SummaryAn isoform of starch synthase from potato tubers which is present both in the stroma of the plastid and tightly bound to starch granules has been identified biochemically and a cDNA has been isolated. The protein encoded by the cDNA is 79.9 kDa and has a putative transit peptide and a distinct N-terminal domain which is predicted to be highly flexible. It is similar in both amino acid sequence and predicted structure to the granule-bound starch synthase II (GBSSII) of pea embryos, When expressed in Eschen'chia coil, the mature protein has starch synthase activity.The importance of the isoform has been' assessed by biochemical measurements and antisense transformation experiments in which the amount of the isoform in the tuber is severely and specifically reduced. Both approaches indicate that the isoform contributes a maximum of 15% of the total starch synthase activity of the tuber. It is suggested that this isoform and the GBSSII of pea embryos represent a widely distributed class of isoforms of starch synthase. The contribution to total starch synthese activity of members of this class probably varies considerably from one type of storage organ to another.

The major isoform of starch synthase from the soluble fraction of developing potato tubers has been purified and used to prepare an antibody and isolate a cDNA. The protein is 140 kD, and it is distinctly different in predicted primary amino acid sequence from other isoforms of the enzyme thus far described. lmmunoinhibition and immunoblotting experiments and analysis of tubers in which activity of the isoform was reduced through expression of antisense mRNA revealed that the isoform accounts for -80% of the activity in the soluble fraction of the tuber and that it is also bound to starch granules. Severe reductions in activity had no discernible effect on starch content or amylose-to-amylopectin ratio of starch in tubers. However, they caused a profound change in the morphology of starch granules, indicative of important underlying changes in the structure of starch polymers within the granule.

Mutants of Pisum sativum L. with seeds containing low‐amylose starch were isolated by screening a population derived from chemically mutagenized material. In all of the mutant lines selected, the low‐amylose phenotype was caused by a recessive mutation at a single locus designated lam. In embryos of all but one mutant line, the 59 kDa granule‐bound starch synthase (GBSSI) was absent or greatly reduced in amount. The granule‐bound starch synthase activity in developing embryos of the mutants was reduced but not eliminated. These results provide further evidence that amylose synthesis is unique to GBSSI. Other granule‐bound isoforms of starch synthase cannot substitute for this protein in amylose synthesis. Examination of iodine‐stained starch granules from mutant embryos by light microscopy revealed large, blue‐staining cores surrounded by a pale‐staining periphery. In this respect, the low‐amylose mutants of pea differ from those of other species. The differential staining may indicate that the structure of amylopectin varies between the core and peripheral regions.

SummaryRight-side-out plasma membrane vesicles isolated from Zea mays roots were used to study membrane potential (Av)-dependent Ca2+ transport. Membrane potentials were imposed on the vesicles using either K+ concentration gradients and valinomycin or SCNconcentration gradients, and the size of the imposed A v was measured with ['4C]tetraphenylphosphonium. Uptake of 45Ca2+ into the vesicles was stimulated by inside-negative Ay. The rate of transport increased to a maximum at a A y of about -80 mV and then declined at more negative Av. When extravesicular Ca2+ concentration was varied, uptake was maximal in the range 100-200 pM Ca2+. Neither dihydropyridine nor phenylalkylamine Ca2+ channel blockers had any effect on Ca2+ uptake but 30 pM ruthenium red was completely inhibitory with half maximal inhibition at 10-15 pM ruthenium red. Calcium transport was also inhibited by inorganic cations. Zn2+, Gd3+ and Mg2+ inhibited by a maximum of 30% while La3+, Nd3+ and Mn2+ inhibited by 70%. The inhibitory effects of La3+ and Gd3+ were additive. Lanthanum-insensitive Ca2+ tive Ca2+ transport was totally inhibited by 80 pM Gd3+ and showed maximum activity at a A~J of -60 mV, with less uptake at both higher and lower Av. Lanthanum and Gd3+ also inhibited Ca2+ uptake into protoplasts isolated from Zea roots and their individual and combined effects were similar in extent to those observed with plasma membrane vesicles. It is concluded that

Transgenic potatoes expressing reduced levels of granulebound starch synthase I (GBSSI) have been used to investigate whether the synthesis of amylose occurs at the surface of the starch granule or within the matrix formed by the synthesis and organization of amylopectin. Amylose in these potatoes is wholly or largely confined to a central region of the granule. Consequently this core region stains blue with iodine whereas the peripheral zone stains red. By making extensive measurements of the relative sizes of the granules and their blue-staining cores in tubers over a range of stages of development, we have established that the blue core increases in size as the granule grows. The extent of the increase in size of the blue core is greater in potatoes with higher levels of GBSSI. These data show that amylose synthesis occurs within the matrix of the granule, and are consistent with the idea that the space available in the matrix may be an important determinant of the amylose content of storage starches.

In this paper we provide further evidence about the nature of a 77-kD starch synthase (SSII) that is both soluble and bound to the starch granules in developing pea (Pisum sativum L.) embryos. Mature SSll gives rise to starch synthase activity when expressed in a strain of Escherichia coli lacking glycogen synthase. In transgenic potatoes (Solanum tuberosum 1.) expressing SSII, the protein is both soluble and bound to the starch granules. These results confirm that We have reported the presence of a 77-kD isoform of starch synthase (referred to as SSII) in developing pea (Pisum sativum L.) embryos both in the stroma of the amyloplast and tightly bound to starch granules (Smith, 1990;Denyer and Smith, 1992: Dry et al., 1992; Denyer et al., 1993). This protein was identified as a granule-bound starch synthase on the grounds that it co-purified with activity solubilized from purified starch with a-amylase, that the solubilized activity was inhibited with an antiserum raised to the protein (referred to as the SSII antiserum), and that the protein had starch synthase activity after renaturation from SDS-polyacrylamide gels of granulebound proteins extracted from starch with SDS (Smith, 1990; Denyer et al., 1993). Evidence that the protein is also soluble in vivo was provided by the co-purification of soluble activity from developing embryos with a 77-kD protein. This protein was strongly recognized by the antiserum raised to the granule-bound isoform and was iden-

Ovine oestrus-associated oviducal glycoprotein (oEGP) is synthesized and secreted specifically by the ampullary region of the ovine oviduct during the peri-ovulatory stages of the oestrous cycle. A cDNA that encodes oEGP was isolated and sequenced. Isolation of oEGP was achieved using the polymerase chain reaction (PCR) with primers based on a bovine oestrus-associated oviducal glycoprotein cDNA (bOGP) sequence. A 1599-bp cDNA encodes, in part, a deduced 519-amino acid sequence of mature protein which carries two potential N-linked glycosylation sites. The deduced amino acid sequence is more than 95% identical to that of bOGP and more than 74% identical to the first 491 amino acids of human oestrogen-dependent oviducal glycoprotein (hOGP). Northern blot hybridizations of RNA from several sheep tissues detected mRNA (2.4 kb) only in an ampulla oviduct sample.