Two low molecular weight pea seed albumins (Mr approximately 6000 and approximately 4000) have been characterized by protein, cDNA, and gene sequencing. Both proteins are encoded by separate regions of the same mRNA species. The initial translation product is a preproprotein from which a signal sequence is removed co-translationally. The resultant proprotein (PA1) is then cleaved post-translationally to yield the mature form of the two albumins (PA1a and PA1b). Comparison of cDNA and protein sequences suggests that at least four different PA1 genes are expressed in the pea genome. Both PA1a and PA1b have an unusually high cysteine content (7.5 and 16.2%, respectively). Pea seeds developing under suboptimal levels of sulfur nutrient supply contain reduced levels of PA1 mRNA and accumulate greatly reduced levels of PA1a and PA1b in the mature seed. In vitro transcription studies showed that this reduced level of PA1 mRNA resulted from reduced post-transcriptional stability rather than an altered rate of transcription of the PA1 gene. In contrast, during normal seed development, the level of PA1 mRNA seems to be under transcriptional control. Sequence comparisons reveal some homology between PA1 and a number of low molecular weight proteins from seeds of a wide range of mono- and dicotyledonous plants.

Cotyledons of developing pea seeds (pisum sativum L.) were labeled with radioactive amino acids and glucosamine, and extracts were prepared and separated into fractions rich in endoplasmic reticulum (ER) or protein bodies, The time-course of synthesis of the polypeptides of legumin and vicilin and the site of their assembly into protein oligomers were studied using immunoaffinity gels and sucrose density gradients. When cotyledons were pulse-labeled (1-2 h), newly synthesized vicilin was present as a series of polypeptides with M(r) 60,000-65,000, and newly synthesized vicilin was present as series of polypeptides with M(r) 75,000, 70,000, 50,000, and 49,000. These radioactive polypeptides were found primarily in the ER (Chrispeels et al., 1982, J Cell Biol., 93:5- 14). During a subsequent chase period, newly synthesized reserve proteins were initially present in the protein bodies in the above-named polypeptides. Between 1 and 20 h later, radioactive legumin subunits (M(r) 40,000 and 19,000) and smaller vicilin polypeptides (M(r) 34,000, 30,000, 25,000, 18,000, 14,000, 13,000, and 12,000) appeared in the protein bodies. The appearance of these labeled polypeptides in the protein bodies was not the result of a slow transport from the ER (or cytoplasm). Newly synthesized legumin and vicilin polypeptides were assembled into oligomers of 8S and 7S, respectively, in the ER. They appeared in the protein bodies in these oligomeric forms before the appearance of the smaller polypeptides (M(r) less than 49,000). These results indicate that the smaller vicilin polypeptides (M(r) less than 49,000) arise delayed posttranslational processing of some or all of the larger vicilin polypeptides. The precursors of legumin are completely processed in the protein bodies 2-3 h after their synthesis. The processing of the vicilin precursors is much slower (6-20 h) and only a fraction of the precursor molecules are processed. As a result both large (M(r) more than 49,000) and small polypeptides of vicilin accumulate in the protein bodies, whereas legumin accumulates only as polypeptides of M(r) 40,000 and 19,000.

Isolation of pea lectin from immature seeds, together with in vivo pulse-chase labeling and in vitro translation of RNA from such seeds, all gave results which indicated strongly that pea lectin is initially synthesized as a large precursor (pre-pro-form) (Mr = 25,000) which is cleaved both co- and post-translationally to yield the alpha and beta subunits of mature lectin. Two overlapping cDNA plasmids complementary to lectin mRNA were sequenced. They coded for both beta and alpha subunits of lectin, in the orientation beta leads to alpha from the NH2 terminus, together with a hydrophobic NH2-terminal leader sequence and included a 3' untranslated region of 124 nucleotides. In the appropriate reading frame, no stop codon was found between the coding sequences for the beta and alpha subunits, confirming the synthesis of pea lectin via a pre-pro-form. The entire pea lectin sequence showed a high degree of homology with the published amino acid sequences of lectins from lentil, broad bean, and, to a lesser extent, with concanavalin A from jack bean. Sequence variation occurred mainly in those regions thought not to be involved in metal- and sugar-binding or in the formation of beta-pleated sheets.

The postruminal supply of the sulfur-containing amino acids, methionine and cysteine, has been reported to be a major limitation to wool growth in sheep. We aim to improve the protein quality of forage for ruminants by introducing into alfalfa chimeric genes encoding a ruminally stable, sulfur amino acid-rich protein from sunflower seeds. Four gene constructs were transferred to Australian commercial cultivars of alfalfa using Agrobacterium tumefaciens-mediated transformation and selection with phosphinothricin (PPT). Modification of the sunflower seed albumin protein-coding region by addition of the coding information for an endoplasmic reticulum (ER) retention signal was found to greatly increase the level to which the sulfur amino acid-rich protein accumulated in the leaves of transgenic alfalfa plants. The Cauliflower Mosaic Virus (CaMV) 35S promoter and two light-regulated plant gene promoter regions were compared for their ability to direct high-level expression of the introduced genes in alfalfa leaves. The highest expression of sunflower seed albumin was found in transformants bearing a gene incorporating the promoter from the Arabidopsis thaliana ats1A gene, which encodes the ribulose bisphosphate carboxylase small subunit. The highest level of sunflower seed albumin found in transgenic alfalfa leaves was estimated to constitute .1% of soluble leaf protein. This level of accumulation of the foreign protein would be predicted to supply an extra 40 mg of sulfur amino acids daily to sheep fed the modified forage. Published studies in which wool growth rates were significantly increased employed supplementation of approximately 1 to 2 g of sulfur amino acids daily. Copyright © . .