Cloning and characterization of full-length cDNA encoding cell-wall ...

Plant Physiol. (1995) 108: 873-874

Plant Gene Register

Cloning and Characterization of Full-Length cDNA Encoding Cell-Wall lnvertase from Maize' Savita Shanker, Reggie W. Salazar, Earl W. Taliercio, and Prem S. Chourey* Department of Plant Pathology (S.S., R.W.S., E.W.T., P.S.C.), and United States Department of Agriculture, Agricultural Research Service (E.W.T., P.S.C.), University of Florida, Gainesville, Florida 3261 1-0680 Invertases (P-fructofuranosidase, EC 3.2.1.26) have been studied in many organisms, including plants. At least two forms of the enzyme, soluble and particulate, are a common feature to a11 organisms. The soluble form is predominantly localized to vacuoles and cytoplasm and the particulate form, in a11 cases tested, is ionically bound to the cell wall (hence, cell-wall-bound form) and is readily extractable with high salt concentration (Sturm and Chrispeels, 1990; von Schaewen et al., 1990; Weil and Rausch, 1990). Each of the two forms of invertase is known to have severa1 isozymes (Jaynes and Nelson, 1971; Unger et al., 1994). Physiological role($ of invertases in various tissues is not well understood, although it is believed to play an important role in Suc mobilization between source and sink organs of the plant. In both tomato and tobacco transgenic plants (Dickinson et al., 1991; von Schaewen et al., 1990, respectively), expression of a chimeric invertase gene in apoplastic regions led to serious interruption in SUC export and much inhibition of growth. These data suggest that cell-wall invertase plays an important role in phloem loading. Consistent with this interpretation are the genetic data from cultivated and wild relatives of tomato that show that accumulation of Suc or hexoses in ripening fruits is entirely dependent on a monogenic trait related to invertase expression in this tissue (Chetelat et al., 1993). Similarly in maize (Zea mays L.), both cell-wall-bound and soluble forms of invertases play a rate-limiting role in the normal development of seed. In particular, a single-gene mutation miniature 1 (mn 1) seed marked by a loss of 80% of the seed weight is associated with invertase deficiency in developing seed (Miller and Chourey, 1992). Genetic data suggest that invertase deficiency is the causal basis of miniature seed phenotype (Miller and Chourey, 1992; Cheng and Chourey, 1994). At the molecular level, the most detailed analyses of invertases in plants are done in carrot (Sturm and

Table 1. Characteristics of the cDNA sequence encoding cell-wallbound invertase in Zea mays L. Organism: Zea mays L. Cenome Location: Nuclear genome, chromosome 5 long arm. Clone Type: cDNA, complete open reading frame. Gene Product, Function: Cell-wall @-fructosidase(EC 3.2.1.26), hydrolysis of SUC. Techniques: Maize Black Mexican Sweet cell-suspension culture cDNA library in AZap vector was screened using a heterologous probe constructed from cDNA clone representing Sorghum cell-wallbound invertase (Salazar and Chourey, 1992); in vivo excision; double-stranded plasmid dideoxy chain termination sequencing. Method of Identification: The deduced amino acid sequence of maize Inv-CW1 showed sequence identity of 57 and 58.3% with tobacco and carrot cell-wall invertases, respectively, and of 43.5% with carrot soluble invertases. In addition, the expressed protein in Escherichia coli cross-reacted with antibodies against carrot cellwall invertase, which identified a polypeptide of approximately 65 k D by western blot analysis. Gene Copy Number: Cenomic Southern blot analyses indicate a low copy number. Expression: Northern blot analyses on total and poly(AC) RNA from Black Mexican Sweet cell-suspension culture and developing kernal showed approximately 2.1 kb transcript in both samples. Features of cDNA Structure: Total length of 2149 bp, with an open reading frame encoding a polypeptide of 590 amino acids with a predicted molecular mass of 64.9 kD. The deduced protein contains two potential glycosylation sites (N-X-S/T) at N604-6'2 and "057-1065

Chrispeels, 1990; Ramloch-Lorenz et al., 1993; Unger et al., 1994). These studies have identified at least two genes encoding the two soluble forms and a single gene for a cell-wall-bound form in this plant. A11 three genes are believed to encode the corresponding preproinvertase sequences with signal peptides and N-terminal propeptides, and no significant homology is seen among the three polypeptides in the leader peptides (Unger et al., 1994). We report here the isolation and characterization of a full-length cDNA clone corresponding to the cell-wall form of invertase in maize. The specific details are shown in

This w o r k was in part supported by U.S. Department of Agriculture, National Research Initiative Competitive Grants Program grant 92001919 and was a cooperative investigation o f U.S. Department of Agriculture, Agricultural Research Service, and Institute o f Food and Agricultural Sciences, University of Florida (Agricultural Experiment journal series No. R-04416). * Corresponding author; e-mail psch8gnv.ifas.ufl.edu; fax

1-904 -392- 6532.

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Table I. Although this cDNA clone maps t o chromosome 5L as a single gene, Inv-CWZ, t w o additional cDNA clones from a developing kernel library h a v e been recently isolated with the Inv-CW1 clone. The present sequence anal-

yses a n d genomic Southern d a t a with these t w o clones suggest that there are a t least t w o additional CW-Inv nuclear genes in t h e maize genome, t h u s making it a small gene family (E.W. Taliercio, S. Shanker, and P.S. Chourey, unpublished data). ACKNOWLEDGMENTS

We are grateful to Drs. E. Dennis, M. Olive, and K. Singh for the Black Mexican Sweet cDNA library, to Dr. A. Sturm for the polyclonal antibodies against carrot CW invertase, and G. Davis for mapping in the maize genome. We are also thankful to the University of Florida DNA Sequencing Core Lab for sequencing DNA, especially to Dr. Ernesto Almira for his help during the project. Received December 20, 1994; accepted January 26, 1995. Copyright Clearance Center: 0032-0889/95/l08/0873/02. The GenBank accession number for the sequence reported in this article is U17695. LITERATURE ClTED

Cheng W-H, Chourey PS (1994) A rate-limiting role of invertases in seed development in maize (abstract No. 918). Plant Physiol 1 0 5 S-165

Plant Physiol. Vol. 108, 1995

Chetelat RT, Klann E, Deverna JW, Yelle S, Bennett AB (1993) Inheritance and genetic mapping of fruit sucrose accumulation in Lycopersicon chmielewskii. Plant J 4: 643-650 Dickinson CD, Atabella T, Chrispeels MJ (1991) Slow growth phenotype of transgenic tomato expressing apoplastic invertase. Plant Physiol 95: 420-425 Jaynes TA, Nelson OE (1971) Invertase activity in normal and mutant maize endosperms during development. Plant Physiol 47: 623-628 Miller ME, Chourey PS (1992) The maize invertase-deficient miniature-2 seed mutation is associated with aberrant pedicel and endosperm development. Plant Cell4: 297-305 Ramloch-Lorenz K, Knudsen S, Sturm A (1993) Molecular characterization of the gene for carrot cell wall P-fructosidase. Plant J 4: 545-554 Salazar RA, Chourey PS (1992). Isolation of a partia1 invertase clone from Sorghum cell suspension cultures (abstract No. 497). Plant Physiol 99: S-84 Sturm A, Chrispeels M (1990) cDNA cloning of carrot extracellular P-fructosidase and its expression in response to wounding and bacterial infection. Plant Cell 2: 1107-1119 Unger C, Hardegger M, Lienhard S, Sturm A (1994) cDNA cloning of carrot soluble acid P-fructofuranosidases and comparison with the cell wall isoenzyme. Plant Physiol 104: 1351-1357 von Schaewen A, Stitt M, Schmidt R, Sonnewald U, Willmitzer L (1990) Expression of yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influentes growth and phenotype of transgenic tobacco plants. EMBO J 9: 3033-3044 Weil M, Rausch T (1990) Cell wall invertase in tobacco. Plant Physiol94 1575-1581