Transgenic Brassica chinensis plants expressing a bacterial codA gene exhibit enhanced tolerance to extreme temperature and high salinity.

Transgenic Brassica compestris L. spp. chinensis plants expressing a choline oxidase (codA) gene from Arthrobacter globiformis were obtained through Agrobacterium tumefaciens-mediated transformation. In the transgenic plants, codA gene expression and its product transportation to chloroplasts were detected by the enzyme-linked immunosorbent assay (ELISA) examination, immunogold localization, and (1)H-nuclear magnetic resonance ((1)H-NMR). Stress tolerance was evaluated in the T(3) plants under extreme temperature and salinity conditions. The plants of transgenic line 1 (L1) showed significantly higher net photosynthetic rate (P(n)) and P(n) recovery rate under high (45 °C, 4 h) and low temperature (1 °C, 48 h) treatments, and higher photosynthetic rate under high salinity conditions (100, 200, and 300 mmol/L NaCl, respectively) than the wild-type plants. The enhanced tolerance to high temperature and high salinity stresses in transgenic plants is associated with the accumulation of betaine, which is not found in the wild-type plants. Our results indicate that the introduction of codA gene from Arthrobacter globiformis into Brassica compestris L. spp. chinensis could be a potential strategy for improving the plant tolerance to multiple stresses.

Role of the aquaporin PIP1 subfamily in the chilling tolerance of rice.

Although an association between chilling tolerance and aquaporins has been reported, the exact mechanisms involved in this relationship remain unclear. We compared the expression profiles of aquaporin genes between a chilling-tolerant and a low temperature-sensitive rice variety using real-time PCR and identified seven genes that closely correlated with chilling tolerance. Chemical treatment experiments, by which rice plants were induced to lose their chilling tolerance, implicated the PIP1 (plasma membrane intrinsic protein 1) subfamily member genes in chilling tolerance. Of these members, changes in expression of the OsPIP1;3 gene suggested this to be the most closely related to chilling tolerance. Although OsPIP1;3 showed a much lower water permeability than members of the OsPIP2 family, OsPIP1;3 enhanced the water permeability of OsPIP2;2 and OsPIP2;4 when co-expressed with either of these proteins in oocytes. Transgenic rice plants (OE1) overexpressing OsPIP1;3 showed an enhanced level of chilling tolerance and the ability to maintain high OsPIP1;3 expression levels under low temperature treatment, similar to that of chilling-tolerant rice plants. We assume that OsPIP1;3, constitutively overexpressed in the leaf and root of transgenic OE1 plants, interacts with members of the OsPIP2 subfamily, thereby improving the plants' water balance under low temperatures and resulting in the observed chilling tolerance of the plants.

Characterization of OsPIP2;7, a water channel protein in rice.

Aquaporins are water channel proteins that facilitate passage of water and other small neutral molecules across biological membranes. There are usually a large number of members of this family in higher plants, which exhibit various physiological functions and are regulated in a time-specific and particular mode. We have previously shown that a rice gene, OsPIP2;7, was generally up-regulated in roots but down-regulated in shoots at the early stage of chilling stress. Here, OsPIP2;7 was cloned and proved to be an aquaporin with high activity in Xenopus oocytes. OsPIP2;7 was localized mainly in mesophyll cells of leaves. In roots it was detected in the vascular tissues, epidermis cells and exodermis cells at the elongation zone, as well as in the epidermis cells, exodermis cells and root hair at the maturation zone. Yeast cells overexpressing OsPIP2;7 showed a higher survival rate after freeze-thaw stress. Furthermore, OsPIP2;7 enhanced the transpiration rate and tolerance to low temperature when overexpressed in rice. These results indicated that OsPIP2;7 was involved in rapid water transport and maintenance of the water balance in cells, and ultimately improves the tolerance of yeast and rice to low temperature stress.

Transport functions and expression analysis of vacuolar membrane aquaporins in response to various stresses in rice.

The vacuole, a multifunctional organelle of most plant cells, has very important roles in space filling, osmotic adjustment, storage and digestion. Previous researches suggested that aquaporins in the tonoplast were involved in vacuolar functions. The rice genome contains 33 aquaporin genes, 10 of which encode tonoplast intrinsic proteins (TIPs). However, the function of each individual TIP isoform and the integrated function of TIPs under various physiological conditions remain elusive. Here, five rice TIP members were characterized with water and/or glycerol transport activities using the Xenopus oocyte expression system. OsTIP1;2, OsTIP2;2, OsTIP4;1 and OsTIP5;1 possessed water transport activity. OsTIP1;2, OsTIP3;2 and OsTIP4;1 were demonstrated with glycerol transport activity. Rice TIP expression patterns under various abiotic stress conditions including dehydration, high salinity, abscisic acid (ABA) and during seed germination were investigated by real-time PCR. OsTIP1s (OsTIP1;1 and OsTIP1;2) were highly expressed during seed germination, whereas OsTIP3s (OsTIP3;1 and OsTIP3;2) were specifically expressed in mature seeds with a decrease in expression levels upon germination. The results of this research provided a functional and expression profiles of rice TIPs.

The role of water channel proteins and nitric oxide signaling in rice seed germination.

Previous studies have demonstrated the possible role of several aquaporins in seed germination. But systematic investigation of the role of aquaporin family members in this process is lacking. Here, the developmental regulation of plasma membrane intrinsic protein (PIP) expression throughout germination and post-germination processes in rice embryos was analyzed. The expression patterns of the PIPs suggest these aquaporins play different roles in seed germination and seedling growth. Partial silencing of the water channel genes, OsPIP1;1 and OsPIP1;3, reduced seed germination while over-expression of OsPIP1;3 promoted seed germination under water-stress conditions. Moreover, spatial expression analysis indicates that OsPIP1;3 is expressed predominantly in embryo during seed germination. Our data also revealed that the nitric oxide (NO) donors, sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO), promoted seed germination; furthermore, the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, inhibited germination and reduced the stimulative effects of SNP and GSNO on rice germination. Exogenous NO stimulated the transcription of OsPIP1;1, OsPIP1;2, OsPIP1;3 and OsPIP2;8 in germinating seeds. These results suggest that water channels play an important role in seed germination, acting, at least partly, in response to the NO signaling pathway.

Water relations and an expression analysis of plasma membrane intrinsic proteins in sensitive and tolerant rice during chilling and recovery.

A symptom of chilling injury is development of water deficit in shoots, resulting from an imbalance of water transport and transpiration. In this work, two rice varieties (Oryza sativa L. var. Wasetoitsu and Somewake) seedlings were chilled at 7 degrees C, followed by recovery at 28 degrees C. Based on the growth phenotype and electrolyte leakage tests, Somewake was shown to be a chilling-tolerant variety, and Wasetoitsu a chilling-sensitive one. The chilling stress reduced markedly the relative water content (RWC) of leaves, accumulative transpiration and osmotic root hydraulic conductivity (Lp) in both varieties. But when returned to 28 degrees C, the water relation balance of Somewake recovered better. The mRNA expression profile of all the 11 plasma membrane intrinsic proteins (PIPs), a subgroup of aquaporins, was subsequently determined by real-time reverse transcription (RT)-PCR with TaqMan-minor grove binder (MGB) probes derived from rice var. Nipponbare during chilling treatment and recovery. Most of the PIP genes was down-regulated at the low temperature, and recovered at the warm temperature. The relative expression of some PIPs in both Somewake and Wasetoitsu decreased in parallel during the chilling. However during the recovery, the relative expression of OsPIP1;1, OsPIP2;1, OsPIP2;7 in shoots and OsPIP1;1, OsPIP2;1 in roots were significantly higher in Somewake than Wasetoitsu. This supports the role of PIPs in re-establishing water balance after chilling conditions. We discuss the diversified roles played by members of the aquaporin PIP subfamily in plant chilling tolerance depending on aquaporin isoforms, plant tissue and the stage of chilling duration.

Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment.

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins (PIPs), was studied at both the protein and mRNA level in upland rice (Oryza sativa L. cv. Zhonghan 3) and lowland rice (Oryza sativa L. cv. Xiushui 63) when they were water stressed by treatment with 20% polyethylene glycol (PEG). Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1;2, OsPIP1;3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1;2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid (ABA) level in upland rice but not in lowland rice under water deficit. Treatment with 60 microM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling pathways during water deficit.

Comparative proteomic analysis provides new insights into chilling stress responses in rice.

Low temperature is one of the major abiotic stresses limiting the productivity and the geographical distribution of many important crops. To gain a better understanding of chilling stress responses in rice (Oryza sativa L. cv. Nipponbare), we carried out a comparative proteomic analysis. Three-week-old rice seedlings were treated at 6 degrees C for 6 or 24 h and then recovered for 24 h. Chilling treatment resulted in stress phenotypes of rolling leaves, increased relative electrolyte leakage, and decreased net photosynthetic rate. The temporal changes of total proteins in rice leaves were examined using two-dimensional electrophoresis. Among approximately 1,000 protein spots reproducibly detected on each gel, 31 protein spots were down-regulated, and 65 were up-regulated at least at one time point. Mass spectrometry analysis allowed the identification of 85 differentially expressed proteins, including well known and novel cold-responsive proteins. Several proteins showed enhanced degradation during chilling stress, especially the photosynthetic proteins such as Rubisco large subunit of which 19 fragments were detected. The identified proteins are involved in several processes, i.e. signal transduction, RNA processing, translation, protein processing, redox homeostasis, photosynthesis, photorespiration, and metabolisms of carbon, nitrogen, sulfur, and energy. Gene expression analysis of 44 different proteins by quantitative real time PCR showed that the mRNA level was not correlated well with the protein level. In conclusion, our study provides new insights into chilling stress responses in rice and demonstrates the advantages of proteomic analysis.

Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase.

We investigated the role that manganese superoxide dismutase (MnSOD), an important antioxidant enzyme, may play in the drought tolerance of rice. MnSOD from pea (Pisum sativum) under the control of an oxidative stress-inducible SWPA2 promoter was introduced into chloroplasts of rice (Oryza sativa) by Agrobacterium-mediated transformation to develop drought-tolerant rice plants. Functional expression of the pea MnSOD in transgenic rice plants (T1) was revealed under drought stress induced by polyethylene glycol (PEG) 6000. After PEG treatment the transgenic leaf slices showed reduced electrolyte leakage compared to wild type (WT) leaf slices, whether they were exposed to methyl viologen (MV) or not, suggesting that transgenic plants were more resistant to MV- or PEG-induced oxidative stress. Transgenic plants also exhibited less injury, measured by net photosynthetic rate, when treated with PEG. Our data suggest that SOD is a critical component of the ROS scavenging system in plant chloroplasts and that the expression of MnSOD can improve drought tolerance in rice.

The role of aquaporin RWC3 in drought avoidance in rice.

Although the discovery of aquaporins in plants has resulted in a paradigm shift in the understanding of plant water relations, the relationship between aquaporins and drought resistance still remains elusive. From an agronomic viewpoint, upland rice is traditionally considered as showing drought avoidance. In the investigation of different morphological and physiological responses of upland rice (Oryza sativa L. spp indica cv. Zhonghan 3) and lowland rice (O. sativa L. spp japonica cv. Xiushui 63) to water deficit, we observed young leaf rolling and the remarkable decline of cumulative transpiration in the upland rice. The expression of water channel protein RWC3 mRNA was increased in upland rice at the early response (up to 4 h) to the 20% polyethylene glycol (PEG) 6000 treatment, whereas there was no significant expression changes in lowland rice. Protein levels were increased in upland rice and decreased in lowland rice at 10 h after the water deficit. The up-regulation of RWC3 in upland rice fits well with the knowledge that upland rice adopts the mechanism of drought avoidance. The physiological significance of this RWC3 up-regulation was then explored with the over-expression of RWC3 in transgenic lowland rice (O. sativa L. spp japonica cv. Zhonghua 11) controlled by a stress-inducible SWPA2 promoter. Compared to the wild-type plant, the transgenic lowland rice exhibited higher root osmotic hydraulic conductivity (Lp), leaf water potential and relative cumulative transpiration at the end of 10 h PEG treatment. These results indicated that RWC3 probably played a role in drought avoidance in rice.

Alkali grass resists salt stress through high [K+] and an endodermis barrier to Na+.

In order to understand the salt-tolerance mechanism of alkali grass (Puccinellia tenuiflora) compared with wheat (Triticum aestivum L.), [K(+)] and [Na(+)] in roots and shoots in response to salt treatments were examined with ion element analysis and X-ray microanalysis. Both the rapid K(+) and Na(+) influx in response to different NaCl and KCl treatments, and the accumulation of K(+) and Na(+) as the plants acclimated to long-term stress were studied in culture- solution experiments. A higher K(+) uptake under normal and saline conditions was evident in alkali grass compared with that in wheat, and electrophysiological analyses indicated that the different uptake probably resulted from the higher K(+)/Na(+) selectivity of the plasma membrane. When external [K(+)] was high, K(+) uptake and transport from roots to shoots were inhibited by exogenous Cs(+), while TEA (tetraethylammonium) only inhibited K(+) transport from the root to the shoot. K(+) uptake was not influenced by Cs(+) when plants were K(+) starved. It was shown by X-ray microanalysis that high [K(+)] and low [Na(+)] existed in the endodermal cells of alkali grass roots, suggesting this to be the tissue where Cs(+) inhibition occurs. These results suggest that the K(+)/Na(+) selectivity of potassium channels and the existence of an apoplastic barrier, the Casparian bands of the endodermis, lead to the lateral gradient of K(+) and Na(+) across root tissue, resulting not only in high levels of [K(+)] in the shoot but also a large [Na(+)] gradient between the root and the shoot.