Differential expression of two genes for sucrose-phosphate synthase in sugarcane: molecular cloning of the cDNAs and comparative analysis of gene expression.

Two cDNA clones, pSoSPS1 and pSoSPS2, encoding sucrose-phosphate synthase (SPS) of sugarcane were isolated from a leaf cDNA library. Northern analysis revealed the transcript of SoSPS1 to be predominant in leaves, but that of SoSPS2 to be distributed not only in leaves but also in roots at a similar level. The transcript of SoSPS1 was markedly accumulated during greening of etiolated leaves, whereas that of SoSPS2 was constitutively expressed. These findings indicate that SPS in sugarcane is encoded by multiple genes, which show organ-specificity and are differentially regulated in response to light.

Glutamine Induces the N-Dependent Accumulation of mRNAs Encoding Phosphoenolpyruvate Carboxylase and Carbonic Anhydrase in Detached Maize Leaf Tissue.

We have used detached leaves to study the N-dependent control of expression of phosphoenolpyruvate carboxylase (PEPC) and carbonic anhydrase (CA) genes in maize (Zea mays L. cv Golden Cross Bantam T51). Following supplementation with an N-source and zeatin, PEPC and CA mRNA levels increased in leaves detached from N-deficient maize plants. Addition of methionine sulfoximine (MSX), a specific inhibitor of glutamine synthetase, inhibited the nitrate-dependent increase of PEPC and CA mRNA but did not affect the glutamine-dependent increase of PEPC and CA mRNA levels. Glutamine levels in detached maize leaves treated with various N sources in the presence or absence of MSX correlated with the levels of PEPC and CA mRNA. We conclude that glutamine is the most likely effector for controlling the N-dependent expression of PEPC and CA in maize plants.

Cytokinin Is Required to Induce the Nitrogen-Dependent Accumulation of mRNAs for Phosphoenolpyruvate Carboxylase and Carbonic Anhydrase in Detached Maize Leaves.

Previous studies with intact maize (Zea mays L.) plants indicated that phosphoenolpyruvate carboxylase (PEPC) levels are controlled by nitrogen (N) availability and that this regulation is presumably at the transcriptional level (B. Sugiharto, K. Miyata, H. Nakamoto, H. Sasakawa, T. Sugiyama [1990] Plant Physiol 92: 963-969; B. Sugiharto, T. Sugiyama [1992] Plant Physiol 98: 1403-1408). In the present study, detached maize leaves were used to investigate further the mechanism of N-dependent regulation of gene expression in C(4) plants. PEPC and carbonic anhydrase (CA) mRNA levels decreased in leaves detached from maize plants. Addition of high nitrate did not prevent this decrease. However, the addition of zeatin to solutions bathing the cut ends of the detached leaves inhibited the decrease of PEPC and CA mRNA levels. Simultaneous addition of high nitrate and zeatin to leaves detached from N-deficient maize plants caused a large and rapid increase in PEPC and CA mRNA levels. Zeatin could be replaced by benzyladenine, but not by indoleacetic acid or abscisic acid. Both CA isozymes were effected and responded in an identical manner. We conclude that detached maize leaves provide an excellent experimental system to study the mechanism(s) of N-mediated regulation of PEPC and CA gene expression. However, zeatin is an essential component of this system.

Effects of Nitrate and Ammonium on Gene Expression of Phosphoenolpyruvate Carboxylase and Nitrogen Metabolism in Maize Leaf Tissue during Recovery from Nitrogen Stress.

We previously showed that the selective accumulation of phosphoenolpyruvate carboxylase (PEPC) in photosynthetically maturing maize (Zea mays L.) leaf cells induced by nitrate supply to nitrogen-starved plants was primarily a consequence of the level of its mRNA (B Sugiharto, K Miyata, H Nakamoto, H Sasakawa, T Sugiyama [1990] Plant Physiol 92: 963-969). To determine the specificity of inorganic nitrogen sources for the regulation of PEPC gene expression, nitrate (16 millimolar) or ammonium (6 millimolar) was supplied to plants grown previously in low nitrate (0.8 millimolar), and changes in the level of PEPC and its mRNA were measured in the basal region of the youngest, fully developed leaves of plants during recovery from nitrogen stress. The exogenous supply of nitrogen selectively increased the levels of protein and mRNA for PEPC. This increase was more pronounced in plants supplemented with ammonium than with nitrate. The accumulation of PEPC during nitrogen recovery increased in parallel with the increase in the activity of glutamine synthetase and/or ferredoxin-dependent glutamate synthase. Among the major amino acids, glutamine was the most influenced during recovery, and its level increased in parallel with the steady-state level of PEPC mRNA for 7 hours after nitrogen supply. The administration of glutamine (12 millimolar) to nitrogen-starved plants increased the steady-state level of PEPC mRNA 7 hours after administration, whereas 12 millimolar glutamate decreased the level of PEPC mRNA. The results indicate that glutamine and/or its metabolite(s) can be a positive control on the nitrogen-dependent regulation of PEPC gene expression in maize leaf cells.

Regulation of expression of carbon-assimilating enzymes by nitrogen in maize leaf.

We have utilized the cellular differentiation gradient of the developed, youngest leaf to examine the regulation by nitrogen of levels of phosphoenolpyruvate carboxylase (PEPCase), pyruvate orthophosphate dikinase (PPDK), and ribulose 1,5-bisphosphate carboxylase in maize (Zea mays L.). The protein whose level regulated most preferentially by N availability was PEPCase, followed by PPDK, and the changes in level occurred most conspicuously at the photosynthetically maturing cells. Pulse and pulse-chase experiments to analyze photosynthetic fixation of [(14)C]CO(2) indicate that maize leaf primarily exploited a C(4)-mode of photosynthetic fixation of carbon dioxide even under a selective reduction in levels of these proteins. The effects of N on the synthesis of these proteins and the accumulation of corresponding mRNAs during recovery from a deficiency were examined by pulse and pulse-chase labeling with [(35)S]Met and by hybridization, respectively. The rate of turnover of PPDK was substantially higher than that of the other proteins. Results also showed that the reduced accumulation of PEPCase, as well as PPDK, under N deficiency could largely be accounted for a reduced level of synthesis of protein with a concomitant reduction in level of their mRNAs. This indicates that the N-dependent selective accumulation of these enzymes is primarily a consequence of level of its mRNAs.

deltaC Values in Maize Leaf Correlate with Phosphoenolpyruvate Carboxylase Levels.

Values of delta(13)C and levels of phosphoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/oxygenase were analyzed in segments from the fourth leaf of young maize (Zea mays L.) plants. The delta(13)C values became significantly more negative from the base to the tip of the leaves. Phosphoenolpyruvate carboxylase levels and ribulose bisphosphate carboxylase levels both increased from the base to the tip. The principal effect of phosphoenolpyruvate carboxylase levels or delta(13)C should arise through its effect on the carboxylation/diffusion balance in the mesophyll. In this case, delta(13)C values should become more negative as phosphoenolpyruvate carboxylase levels increase, unless there are offsetting changes in stomatal aperture. The principal effect of ribulose bisphosphate carboxylase/oxygenase on delta(13)C should occur through its effect on the extent of leakage of CO(2) from the bundle sheath cells. In this case, delta(13)C values should become more positive as ribulose bisphosphate carboxylase levels increase. Accordingly, the variation in delta(13)C values seen in maize leaves appears to be the result of variations in the level of phosphoenolpyruvate carboxylase.