Proteomic and Glycomic Characterization of Rice Chalky Grains Produced Under Moderate and High-temperature Conditions in Field System.

BACKGROUND: Global climate models predict an increase in global mean temperature and a higher frequency of intense heat spikes during this century. Cereals such as rice (Oryza sativa L.) are more susceptible to heat stress, mainly during the gametogenesis and flowering stages. During periods of high temperatures, grain filling often causes serious damage to the grain quality of rice and, therefore, yield losses. While the genes encoding enzymes involved in carbohydrate metabolism of chalky grains have been established, a significant knowledge gap exists in the proteomic and glycomic responses to warm temperatures in situ. Here, we studied the translucent and opaque characters of high temperature stressed chalky grains of 2009 and 2010 (ripening temperatures: 24.4 and 28.0 °C, respectively). RESULTS: Appearance of chalky grains of both years showed some resemblance, and the high-temperature stress of 2010 remarkably extended the chalking of grain. Scanning electron microscopic observation showed that round-shaped starch granules with numerous small pits were loosely packed in the opaque part of the chalky grains. Proteomic analyzes of rice chalky grains revealed deregulations in the expression of multiple proteins implicated in diverse metabolic and physiological functions, such as protein synthesis, redox homeostasis, lipid metabolism, and starch biosynthesis and degradation. The glycomic profiling has shown slight differences in chain-length distributions of starches in the grains of 2009-to-2010. However, no significant changes were observed in the chain-length distributions between the translucent and opaque parts of perfect and chalky grains in both years. The glucose and soluble starch contents in opaque parts were increased by the high-temperature stress of 2010, though those in perfect grains were not different regardless of the environmental changes of 2009-to-2010. CONCLUSION: Together with previous findings on the increased expression of α-amylases in the endosperm, these results suggested that unusual starch degradation rather than starch synthesis is involved in occurring of chalky grains of rice under the high-temperature stress during grain filling period.

Golgi/plastid-type manganese superoxide dismutase involved in heat-stress tolerance during grain filling of rice.

Superoxide dismutase (SOD) is widely assumed to play a role in the detoxification of reactive oxygen species caused by environmental stresses. We found a characteristic expression of manganese SOD 1 (MSD1) in a heat-stress-tolerant cultivar of rice (Oryza sativa). The deduced amino acid sequence contains a signal sequence and an N-glycosylation site. Confocal imaging analysis of rice and onion cells transiently expressing MSD1-YFP showed MSD1-YFP in the Golgi apparatus and plastids, indicating that MSD1 is a unique Golgi/plastid-type SOD. To evaluate the involvement of MSD1 in heat-stress tolerance, we generated transgenic rice plants with either constitutive high expression or suppression of MSD1. The grain quality of rice with constitutive high expression of MSD1 grown at 33/28 °C, 12/12 h, was significantly better than that of the wild type. In contrast, MSD1-knock-down rice was markedly susceptible to heat stress. Quantitative shotgun proteomic analysis indicated that the overexpression of MSD1 up-regulated reactive oxygen scavenging, chaperone and quality control systems in rice grains under heat stress. We propose that the Golgi/plastid MSD1 plays an important role in adaptation to heat stress.

Identification of oxidatively modified proteins in salt-stressed Arabidopsis: a carbonyl-targeted proteomics approach.

In plants, environmental stresses cause an increase in the intracellular level of reactive oxygen species (ROS), leading to tissue injury. To obtain biochemical insights into this damage process, we investigated the protein carbonyls formed by ROS or by the lipid peroxide-derived α,ß-unsaturated aldehydes and ketones (i.e. reactive carbonyl species, or RCS) in the leaves of Arabidopsis thaliana under salt stress. A. thaliana Col-0 plants that we treated with 300 mM NaCl for 72 h under continuous illumination suffered irreversible leaf damage. Several RCS such as 4-hydroxy-(E)-2-nonenal (HNE) were increased within 12 h of this salt treatment. Immunoblotting using distinct antibodies against five different RCS, i.e. HNE, 4-hydroxy-(E)-2-hexenal, acrolein, crotonaldehyde and malondialdehyde, revealed that RCS-modified proteins accumulated in leaves with the progress of the salt stress treatment. The band pattern of Western blotting suggested that these different RCS targeted a common set of proteins. To identify the RCS targets, we collected HNE-modified proteins via an anti-HNE antiserum affinity trap and performed an isobaric tag for relative and absolute quantitation, as a quantitative proteomics approach. Seventeen types of protein, modified by 2-fold more in the stressed plants than in the non-stressed plants, were identified as sensitive RCS targets. With aldehyde-reactive probe-based affinity trapping, we collected the oxidized proteins and identified 22 additional types of protein as sensitive ROS targets. These RCS and ROS target proteins were distributed in the cytosol and apoplast, as well as in the ROS-generating organelles the peroxisome, chloroplast and mitochondrion, suggesting the participation of plasma membrane oxidation in the cellular injury. Possible mechanisms by which these modified targets cause cell death are discussed.

Quantitative proteomic analysis of intact plastids.

Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described.

Rapid and high-throughput N-glycomic analysis of plant glycoproteins.

Glycoprotein is a major element in higher organisms including mammalians and plants. It is widely accepted that variation in cellular N-glycome is related to modulation in dynamic cellular mechanisms such as cell-cell adhesion, cell activation, and malignant alterations in mammalian cells. However, the physiological importance of glycan modification of glycoproteins in plant cells is still a matter of dispute. Therefore, a comprehensive and high-throughput analysis of N-glycome in plant glycoproteins is needed. Here, an application of the glycoblotting-mass spectrometry technique to plant glycoprotein research is described.

Proteomics of rice grain under high temperature stress.

Recent proteomic analyses revealed dynamic changes of metabolisms during rice grain development. Interestingly, proteins involved in glycolysis, citric acid cycle, lipid metabolism, and proteolysis were accumulated at higher levels in mature grain than those of developing stages. High temperature (HT) stress in rice ripening period causes damaged (chalky) grains which have loosely packed round shape starch granules. The HT stress response on protein expression is complicated, and the molecular mechanism of the chalking of grain is obscure yet. Here, the current state on the proteomics research of rice grain grown under HT stress is briefly overviewed.

Salicylic acid is involved in the regulation of starvation stress-induced flowering in Lemna paucicostata.

The short-day plant, Lemna paucicostata (synonym Lemna aequinoctialis), was induced to flower when cultured in tap water without any additional nutrition under non-inductive long-day conditions. Flowering occurred in all three of the tested strains, and strain 6746 was the most sensitive to the starvation stress conditions. For each strain, the stress-induced flowering response was weaker than that induced by short-day treatment, and the stress-induced flowering of strain 6746 was completely inhibited by aminooxyacetic acid and l-2-aminooxy-3-phenylpropionic acid, which are inhibitors of phenylalanine ammonia-lyase. Significantly higher amounts of endogenous salicylic acid (SA) were detected in the fronds that flowered under the poor-nutrition conditions than in the vegetative fronds cultured under nutrition conditions, and exogenously applied SA promoted the flowering response. The results indicate that endogenous SA plays a role in the regulation of stress-induced flowering.

Flowering and dwarfism induced by DNA demethylation in Pharbitis nil.

Flowering and dwarfism induced by 5-azacytidine and zebularine, which both cause DNA demethylation, were studied in a short-day (SD) plant Pharbitis nil (synonym Ipomoea nil), var. Violet whose photoinduced flowering state does not last for a long period of time. The DNA demethylating reagents induced flowering under non-inductive long-day (LD) conditions. The flower-inducing effect of 5-azacytidine did not last for a long period of time, and the plants reverted to vegetative growth. The progeny of the plants that were induced to flower by DNA demethylation did not flower under the non-inductive photoperiodic conditions. These results suggest that the flowering-related genes were activated by DNA demethylation and then remethylated again in the progeny. The DNA demethylation also induced dwarfism. The dwarfism did not last for a long period of time, was not heritable and was overcome by gibberellin A3 but not by t-zeatin or kinetin. The change in the genome-wide methylation state was examined by methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis. The analysis detected many more polymorphic fragments between the DNA samples isolated from the cotyledons treated with SD than from the cotyledons under LD conditions, indicating that the DNA methylation state was altered by photoperiodic conditions. Seven LD-specific fragments were extracted from the gel of the MS-AFLP and were sequenced. One of these fragments was highly homologous with the genes encoding ribosomal proteins.

Salicylic acid and the flowering gene FLOWERING LOCUS T homolog are involved in poor-nutrition stress-induced flowering of Pharbitis nil.

The short-day plants Pharbitis nil (synonym Ipomoea nil), var. Violet and Tendan were grown in a diluted nutrient solution or tap water for 20 days under long-day conditions. Violet plants were induced to flower and vegetative growth was inhibited, whereas Tendan plants were not induced to flower, although vegetative growth was inhibited under these conditions. The Violet plants induced to flower by poor-nutrition stress produced fertile seeds and their progeny developed normally. Defoliated Violet scions grafted onto the rootstocks of Violet or Tendan were induced to flower under poor-nutrition stress conditions, but Tendan scions grafted onto the Violet rootstocks were not induced to flower. These results indicate that a transmissible flowering stimulus is involved in the induction of flowering by poor-nutrition stress. The poor-nutrition stress-induced flowering was inhibited by aminooxyacetic acid, a phenylalanine ammonia-lyase inhibitor, and this inhibition was almost completely reversed by salicylic acid (SA). However, exogenously applied SA did not induce flowering under non-stress conditions, suggesting that SA may be necessary but not sufficient to induce flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the Violet plants were induced to flower by growing in tap water, but expression of PnFT1, another ortholog of FT, was not induced, suggesting the specific involvement of PnFT2 in stress-induced flowering.