| During development and under biotic and abiotic stress conditions, the production of reactive oxygen species (ROS) in plants is inevitable. ROS affect the cells in many aspects. For example, it can cause lipid peroxidation, DNA damage and protein oxidation. In addition to its harmful effects on cells, ROS could also function as an important signaling molecule. As a kind of redox active protein, Thioredoxins (Trxs) can regulate the effect of ROS at all the above levels. Until now, only two kinds of Trxs have been found in mammals. In contrast, a large Trxs family has been identified in plants. In the plant model Arabidopsis thaliana, there are at least 42 Trx isoforms. The H-type Trxs is a multigenic family in plants. Recently, some of their functions have been characterized in different plant species. However, there are 10 members of H-type Trxs in rice and none of them have been clearly characterized.Previously, we analyzed the soluble apoplast proteins of rice roots by two-dimensional electrophoresis. An h-type thioredoxin, OsTRXhl, which was identified by mass spectrometry, increased significantly in abundance under salt stress. In this paper, OsTRXh1 was studied further. Based on the multiple sequence alignment, we found that OsTRXhl belong to the h-type thioredoxin subgroup I. To determine the redox activity of OsTRXh1 in vitro, we expressed and purified OsTRXhl in bacteria and proved it has a high insulin reduction activity in vitro. To verify that OsTRXh1 is also a functional Trx in vivo, its ability to complement the trxlA trx2A yeast mutant was tested. Our results showed that the wild-type OsTRXhl complements the hydrogen peroxide sensitivity of the trx1Δtrx2Δyeast mutant but the OsTRXh1C43S mutant could not. Overall, these results suggest that OsTRXhl is a typical and redox-active thioredoxin.In order to study the function of OsTRXhl in rice, we first examined its expression pattern in rice. Through real-time quantitative PCR we found that OsTRXhl expression was induced by salt and ABA. Using GUS staining analysis of ProOsTRXh1:GUS transgenic rice plants, we found that OsTRXhl was expressed abundantly and extensively. In this study, we demonstrated in vivo that OsTRXhl is secreted into the apoplast in onion epidermal cells and tobacco leaf cells using transient expression systems. We also demonstrated the expression of OsTRXh1 at the protein level was induced by salt stress in apoplast of rice. To further examine the function of OsTRXh1 in rice, we carried out RNAi and over-expression of OsTRXh1 in rice. Knockdown of OsTRXh1 by RNAi led to dwarf and low-tilling phenotypes; Over-expression of OsTRXh1 led to salt-sensitive phenotype; Knockdown and over-expression of OsTRXh1 led to ABA-insensitive phenotype. These results suggest that OsTRXh1 may be involved in the regulation of rice growth, development and stress responses. Measurements of ROS in the apoplast with a cell-impermeable sensitive fluorogenic dye revealed that salt treatment caused lower ROS accumulation in the apoplast of overexpression plants than that in wild-type plants, while the RNAi plants contained more hydrogen peroxide in the apoplast. These results indicate that OsTRXh1 regulated the content of hydrogen peroxide in the rice apoplast.In order to reveal the mechanism of OsTRXh1 function, we purified OsTRXh1 target proteins by point mutant OsTRXh1. Through mass spectrometry we identified 11 target proteins, of which 7 were the previously reported targets,4 were previously unrecognized targets. The target protein identification and validation will give us a better understanding of the molecular mechanism of OsTRXh1 function.For the first time, these results reveal that OsTRXh1 as an apoplastic h-type Trx influences plant architecture and is involved in stress response through regulation of the apoplastic ROS balance in rice. These results also indicated that the apoplast is an important component of plant cells and plays vital roles in plant growth and development as well as feelings of environmental stimulation. |
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