A Tobacco cDNA Coding for Cell-Wall Invertase

The CWI of higher plants is a structurally well-characterized basic protein (pI approximately 9.5) showing a complex glycosylation pattern. CWIs of severa1 species have been characterized at the protein leve1 (Laurière et al., 1988; Weil and Rausch, 1994, and refs. cited therein). Recently, complete cDNA clones have been obtained for CWIs of carrot (Sturm and Chrispeels, 1990), Arabidopsis thaliana (Schwebel-Dugué et al., 1994), and potato (Hedley et al., 1993). Sequencing of vacuolar invertase isoform cDNAs of different plant species has revealed high homology to CWIs; however, upon alignment of protein sequences, vacuolar invertase isoforms form a clearly separated group (Unger et al., 1994, and refs. cited therein). In contrast to the detailed knowledge about the structure of CWI, its physiological functions are not entirely clear. CWI has been proposed to play a role in phloem unloading during (a) seed development in maize (Miller and Chourey, 19921, (b) gravistimulation of oat shoot pulvinus (Wu et al., 1993), and (c) assimilate accumulation in tobacco (Nicotiana tabacum L.) crown gall tumors (Weil and Rausch, 1990). Apart from its putative role in phloem unloading (Eschrich, 1980) CWI may be involved in osmotic adjustment (regulation of apoplastic water potential) and pathogen defense (Sturm and Chrispeels, 1990). With the aim to characterize further the role of CWI for the growth of transformed suspension-cultured cells and tobacco crown gall tumors (Weil and Rausch, 1990, 1994) we have cloned a cDNA from a tobacco cell culture (Table I). For the tobacco CWI clone, a hydrophobic stretch of 15 amino acids, located near the N terminus of the open reading frame (position 9-23), may represent part of the putative signal sequence for entry into the ER (Sturm and Chrispeels, 1990). In carrot the mature protein starts with Ser49, i.e. 18 or 10 amino acids downstream of the two putative cleavage sites, suggesting a posttranslational remova1 of a prosequence (Sturm and Chrispeels, 1990). It is noteworthy that this Ser residue is conserved in a11 CWIs sequenced thus far. If the corresponding Ser residue 41 in tobacco CWI represents the N-terminal amino acid of the mature protein, the processed polypeptide will have a molecular mass of 61 kD, as has been previously determined for the completely deglycosylated protein by SDSPAGE, and a pI of 9.51, identical with the value obtained by IEF of native CWI (Weil and Rausch, 1994). Among a11