediated hyponastic leaves and spontaneous lesions.In summary, our data indicate that H2O2, as one of the major ROS generated by cells, plays an important role in determining leaf morphology. H2O2regulates the level of auxin by altering the expression of auxin synthesis-related genes through modulating the GSH redox status in cat2-1mutant. Furthermore, the expression of genes involved in leaf curvature regulation, including TCP3, TCP4, TCP10and HASTY, is down-regulated in cat2-1mutant. Finally, we showed that the GSH redox status affects the H2O2-mediated changes in auxin level specifically in hyponastic leaves.