The spatial and temporal distribution of sucrose synthase (RSuS) in rice (Oryza sativa L.) was studied by Western and immunohistochemical analyses using the monospecific antibodies for three RSuS isoforms. In leaf tissues, RSuS1 was localized in the mesophyll while RSuS2 was in the phloem in addition to the mesophyll. In the roots, only RSuS1 was found in the phloem. No RSuS3 could be detected in any parts of etiolated seedlings. The expression of each RSus gene is closely linked to the seed development. RSuS1 was present in the aleurone layers of developing seeds, and at a low level in endosperm cells. RSuS2 was evenly distributed in seed tissues other than the endosperm. RSuS3 was localized predominantly in the endosperm cells. The tissue specific localizations of the three gene products suggest that RSuS1 plays a role in sugar transport into endosperm cells where the reaction catalyzed by RSuS3 provides the precursor of starch synthesis. RSus2, which is ubiquitously expressed, may play a housekeeping role.

Phenylalanine ammonia-lyase is the first enzyme of general phenylpropanoid pathway. A PAL gene, designated as BoPAL1, was cloned from a Bambusa oldhamii cDNA library. The open reading frame of BoPAL1 was 2,139 bp in size and predicted to encode a 712-amino acid polypeptide. BoPAL1 was the first intronless PAL gene found in angiosperm plant. Several putative cis-acting elements such as P box, GT-1motif, and SOLIPs involved in light responsiveness were found in the 5'-flanking sequence of BoPAL1 which was obtained by TAIL-PCR method. Recombinant BoPAL1 protein expressed in Pichia pastoris was active. The optimum temperature and pH for BoPAL1 activity was 50°C and 9.0, respectively. The molecular mass of recombinant BoPAL1 was estimated as 323 kDa using gel filtration chromatography and the molecular mass of full-length BoPAL was about 80 kDa, indicating that BoPAL1 presents as a homotetramer. The Km and kcat values of BoPAL1 for L-Phe were 1.01 mM and 10.11 s(-1), respectively. The recombinant protein had similar biochemical properties with PALs reported in other plants.

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Sweet potato (Ipomoea batatas) starch phosphorylase cDNA clones were isolated by screening an expression library prepared from the young root poly(A)+ RNA successively with an antiserum, a monoclonal antibody, and a specific oligonucleotide probe. One cDNA clone had 3292 nucleotide residues in which was contained an open reading frame coding for 955 amino acids. This sequence was compared with those of potato (916 residues plus 50-residue putative transit peptide) and rabbit muscle (841 residues) phosphorylases. The sweet potato phosphorylase has an overall structural feature highly homologous to that reported for potato phosphorylase, in conformity with the finding that they belong to the same class of plant phosphorylase. High divergencies of the two enzymes are found in the about 70 residue N-termini each including a putative transit peptide, and the midchain 78 residue insert typical of type I plant phosphorylase. We consider that the very high dissimilarity found in the midchain inserts is related to the difference in proteolytic lability of the two plant phosphorylases. Some structural features of the cDNA clone were also discussed.The biochemical mechanism of starch synthesis in plants, at least in the photosynthetic tissue, has been shown to center around ADPG pyrophosphorylase (17). SP2 has been regarded as a starch degrading enzyme in the diurnal starch accumulation cycle ofchloroplasts (2) although a synthetic role played by it in the starch accumulating amyloplast has not been ruled out (3). We have shown that, in the young sweet potato root, the SP protein content is proportional to the starch content that ranges from 7 to 23% in about 60 varieties (7), and that SP and ,B-amylase, the two enzymes that share a common substrate amylose, are both localized in the starch accumulating amyloplast, and the latter is a noncompetitive inhibitor of the former (5,6,16 might play a hitherto unknown regulatory role in that pathway. The sweet potato SP has multiple forms (5) and shows a complex pattern of subunits on SDS-PAGE analyses. These are attributed to the presence of heterologous subunits as well as the persistent contamination of inhibitor-resistant protease(s) in the enzyme preparation. Because of this situation, we have not been able to obtain pure peptide fragments for amino acid sequencing. Of all plant SP, the only known total amino acid sequence has been that of potato established by the protein sequencing (15). Recently, the presence of a putative transit peptide in the potato SP sequence was deduced from a partial cDNA sequence, and a role ofthe transit peptide in the growth associated change in enzyme distribution was suggested (4). Apparently, we need more information on the primary structure of SP from different sources for elucidating the mechanisms of SP-,-amylase interaction, SP protein metabolism in vivo, etc. But for an enzyme that is not easily isolated intact such as sweet potato SP, cDNA sequencing is apparently the method of choice. We report here the base sequence of a cDNA c...