Shihua Shen

Ascending Migration of Endophytic Rhizobia, from Roots to Leaves, inside Rice Plants and Assessment of Benefits to Rice Growth Physiology

ABSTRACT Rhizobia, the root-nodule endosymbionts of leguminous plants, also form natural endophytic associations with roots of important cereal plants. Despite its widespread occurrence, much remains unknown about colonization of cereals by rhizobia. We examined the infection, dissemination, and colonization of healthy rice plant tissues by four species of gfp-tagged rhizobia and their influence on the growth physiology of rice. The results indicated a dynamic infection process beginning with surface colonization of the rhizoplane (especially at lateral root emergence), followed by endophytic colonization within roots, and then ascending endophytic migration into the stem base, leaf sheath, and leaves where they developed high populations. In situ CMEIAS image analysis indicated local endophytic population densities reaching as high as 9 × 1010 rhizobia per cm3 of infected host tissues, whereas plating experiments indicated rapid, transient or persistent growth depending on the rhizobial strain and rice tissue examined. Rice plants inoculated with certain test strains of gfp-tagged rhizobia produced significantly higher root and shoot biomass; increased their photosynthetic rate, stomatal conductance, transpiration velocity, water utilization efficiency, and flag leaf area (considered to possess the highest photosynthetic activity); and accumulated higher levels of indoleacetic acid and gibberellin growth-regulating phytohormones. Considered collectively, the results indicate that this endophytic plant-bacterium association is far more inclusive, invasive, and dynamic than previously thought, including dissemination in both below-ground and above-ground tissues and enhancement of growth physiology by several rhizobial species, therefore heightening its interest and potential value as a biofertilizer strategy for sustainable agriculture to produce the worlds most important cereal crops.

Abscisic acid pretreatment enhances salt tolerance of rice seedlings: proteomic evidence.

Enhanced salt tolerance of rice seedlings by abscisic acid (ABA) pretreatment was observed from phenotypic and physiological analyses. Total proteins from rice roots treated with ABA plus subsequent salt stress were analyzed by using proteomics method. Results showed that, 40 protein spots were uniquely upregulated in the seedlings under the condition of ABA pretreatment plus subsequent salt stress, whereas only 16 under the condition of salt treatment. About 78% (31 spots) of the 40 protein spots were only upregulated in the presence of the subsequent salt stress, indicating that plants might have an economical strategy to prevent energy loss under a false alarm. The results also showed that more enzymes involved in energy metabolism, defense, primary metabolism, etc. were upregulated uniquely in ABA-pretreated rice seedlings, suggesting more abundant energy supply, more active anabolism (nitrogen, nucleotide acid, carbohydrate, etc), and more comprehensive defense systems in ABA-pretreated seedlings than in salt stressed ones.

Constructing the metabolic and regulatory pathways in germinating rice seeds through proteomic approach

Construction of metabolic and regulatory pathways from proteomic data can contextualize the large‐scale data within the overall physiological scheme of an organism. It is an efficient way to predict metabolic phenotype or regulatory style. We did protein profiling in the germinating rice seeds through 1‐DE via LC MS/MS proteomic shotgun strategy. In total, 673 proteins were identified, and could be sorted into 14 functional groups. The largest group was metabolism related. The metabolic proteins were integrated into different metabolic pathways to show the style of reserves mobilization and precursor preparation during the germination. Analysis of the regulatory proteins indicated that regulation of redox homeostasis and gene expression also play important roles for the rice seed germination. Although transcription is unnecessary for the germination, it could ensure the rapidity and uniformity of germination. On the contrary, translation with the stored mRNA is required for the germination. This study will help us to further understand the metabolic style, regulation of redox homeostasis, and gene expression during rice seed germination.

Drought-responsive mechanisms in rice genotypes with contrasting drought tolerance during reproductive stage.

Water status is the main factor affecting rice production. In order to understand rice strategies in response to drought condition in the field, the drought-responsive mechanisms at the physiological and molecular levels were studied in two rice genotypes with contrasting susceptibility to drought stress at reproductive stage. After 20 d of drought treatment, the osmotic potential of leaves reduced 78% and 8% in drought susceptible rice cultivar Zhenshan97B and tolerant rice cultivar IRAT109, respectively. The panicle lengths had no obvious changes in drought stressed Zhenshan97B and IRAT109, suggesting that drought stress impose less effect on assimilate translocation from leaf to vegetative growth of panicles. IRAT109 showed more extensive deeper root growth that could be considered a second line of defense against drought stress. The C(i)/C(a) ratio exhibited enhancement over reduction of g(s) in both cultivars, reflecting the non-stomatal limitation to photosynthesis occurred during drought stress. Orthophosphate dikinase, glycine dehydrogenase, ribulose bisphosphate carboxylase (Rubisco), glycine hydroxymethyltransferase and ATP synthase were down-regulated for Zhenshan97B in response to drought stress, suggesting the reduction of capacity of carbon assimilation in this rice cultivar. In drought-stressed IRAT109, transketolase, Rubisco were down-regulated, however, Rubisco activase and peptidyl-prolyl cis-trans isomerase, which might alleviate the damage on Rubisco by drought stress, were up-regulated. The increased abundances of chloroplastic superoxide dismutase [Cu-Zn] and dehydroascorbate reductase might provide antioxidant protection for IRAT109 against damage by dehydration.

Exploring the Mechanism of Physcomitrella patens Desiccation Tolerance through a Proteomic Strategy

The moss Physcomitrella patens has been shown to tolerate abiotic stresses, including salinity, cold, and desiccation. To better understand this plants mechanism of desiccation tolerance, we have applied cellular and proteomic analyses. Gametophores were desiccated over 1 month to 10% of their original fresh weight. We report that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling, and microtubule depolymerization. Despite the severe desiccation, the membrane system maintains integrity. Through two-dimensional gel electrophoresis and image analysis, we identified 71 proteins as desiccation responsive. Following identification and functional categorization, we found that a majority of the desiccation-responsive proteins were involved in metabolism, cytoskeleton, defense, and signaling. Degradation of cytoskeletal proteins might result in cytoskeletal disassembly and consequent changes in the cell structure. Late embryogenesis abundant proteins and reactive oxygen species-scavenging enzymes are both prominently induced, and they might help to diminish the damage brought by desiccation.Bryophytes as the first land plants are believed to have colonized the land from a fresh water origin, requiring adaptive mechanisms that survival of dehydration. Physcomitrella patens is such a non-vascular bryophyte and shows rare desiccation tolerance in its vegetative tissues. Previous studies showed that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling and microtubule depolymerization. And proteomic alteration supported the cellular structural changes in respond to desiccation stress. In this addendum, we report that Golgi bodies are absent and adaptor protein complex AP-1 large subunit is downregulated during the course of dehydration. Those phenomena may be adverse in protein posttranslational modification, protein sorting and cell walls synthesis under the desiccation condition.

Proteomic Analysis of Oil Mobilization in Seed Germination and Postgermination Development of Jatropha curcas

To understand oil mobilization in germinating seeds, we performed ultrastructural observation and proteomic analysis of endosperm in germinating Jatropha curcas seeds. Results showed that the oil mobilization was initiated during germination, and then the oil was consumed for early seedling development. The significant change in abundance of 50 protein spots during germination indicated that several pathways including beta-oxidation, glyoxylate cycle, glycolysis, citric acid cycle, gluconeogenesis, and pentose phosphate pathway were involved in the oil mobilization.

Isolation and functional characterization of the JcERF gene, a putative AP2/EREBP domain-containing transcription factor, in the woody oil plant Jatropha curcas

A cDNA clone, named JcERF, was isolated from Jatropha curcas seedlings (a woody oil plant). It was classified as an ERF subfamily member based on multiple sequence alignment and phylogenetic characterization. The deduced amino acid sequences of the JcERF clone showed no significant sequence similarity with other known ERF proteins except for the conserved AP2/EREBP DNA-binding domain. Expression of the JcERF gene was rapidly induced upon salinity, drought, ethylene and mechanical wounding treatments. No significant changes in the JcERF expression were observed under ABA stress. Gel retardation assay revealed that the JcERF protein could bind specifically to the GCC box as well as to the C/DRE motif. Also it can be inferred from the gel-shift that there is a possibility that the near sequence of the GCC box has an important effect on the DNA-binding activity. In yeast, the JcERF protein specifically bound to the DRE sequence and activated the transcription of two reporter genes His3 and LacZ driven by the DRE sequence. When fused to the LexA DNA-binding domain, the full-length JcERF functioned effectively as a trans-activator in the yeast one-hybrid assay. Overexpression of JcERF cDNA in transgenic Arabidopsis enhanced the salt and freezing tolerance. Meanwhile the seed germination of JcERF transgenic plants was not affected by various concentrations ABA in MS medium. Taken together, the results showed that JcERF functioned as a novel transcription factor and it exhibited a mechanism of plant response to environmental factors like the other AP2/EREBP regulons that also exist in tropical woody plants.

Rice Proteomics A Step Toward Functional Analysis of the Rice Genome

The technique of proteome analysis with two-dimensional PAGE has the power to monitor global changes that occur in the protein expression of tissues and organisms and/or expression that occurs under stresses. In this study, the catalogues of the rice proteome were constructed, and a functional characterization of some of these proteins was examined. Proteins extracted from tissues of rice and proteins extracted from rice under various kinds of stress were separated by two-dimensional PAGE. An image analyzer was used to reveal a total of 10,589 protein spots on 10 kinds of two-dimensional PAGE gels stained by Coomassie Brilliant Blue. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and the N-terminal amino acid sequences of 272 of 905 proteins were determined. The internal amino acid sequences of 633 proteins were determined using a protein sequencer or mass spectrometry after enzyme digestion of the proteins. Finally, a data file of rice proteins that included information on amino acid sequences and sequence homologies was constructed. The major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that turned out to be calreticulin and a gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have functions in the signal transduction pathway. The information thus obtained from the rice proteome will be helpful in predicting the function of the unknown proteins and will aid in their molecular cloning.

Proteomic analysis of rice seedlings infected by Sinorhizobium meliloti 1021

Rhizobial endophytes infect and colonize not only leguminous plants, but several non‐leguminous species as well. Using green fluorescent protein tagging technique, it has been shown that Rhizobia infect different varieties of rice species and migrate from plant roots to aerial tissues such as leaf sheaths and leaves. The interaction between them was found to promote the growth of rice. The growth promotion is the cumulative result of enhanced photosynthesis and stress resistance. In addition, indole‐3‐acetic acid also contributes to the promotion. Gel‐based comparative proteomic approaches were applied to analyze the protein profiles of three different tissues (root, leaf sheath and leaf) of Sinorhizobium meliloti 1021 inoculated rice in order to get an understanding about the molecular mechanism. Upon the inoculation of rhizobia, proteins involved in nine different functional categories were either up‐regulated or down‐regulated. Photosynthesis related proteins were up‐regulated only in leaf sheath and leaf, while the up‐regulated proteins in root were exclusively defense related. The results implied that there might have been an increase in the import and transport of proteins involved in light and dark reactions to the chloroplast as well as more efficient distribution of nutrients, hence enhanced photosynthesis. Although the initiation of defensive reactions mainly occurred in roots, some different defense mechanisms were also evoked in the aerial tissues.

A comparative proteomic analysis of rice seedlings under various high-temperature stresses

To understand the responses of rice seedlings to different high-temperature stresses, seven-day-old rice seedlings were exposed to different high temperatures for 48 h, and the maximal quantum yield of PS II photochemistry measurements, ascorbate peroxidase activity assays and proteomic analyses in leaf tissue were performed. The results showed that when rice seedlings were exposed to high temperatures at 35 degrees C, 40 degrees C and 45 degrees C, the maximal quantum yield of photosystem II photochemistry, the activity of ascorbate peroxidase and the proteome changed greater at higher temperature. The proteomics analysis showed that proteins such as lignification-related proteins were regulated by high temperature and distinct proteins related to protection were up-regulated at different high temperatures. All the results indicated that different strategies were adopted at different levels of high temperature: the higher the temperature, the more protection machineries were involved. At 35 degrees C, some protective mechanisms were activated to maintain the photosynthetic capability. At 40 degrees C, antioxidative pathways were also active. When rice seedlings encountered high-temperature stress at 45 degrees C, in addition to those induced at 35 degrees C and 40 degrees C, heat shock proteins were effectively induced.