The apoplast of plant cells is a dynamic compartment involved in many processes, including maintenance of tissue shape, development, nutrition, signalling, detoxification and defence. In this work we used Nicotiana tabacum plants as a model to investigate changes in the soluble apoplast composition induced in response to salt stress. Apoplastic fluid was extracted from leaves of control plants and plants exposed to salt stress, using a vacuum infiltration procedure. Two-dimension electrophoretic analyses revealed about 150 polypeptide spots in the pH range of 3.0 to 10.0, in independent protein extracts, with a high level of reproducibility between the two sample sets. Quantitative evaluation and statistical analyses of the resolved spots in treated and untreated samples revealed 20 polypeptides whose abundance changed in response to salt stress. Mass spectroscopic peptide separation and sequencing was used to identify polypeptides affected by salt stress. While the levels of some proteins were reduced by salt-treatment, an enhanced accumulation of protein species known to be induced by biotic and abiotic stresses was observed. In particular, two chitinases and a germin-like protein increased significantly and two lipid transfer proteins were expressed entirely de novo. Some apoplastic polypeptides, involved in cell wall modifications during plant development, remained largely unchanged. The significance of these components is discussed in the context of stress responses in plants.

Maturing pea cotyledons accumulate large quantities of storage proteins at a specific time in seed development. To examine the sequences responsible for this regulated expression, a series of deletion mutants of the legA major seed storage protein gene were made and transferred to tobacco using the Bin19 disarmed Agrobacterium vector system. A promoter sequence of 97 bp including the CAAT and TATA boxes was insufficient for expression. Expression was first detected in a construct with 549 bp of upstream flanking sequence which contained the the leg box element, a 28 bp conserved sequence found in the legumin-type genes of several legume species. Constructs containing -833 and -1203 bp of promoter sequence significantly increased levels of expression. All expressing constructs preserved seed specificity and temporal regulation. The results indicate that promoter sequences between positions -97 and -549 bp are responsible for promoter activity, seed specificity, and temporal regulation of the pea legA gene. Sequences between positions -549 and -1203 bp appear to function as enhancer-like elements, to increase expression.

One of several genes coding for the major pea storage protein, legumin, has been completely sequenced. The sequence covers the whole of the transcribed region, plus 5' and 3' untranscribed sequences. The predicted protein sequence starts with a signal peptide and is followed by the legumin alpha polypeptide sequence of 36. 44kd and the beta polypeptide sequence of 20. 19kd . Compared to other legume storage proteins, the alpha and beta polypeptide sequences encoded by this legumin gene, which contain 3 met and 5 cys residues, are relatively rich in the sulphur amino acids. The coding sequence is interrupted by three introns which show boundary sequences typical of higher plant genes. The 5' end of the gene sequence contains a 'TATA box', a ' CAAT box' and a sequence showing some homology to an ' AGGA box'. An extra sequence, identical to the normal polyadenylation signal of the legumin message is seen in the 3' untranscribed region. The structure of the gene and the possible significance of secondary structures in the nascent RNA transcript in affecting the choice of polyadenylation site is discussed.

A third storage protein, distinct from legumin and vicilin, has been purified from the seeds of pea (Pisum sativum L.). This protein has been named 'convicilin' and is present in protein bodies isolated from pea seeds. Convicilin has a subunit mol.wt. of 71 000 and a mol.wt. in its native form of 290 000. Convicilin is antigenically dissimilar to legumin, but gives a reaction of identity with vicilin when tested against antibodies raised against both proteins. However, convicilin contains no vicilin subunits and may be clearly separated from vicilin by non-dissociating techniques. Unlike vicilin, convicilin does not interact with concanavalin A, and contains insignificant amounts of carbohydrates. Limited heterogeneity, as shown by isoelectric focusing, N-terminal analysis, and CNBr cleavage, is present in convicilin isolated from a single pea variety; genetic variation of the protein between pea lines has also been observed.

Investigations of the vicilin fraction of the storage proteins of pea (Pisum sativum L.) have shown that its major components are a number of protein species of M , 170000. Convicilin ( M , 280000, composed of 71 000-M, subunits) is a separable component of this fraction. The vicilin proteins are composed principally of z 50000-M, polypeptides, but also contain a number of smaller polypeptides. The sub-unit polypeptide composition of vicilin changes during seed development quantitatively and qualitatively. Vicilin sub-units have been shown to be synthesised as polypeptides of M , z 50000 by means of pulse-labelling experiments in vivo, and synthesis of vicilin in vitro directed by mRNA, polysomes and microsomes extracted from pea cotyledons in cell-free translation systems. Polypeptides then undergo two distinct types of proteolytic modification : (a) co-translational removal of a small polypeptide ( M , less than 1000); (b) 'nicking' of polypeptide chains in assembled vicilin molecules, which occurs more than 4 h after their initial synthesis. The basic structure of the vicilin molecule is thus a multimer, possibly a trimer, of z 50000-M, subunits. The heterogeneity of the initially synthesised SOOOO-M, subunits accounts not only for the several different SOOOO-M, polypeptides found in vicilin, but also for the range of minor polypeptides, since the 'nicking' points will differ among subunits. It also accounts for the observed partial separation of vicilin into different molecular species, since different subunit combinations will give rise to molecules with different properties. Vicilin is also glycosylated and this is a source of further variation.The storage proteins of pea (Pisum sativum L.) have been conventionally divided into two fractions, legumin and vicilin [l]. Depending on the variety, vicilin is usually the quantitatively smaller fraction and is thought to contain more than one protein [2], since it is heterogeneous when analysed in the ultracentrifuge [3] by ion-exchange chromatography [4] or when fractionated by solubility under varying conditions of pH, ionic strength and temperature [5,6] or by carbohydrate affinity chromatography [7]. The vicilin fraction contains major polypeptides of M ,