Atsushi Oyanagi

Cell wall proteome of wheat roots under flooding stress using gel-based and LC MS/MS-based proteomics approaches.

Cell wall proteins (CWPs) are important both for maintenance of cell structure and for responses to abiotic and biotic stresses. In this study, a destructive CWP purification procedure was adopted using wheat seedling roots and the purity of the CWP extract was confirmed by minimizing the activity of glucose-6-phosphate dehydrogenase, a cytoplasmic marker enzyme. To determine differentially expressed CWPs under flooding stress, gel-based proteomic and LC-MS/MS-based proteomic techniques were applied. Eighteen proteins were found to be significantly regulated in response to flood by gel-based proteomics and 15 proteins by LC MS/MS-based proteomics. Among the flooding down-regulated proteins, most were related to the glycolysis pathway and cell wall structure and modification. However, the most highly up-regulated proteins in response to flooding belong to the category of defense and disease response proteins. Among these differentially expressed proteins, only methionine synthase, beta-1,3-glucanases, and beta-glucosidase were consistently identified by both techniques. The down-regulation of these three proteins suggested that wheat seedlings respond to flooding stress by restricting cell growth to avoid energy consumption; by coordinating methionine assimilation and cell wall hydrolysis, CWPs played critical roles in flooding responsiveness.

Ethylene and reactive oxygen species are involved in root aerenchyma formation and adaptation of wheat seedlings to oxygen-deficient conditions

Ethylene-mediated reactive oxygen species signalling is involved in adaptive responses of wheat seedlings to waterlogged conditions through controlling formation of lysigenous aerenchyma and expression of genes encoding ethanol fermentation enzymes in roots

Cadmium Concentration in Grains of Japanese Wheat Cultivars : Genotypic Difference and Relationship with Agronomic Characteristics

The contamination of cadmium (Cd) into the food chain can be harmful because Cd causes chronic health problems. To evaluate the breeding potential reducing the Cd concentration in wheat grain, we compared Cd concentrations in 237 wheat genotypes including Japanese landraces, Japanese cultivars and introduced alien cultivars for breeding using grain samples collected from upland fields in 2004−5 and 2005−6 growing seasons. The Cd concentration in wheat grain significantly varied with the growing seasons and with the experimental fields. Cultivars bred in northern Japan, including the recent Japanese leading cultivar ‘Hokushin’, tended to have a low Cd concentration in grain compared with that bred in central and southern Japan. Simple correlation analysis between Cd concentration in grain and agronomic characteristics revealed that the Cd concentration in grain showed significant negative correlations with stem number, culm length and spikelet number per spike, and showed significant positive correlation with SPAD value (chlorophyll content) of flag leaf. Stepwise multipleregression analysis showed that the genotypic variation of Cd concentration in grain was associated with the culm length and spiklet number per spike. This study clarified the geographical pattern of genotypes with different Cd concentrations in grain in Japanese wheat cultivars. Cultivars originating from northern Japan may be useful genetic resources to develop cultivars with a low Cd concentration in grain to be grown in the areas where Cd accumulation in wheat grain is a problem.

Adventitious roots of wheat seedlings that emerge in oxygen-deficient conditions have increased root diameters with highly developed lysigenous aerenchyma

Exposing roots of plants to hypoxic conditions is known to greatly improve their anoxic stress tolerance. We previously showed that pre-treatment of wheat seedlings with an ethylene precursor, 1-aminocyclopropanecarboxylic acid (ACC), enhanced their tolerance of oxygen-deficient conditions. Although ACC-pretreated seminal roots of wheat seedlings grown under oxygen-deficient conditions avoided root tip death, they elongated very little. In the present study, we assessed the effects of ethylene on the responses of adventitious roots of wheat seedlings to oxygen-deficient conditions. Lysigenous aerenchyma formation in the adventitious roots of wheat seedlings pretreated with ACC appeared to reduce tip death under oxygen-deficient conditions, but the adventitious roots, like the seminal roots, hardly elongated. We also found that adventitious roots that emerge in oxygen-deficient conditions continued to elongate even under such conditions. The adventitious roots emerged in oxygen-deficient conditions were found to have thicker root diameters than those emerged in aerated conditions. These results suggest that the adventitious roots with thicker root diameters can better cope with oxygen-deficient conditions. Measurements of the area of the lysigenous aerenchyma confirmed that the increased root diameters have a greater amount of air space generated by lysigenous aerenchyma formation.

Aerenchyma formation in the seminal roots of Japanese wheat cultivars in relation to growth under waterlogged conditions.

Abstract Morphological adaptation of roots is critical for plants to survive under waterlogging. In this study, we evaluated the capacity of wheat to form aerenchyma in seminal roots in combination with the growth angle of the roots. We used five Japanese cultivars from the waterlogging-prone Kanto-Kyushu region in Japan, and a non-Japanese cultivar, Bobwhite for comparison. Seedlings in pot culture were waterlogged at a 3-cm depth for 7 days. The first adverse effect of waterlogging on plant growth was a significant reduction of root dry mass. The reduction rate varied with the cultivar, and it was 19.2% in cv. Shiroganekomugi and 40.0% in cv. Norin 61. Root aerenchyma was initially observed on the 2nd day of waterlogging and developed until the 7th day, in all 6 cultivars. Quantitative analysis of the aerenchyma development revealed no significant difference in radial distribution among the cultivars, whereas a slight difference was found in the axial distribution. As a consequence, the heavier root weight of Shiroganekomugi was not related to either the radial or axial developing capacity of aerenchyma but might be due to the effect of its shallow root angle in the soil. These results suggest that the capacity to form aerenchyma in the seminal root is not sufficient for expression of waterlogging tolerance in the Japanese wheat cultivars.

Characterization of a wheat pathogenesis-related protein, TaBWPR-1.2, in seminal roots in response to waterlogging stress.

We examined the role of pathogenesis-related protein TaBWPR-1.2 in the context of molecular and physiological responses of wheat (Triticum aestivum) seminal roots under waterlogging stress. Two cDNAs corresponding to the TaBWPR-1.2 gene, TaBWPR-1.2#2 and TaBWPR-1.2#13 were cloned from seminal roots. These cDNAs were predicted to encode proteins of 173 and 172 amino acids, respectively. In a time-course experiment, TaBWPR-1.2 gene expression was highest in whole seminal roots after 1 day of waterlogging treatment and higher than the control for at least 10 days; significantly increased protein abundance was observed after 7 days of waterlogging. Drought, another abiotic stress, did not influence TaBWPR-1.2 gene expression in wheat seminal roots at 5-d-old seedlings. Tissue-specific studies revealed that the highest TaBWPR-1.2 gene expression and protein levels were in the aerenchymatous root zone. TaBWPR-1.2 expression in seminal roots was also increased by the signalling molecules 1-aminocyclopropane-1-carboxylic acid (ACC; an ethylene precursor), H2O2, jasmonic acid (JA), and nitric oxide (NO); however, treatment with abscisic acid (ABA), salicylic acid (SA), and ethanol did not alter its expression. Interestingly, aerenchyma formation in the seminal root cortex was induced only by ACC and H2O2. Taken together, these results indicate that TaBWPR-1.2 is a waterlogging-responsive gene that might be associated with root cortex tissue alteration in wheat plants through ACC and/or H2O2 regulatory mechanisms.

Quantitative Proteomics of the Root of Transgenic Wheat Expressing TaBWPR-1.2 Genes in Response to Waterlogging

Once candidate genes are available, the application of genetic transformation plays a major part to study their function in plants for adaptation to respective environmental stresses, including waterlogging (WL). The introduction of stress-inducible genes into wheat remains difficult because of low transformation and plant regeneration efficiencies and expression variability and instability. Earlier, we found two cDNAs encoding WL stress-responsive wheat pathogenesis-related proteins 1.2 (TaBWPR-1.2), TaBWPR-1.2#2 and TaBWPR-1.2#13. Using microprojectile bombardment, both cDNAs were introduced into “Bobwhite”. Despite low transformation efficiency, four independent T2 homozygous lines for each gene were isolated, where transgenes were ubiquitously and variously expressed. The highest transgene expression was obtained in Ubi:TaBWPR-1.2#2 L#11a and Ubi:TaBWPR-1.2#13 L#4a. Using quantitative proteomics, the root proteins of L#11a were analyzed to explore possible physiological pathways regulated by TaBWPR-1.2 under normal and waterlogged conditions. In L#11a, the abundance of proteasome subunit alpha type-3 decreased under normal conditions, whereas that of ferredoxin precursor and elongation factor-2 increased under waterlogged conditions in comparison with normal plants. Proteomic results suggest that L#11a is one of the engineered wheat plants where TaBWPR-1.2#2 is most probably involved in proteolysis, protein synthesis and alteration in the energy pathway in root tissues via the above proteins in order to gain metabolic adjustment to WL.