Masataka Wakayama

Introduction of the ZmDof1 gene into rice enhances carbon and nitrogen assimilation under low-nitrogen conditions

The excessive application of nitrogen fertilizer to maximize crop yields causes negative environmental effects such as pollution and ecological imbalance. To overcome this problem, researchers have attempted to improve the nitrogen assimilation capacity of crops. Maize Dof1 (ZmDof1) is a plant-specific transcription factor shown to promote nitrogen assimilation in Arabidopsis thaliana (Arabidopsis) even under nitrogen-deficient conditions. The present study examines the effect of the introduction of the ZmDof1 gene on carbon and nitrogen assimilation in rice. ZmDof1 induced the expression of phosphoenolpyruvate carboxylase (PEPC) genes in transgenic rice plants and transactivated the PEPC promoters in protoplast transient assays, showing similar effects in rice as in Arabidopsis. Transgenic rice expressing ZmDof1 and grown in the presence of 360 μm (nitrogen-sufficient) or 90 μm (nitrogen-deficient) of nitrogen concentrations showed modulation of metabolite content and gene expression associated with the anaplerotic pathway for the TCA cycle, suggesting an increased carbon flow towards nitrogen assimilation. Furthermore, increases in carbon and nitrogen amounts per seedling were found in Dof1 rice grown under nitrogen-deficient conditions. Nitrogen deficiency also resulted in the predominant distribution of nitrogen to roots, accompanied by significant increases in root biomass and modification of the shoot-to-root ratio. Measurement of the CO₂ gas exchange rate showed a significant increase in the net photosynthesis rate in Dof1 rice under nitrogen-deficient conditions. Taken these together, the present study displayed that ZmDof1 expression in rice could induce gene expressions such as PEPC genes, modulate carbon and nitrogen metabolites, increase nitrogen assimilation and enhance growth under low-nitrogen conditions.

Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA)

In plants, glutamine synthetase (GS) is the enzyme that is mainly responsible for the assimilation of ammonium. Conversely, in microorganisms such as bacteria and Ascomycota, NADP(H)-dependent glutamate dehydrogenase (GDH) and GS both have important roles in ammonium assimilation. Here, we report the changes in nitrogen assimilation, metabolism, growth, and grain yield of rice plants caused by an ectopic expression of NADP(H)-GDH (gdhA) from the fungus Aspergillus niger in the cytoplasm. An investigation of the kinetic properties of purified recombinant protein showed that the fungal gdhA had 5.4–10.2 times higher Vmax value and 15.9–43.1 times higher Km value for NH4+, compared with corresponding values for rice cytosolic GS as reported in the literature. These results suggested that the introduction of fungal GDH into rice could modify its ammonium assimilation pathway. We therefore expressed gdhA in the cytoplasm of rice plants. NADP(H)-GDH activities in the gdhA-transgenic lines were markedly higher than those in a control line. Tracer experiments by feeding with 15NH4+ showed that the introduced gdhA, together with the endogenous GS, directly assimilated NH4+ absorbed from the roots. Furthermore, in comparison with the control line, the transgenic lines showed an increase in dry weight and nitrogen content when sufficient nitrogen was present, but did not do so under low-nitrogen conditions. Under field condition, the transgenic line examined showed a significant increase in grain yield in comparison with the control line. These results suggest that the introduction of fungal gdhA into rice plants could lead to better growth and higher grain yield by enhancing the assimilation of ammonium.

Simultaneous analysis of amino acids and carboxylic acids by capillary electrophoresis-mass spectrometry using an acidic electrolyte and uncoated fused-silica capillary.

A simple, low-cost capillary electrophoresis-mass spectrometry (CE-MS) method is demonstrated for the simultaneous analysis of amino acids and small carboxylic acids (glycerate, lactate, fumarate, succinate, malate, tartrate, citrate, iso-citrate, cis-aconitate, and shikimate). All CE-MS experiments were performed using a single uncoated fused-silica capillary and with a single separation electrolyte, formic acid. For CE polarity, the CE inlet was set as the anode, and the MS side was set as the cathode. By using high-speed sheath gas flow, the apparent mobilities of all compounds were sped up; thus, the migration times of the carboxylic acids were reduced. In positive ion mode ESI-MS detection, small carboxylic acids were detected faintly as m/z = [M + 18](+) or [M + 23](+), after protonated molecule detection (m/z = [M + 1](+)) of the amino acids. In negative ion mode, all of these small carboxylic acids were detected clearly as deprotonated molecules (m/z = [M - 1](-)), after detection of the amino acids. By changing the polarity of the MS during CE separation, both amino acids and small carboxylic acids were detectable in a single electrophoresis analysis run. With this method, the diurnal metabolic changes of pineapple leaves were observed as reflecting Crassulacean acid metabolism.

Structure and enzyme expression in photosynthetic organs of the atypical C4 grass Arundinella hirta

In its leaf blade, Arundinella hirta has unusual Kranz cells that lie distant from the veins (distinctive cells; DCs), in addition to the usual Kranz units composed of concentric layers of mesophyll cells (MCs) and bundle sheath cells (BSCs; usual Kranz cells) surrounding the veins. We examined whether chlorophyllous organs other than leaf blades—namely, the leaf sheath, stem, scale leaf, and constituents of the spike—also have this unique anatomy and the C4 pattern of expression of photosynthetic enzymes. All the organs developed DCs to varying degrees, as well as BSCs. The stem, rachilla, and pedicel had C4-type anatomy with frequent occurrence of DCs, as in the leaf blade. The leaf sheath, glume, and scale leaf had a modified C4 anatomy with MCs more than two cells distant from the Kranz cells; DCs were relatively rare. An immunocytochemical study of C3 and C4 enzymes revealed that all the organs exhibited essentially the same C4 pattern of expression as in the leaf blade. In the scale leaf, however, intense expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in the MCs as well as in the BSCs and DCs. In the leaf sheath, the distant MCs also expressed Rubisco. In Arundinella hirta, it seems that the ratio of MC to Kranz cell volumes, and the distance from the Kranz cells, but not from the veins, affects the cellular expression of photosynthetic enzymes. We suggest that the main role of DCs is to keep a constant quantitative balance between the MCs and Kranz cells, which is a prerequisite for effective C4 pathway operation.

Cellular expression of C3 and C4 photosynthetic enzymes in the amphibious sedge Eleocharis retroflexa ssp. chaetaria

The amphibious leafless sedge Eleocharis retroflexa ssp. chaetaria expresses C4-like biochemical characteristics in both the terrestrial and submerged forms. Culms of the terrestrial form have Kranz anatomy, whereas those of the submerged form have Kranz-like anatomy combined with anatomical features of aquatic plant leaves. We examined the immunolocalization of C3 and C4 enzymes in culms of the two forms. In both forms, phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase; and NAD-malic enzyme were compartmentalized between the mesophyll (M) and Kranz cells, but their levels were somewhat reduced in the submerged form. In the terrestrial form, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) occurred mainly in the Kranz cells, and weakly in the M chloroplasts. In the submerged form, the rubisco occurred at higher levels in the M cells than in the terrestrial form. In both forms, the C4 pattern of enzyme expression was clearer in the M cells adjacent to Kranz cells than in distant M cells. During the transition from terrestrial to submerged conditions, the enzyme expression pattern changed in submerged mature culms that had been formed in air before submergence, and matched that in culms newly developed underwater. It seems that effects of both environmental and developmental factors overlap in the C4 pattern expression in this plant.

Structure and immunocytochemical localization of photosynthetic enzymes in the lamina joint and sheath pulvinus of the C4 grass Arundinella hirta

The C4 grass Arundinella hirta exhibits a unique C4 anatomy, with isolated Kranz cells (distinctive cells) and C4-type expression of photosynthetic enzymes in the leaf sheath and stem as well as in the leaf blade. The border zones between these organs are pale green. Those between the leaf blade and sheath and between the sheath and stem are called the lamina joint and sheath pulvinus, respectively, and are involved in gravity sensing. We investigated the structure and localization of C3 and C4 photosynthetic enzymes in these tissues. In both zones the epidermis lacked stomata. The inner tissue was composed of parenchyma cells and vascular bundles. The parenchyma cells were densely packed with small intercellular spaces and contained granal chloroplasts with large starch grains. No C4-type cellular differentiation was recognized. Western blot analysis showed that the lamina joint and pulvinus accumulated substantial amounts of phosphoenolpyruvate carboxylase (PEPC), pyruvate,Pi dikinase (PPDK), and ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco). Immunogold electron microscopy revealed PEPC in the cytosol and both PPDK and rubisco in the chloroplasts of parenchyma cells, suggesting the occurrence of C3 and C4 enzymes within a single type of chlorenchyma cell. These data indicate that the lamina joint and pulvinus have unique expression patterns of C3 and C4 enzymes, unlike those in C4-type anatomy.