Role of the transcriptional regulator opaque2 in carbon partitioning between starch and proteins in the sink
--M. Maddaloni, G. Donini, F. Forlani, L. Stasse and M. Motto

The endosperm is considered the primary sink for carbon (C) and nitrogen (N) assimilates in maize. Starch and zein protein are major storage components in the endosperm sink that affect grain yield and nutritional quality. The synthesis of these two storage components requires sucrose and amino acids, which are provided by vegetative tissues. Previous studies indicate that the relative level of C and N in kernels may vary among maize hybrids. Moreover, evidence has suggested that the amount and the relative proportion of starch and proteins in the endosperm, i.e. the C/N ratio, are determined by the nutrient supply, the sink demand and the interaction between them (Balconi et al., Plant Sci. 73:1-9, 1991). The coordinate regulation of C and N supply is a subject of great interest because it is responsible for the accumulation of starch, protein and corresponding increases in dry weight of the kernel. However, the biochemical and physiological background of this relationship is complex and not fully understood.

The maize o2 locus, which is known by classical genetic and molecular studies to activate the 22 kD zein and b-32 genes in trans, encodes a protein which belongs to the basic/leucine zipper (bZIP) class of transcription factors (Lohmer et al., EMBO J. 10:617-624, 1991). We have recently shown that in transformed yeast cells the O2 protein could substitute for GCN4 protein, a yeast transcriptional activator of amino acids biosynthetic genes which are subjected to general amino acid control. It is conceivable that o2 may play a similar role in maize endosperm, namely that it regulates amino acids biosynthetic genes and/or genes involved in C partitioning between proteins and starch.

The gene encoding the cytosolic form of pyruvate orthophosphate dikinase (PPDK:EC2.7.9.1), which catalyzes the conversion of pyruvate, ATP and Pi to PEP, AMP, and PPi. Three genes have been described in maize by Sheen (Plant Cell 3:225-245, 1991): one encoding the C4, chloroplastic PPDK (C4PPDK) and two encoding cytosolic PPDK activities, cyPPDK1 and cyPPDK2, the last being poorly expressed in all tissues examined.

An assay for transient gene expression in tobacco protoplast has been employed to investigate the possible activation of two different PPDK promoters by the O2 product. The assay was based on cotransfection of tobacco mesophyll protoplasts with an expression and a reporter plasmid. The expression plasmid, pCaMVO2, consisted of the full length O2 cDNA placed as a transcriptional fusion under the control of the 35S gene promoter from CaMV. The reporter plasmids consisted of different PPDK gene promoters fused to the coding region of chloramphenicol acetyltransferase (CAT) gene. These constructs were generously provided by J. Sheen, Massachusetts General Hospital.

The results of the transient expression experiments showed that the promoter of the C4PPDK gene is not transactivated by O2, while the promoter of cyPPDK1 was strongly activated by O2, suggesting that cytosolic PPDK1 expression is under the control of O2.

Such observation is congruent with the hypothesis that O2 may be active in the diversion of C flux from sugars to amino acids, because PEP is generally believed to play a crucial role in amino acid biosynthesis. In fact, PEP can be carboxylated by cytosolic PEPcase to generate oxaloacetate, which is easily transformed into aspartate. This molecule is in turn the first compound for the synthesis of branched amino acids. PEP itself is also the first compound common to the aromatic amino acids biosynthetic pathway. Experiments are in press to assess the real impact of this diversion route in the in vivo partitioning of photosynthates. 

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