Plant ascorbate peroxidases (APXs), enzymes catalyzing the dismutation of H2O2 into H2O and O2, play an important role in reactive oxygen species homeostasis in plants. The rice genome has eight OsAPXs, but their physiological functions remain to be determined. In this report, we studied the function of OsAPX2 gene using a T-DNA knockout mutant under the treatment of drought, salt and cold stresses. The Osapx2 knockout mutant was isolated by a genetic screening of a rice T-DNA insertion library under 20% PEG-2000 treatment. Loss of function in OsAPX2 affected the growth and development of rice seedlings, resulting in semi-dwarf seedlings, yellow-green leaves, leaf lesion mimic and seed sterility. OsAPX2 expression was developmental- and spatial-regulated, and was induced by drought, salt, and cold stresses. Osapx2 mutants had lower APX activity and were sensitive to abiotic stresses; overexpression of OsAPX2 increased APX activity and enhanced stress tolerance. H2O2 and MDA levels were high in Osapx2 mutants but low in OsAPX2-OX transgenic lines relative to wild-type plants after stress treatments. Taken together, the cytosolic ascorbate peroxidase OsAPX2 plays an important role in rice growth and development by protecting the seedlings from abiotic stresses through scavenging reactive oxygen species.
High-level expression of mGFP4, a GFP variant suitable for higher plants, is obtained through bombardment transformation into rice calli. Bright green fluorescence is visualized by fluorescence and confocal microscopy. The formation of mGFP4 chromophore is swift and green fluorescence is visualized within 4 h following bombardment. Also, the fluorescence emission lasts for about 2 d. The transformation and expression of mGFP4 are demonstrated by Southern blotting and Kanamycin selection experiments. Taken together, rice calli are suitable materials for the transformation and expression of mGFP4, and the success of mGFP4 transformation into rice calli will be valuable to our future research in fusing proteins to GFP and their function analysis in rice cells.
Electroneutral monovalent cation/proton antiport across the chloroplast envelope has been shown previously to have an important regulatory effect on stromal pH and thereby on photosynthetic carbon reduction. Here we report that an Arabidopsis nuclear gene, AtCHX23 , encodes a putative Na + (K + )/H + exchanger and functions in the adjustment of pH in the cytosol and possibly in maintaining a high pH level in the chloroplast stroma. The AtCHX23 protein is localized in the chloroplast envelope. Plastids from chx23 mutants had straight thylakoids but lacked grana lamellae. chx23 mutant leaves were pale yellow and had a much reduced chlorophyll content. The chlorophyll content of chx23 was increased by growing in medium at low (4.0) pH and decreased by growing at high (7.0) pH. The cytosolic pH in the leaves of the mutant was significantly higher than that in the wild type. chx23 mutants displayed a high sensitivity to NaCl. Together, these data indicate that CHX23 is a probable chloroplast Na + (K + )/H + exchanger important for pH homeostasis and chloroplast development and function.
The Arabidopsis NHX antiporter family contains eight members that are divided into three subclasses: vacuolar, endosomal, and plasma membrane. While the plasma membrane and vacuolar NHXs have been studied extensively, the activity and function of the endosomal NHXs are beginning to be discovered. AtNHX5 and AtNHX6 are endosomal Na(+),K(+)/H(+) antiporters that share high sequence similarity. They are localized in the Golgi, trans-Golgi network (TGN), and prevacuolear compartment (PVC). Studies have shown that AtNHX5 and AtNHX6 mediate K(+) and Na(+) transport, and regulate cellular pH homeostasis. Sequence alignment has found that AtNHX5 and AtNHX6 contain four conserved acidic amino acid residues in transmembrane domains that align with yeast and human NHXs. Three of these conserved acidic residues are critical for K(+) transport and seedling growth in Arabidopsis. Moreover, studies have shown that the precursors of the seed storage proteins are missorted to the apoplast in the nhx5 nhx6 knockout mutant, suggesting that AtNHX5 and AtNHX6 regulate protein transport into the vacuole. Further analysis found that AtNHX5 and AtNHX6 regulated the binding of VSR to its cargoes. Taken together, AtNHX5 and AtNHX6 play an important role in cellular ion and pH homeostasis, and are essential for protein transport into the vacuole.
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