Fructose-bisphosphate aldolase is a glycolytic enzyme whose activity increases in rice roots treated with gibberellin (GA). To investigate the relationship between aldolase and root growth, GA-induced root aldolase was characterized. GA3 promoted an increase in aldolase accumulation when 0.1 microM GA3 was added exogenously to rice roots. Aldolase accumulated abundantly in roots, especially in the apical region. To examine the effect of aldolase function on root growth, transgenic rice plants expressing antisense aldolase were constructed. Root growth of aldolase-antisense transgenic rice was repressed compared with that of the vector control transgenic rice. Although aldolase activity increased by 25% in vector control rice roots treated with 0.1 microM GA3, FBPA activity increased very little by 0.1 microM GA3 treatment in the root of aldolase-antisense transgenic rice. Furthermore, aldolase co-immunoprecipitated with antibodies against vacuolar H+ -ATPase in rice roots. In the root of OsCDPK13-antisense transgenic rice, aldolase did not accumulate even after treatment with GA3. These results suggest that the activation of glycolytic pathway function accelerates root growth and that GA3-induced root aldolase may be modulated through OsCDPK13. Aldolase physically associates with vacuolar H-ATPase in roots and may regulate the vacuolar H-ATPase mediated control of cell elongation that determines root length.

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Protein databases constructed using high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) were used to explore the proteome expressed in various rice tissues. Proteins from leaf sheath, root, and cultured suspension cells were systematically analyzed using 2D-PAGE, mass spectrometry and Edman sequencing, followed by database searching. In all, 79 of the 431 spots detected by 2D-PAGE in the leaf sheath, 73 of the 508 spots in the root and 140 of the 962 spots in the cultured suspension cells could be identified. Protein lists were constructed for each tissue and used to investigate the effects of gibberellin (GA) treatment. In the leaf sheath, root and cultured suspension cells, 8, 21, and 14 of the identified proteins, respectively, were regulated by GA. These proteins included polypeptides involved in general metabolism, energy production, transcriptional regulation and signal transduction in the leaf sheath; in metabolism and defense in the root; and in metabolism, energy production, cell growth, defense and signal transduction in the cultured suspension cells. These results indicate that the proteome databases assembled in these studies will be useful for the rapid assessment of changes in protein content in specific tissues, and that proteins regulated by GA may play a significant role in tissue growth.

Proteins extracted from leaf blades of rice plants infected with blast fungus, Magnaporthe grisea, were separated by two-dimensional polyacrylamide gel electrophoresis. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and 63 proteins were analyzed by a gas-phase protein sequencer. The N-terminal amino acid sequences of 33 out of 63 proteins were determined in this manner. N-terminal regions of the remaining proteins could not be sequenced. The internal amino acid sequences of 12 proteins were determined by sequence analysis of peptides obtained by the Cleveland peptide mapping method. The amino acid sequences were compared with those of known plant and animal protein sequences to understand the nature of these proteins. As expected, leaf blades revealed predominantly the presence of photosynthetic proteins. Using this experimental approach named as proteome analysis, the functional proteins during blast fungus infection of rice with different levels of nitrogen nutrient were analyzed. Twelve proteins which appeared to change with different levels of nitrogen nutrient were identified. It was revealed that the level of ribulose-1,5-bisphosphate carboxylase/oxygenase was increased by top-dressing with nitrogen nutrient. Additionally, the pathogenesis related protein were observed following blast fungus infection using immunoblot analysis. It was conjectured that these proteins might be involved in incompatible interaction in rice plants following blast fungus infection. The information obtained on the amino acid sequences and antibodies interaction is expected to be helpful in predicting the function of these proteins.

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