Virginia Stiefel

Molecular cloning of cDNAs encoding a putative cell wall protein from Zea mays and immunological identification of related polypeptides

Copy DNAs corresponding to a highly repetitive, proline-rich protein from maize have been cloned by differential screening of a coleoptile cDNA library. The deduced amino acid sequence contains a single repetitive element of carrot extensin (Ser-Pro-Pro-Pro-Pro). The related mRNAs have a defined distribution in tissues of the plant and are accumulated mainly in the coleoptile node and root tip. A peptide that corresponds to one of the repetitive elements of the protein has been synthesized and antisera have been obtained in rabbits. These antibodies react against crude preparations of coleoptile cell wall and against polypeptides extracted following the protocols described for the extraction of extensin. From these data it is concluded that the cDNAs correspond to a family of cell wall glycoproteins from maize.

Expression of genes for cell-wall proteins in dividing and wounded tissues of Zea mays L.

Hydroxyproline-rich glycoproteins (HRGPs) fromZea mays have been immunolocalized in the cell wall of root tip cells using ultrathin sections and antibodies ellicited against the purified protein. The accumulation of mRNA corresponding to this protein was studied using the cDNA probe. Maximum accumulation of the mRNA was found in tissues with a high proportion of dividing cells such as those in the root tip of young maize seedlings and a close relationship with cellular division was also observed in in-vitro cultures. However, the level of the mRNA in elongating tissues was minimal, as shown by studies carried out on the elongation zones of root tips and coleoptiles. The mRNA was induced by stress conditions, particularly by wounding young leaves and coleoptiles. It is concluded that in maize this group of proline-rich cell-wall proteins accumulates during cell division and not during cell elongation or differentiation, and participates in the stress-response mechanisms of the plant.

The maize caffeic acid O-methyltransferase gene promoter is active in transgenic tobacco and maize plant tissues

The pattern of expression directed by the promoter of the maize caffeic acid O-methyltransferase (COMT) gene was studied by histochemical and fluorometric β-glucuronidase (GUS) analysis in transgenic maize and tobacco plants. The COMT promoter directs GUS expression to the xylem and the other tissues undergoing lignification, and it responds to wounding and to elicitors. In transgenic maize plants, expression of GUS corresponds to the pattern of expression of the endogenous COMT gene as determined by northern analysis and in situ hybridization. The pattern in transgenic tobacco plants clearly shows that the maize promoter sequence is recognized by tobacco transcriptional factors, in spite of the anatomical differences and the evolutionary distance between these two species. The results suggest that the most significant promoter signals that induce the specific expression of the lignin COMT are conserved in different species.

Maize C4 and non-C4 NADP-dependent malic enzymes are encoded by distinct genes derived from a plastid-localized ancestor

NADP-dependent malic enzymes (NADP-ME; EC1.1.1.40) have been implicated in a wide range of metabolic pathways in the plastids and cytosol of plant cells. In maize, an NADP-ME type C4 plant, the most abundant NADP-ME form is the chloroplastic leaf isoform that delivers CO2 intracellularly to ribulose bisphosphate carboxylase (RuBPCase). A second NADP-ME isoform predominates in maize roots and exhibits distinct C3-like enzymatic characteristics. We show that the C3-like isoform is encoded by a pair of nearly identical genes that encode precursor proteins with functional chloroplast transit peptides. Using RT-PCR, we also show that the messages encoding the C4 and C3-like NADP-ME isoforms are differentially regulated with respect to the developmental stage of the leaf, light conditions, and tissue type. Based on these characteristics and on sequence comparison of ME families in other species, we propose a scheme for the origin of the C4-specific NADP-ME gene.

The direct activation of MIK, a germinal center kinase (GCK)-like kinase, by MARK, a maize atypical receptor kinase, suggests a new mechanism for signaling through kinase-dead receptors

Signaling by receptor protein kinases (RPKs) involves their dimerization and transphosphorylation. However, atypical RPKs with kinase-defective domains have been described recently. Some of them are essential for proper signaling in animal systems, although the precise mechanism involved is unknown in most cases. Here we describe the cloning and characterization of an atypical plant receptor kinase from maize, MARK, which does not phosphorylate in vitro. A yeast two-hybrid approach has allowed us to identify a new germinal center kinase (GCK)-related protein, MIK, that interacts with MARK. Interestingly, the interaction of the intracellular domain of MARK with the regulator domain of MIK strongly induces MIK kinase activity. As some GCK-related proteins connect cell-surface receptors to the intracellular MAPK cascades, the activation of MIK by direct interaction with MARK could illustrate a new mechanism for signaling through atypical RPKs.

Molecular characterization of the gene coding for GPRP, a class of proteins rich in glycine and proline interacting with membranes in Arabidopsis thaliana

The gene coding for a new class of proteins rich in glycine and proline (GPRP) was cloned in Arabidopsis thaliana. In the protein sequence, five amino acids-glycine, proline, alanine, tyrosine and histidine-account for 79.4% of the total composition. The protein has two different glycine-rich domains interrupted by a hydrophobic segment having a high probability of helix formation. The protein synthesized in vitro interacts with microsomes possibly through the hydrophobic domain. The gene in Arabidopsis has two introns, one in the coding region and the other one in the 5′ non-coding region. The later one is 778 bp long. Homologous sequences are found in carrot, tomato and tobacco. GPRP mRNA is found in the different organs of the plant analyzed except in mature seeds and anthers, and mostly in epidermal and vascular tissues. Possible hypotheses about the function of GPRP are discussed.

Characterization of the sequence coding for the clathrin coat assembly protein AP17 (σ2) associated with the plasma membrane from Zea mays and constitutive expression of its gene

The cDNA and genomic sequences coding for the clathrin coat assembly protein AP17 (sigma2) from maize and its corresponding mRNA accumulation have been analyzed. This protein in yeast and mammals has been shown to be part of the associated protein (AP) complex of clathrin in the plasma membrane. The availability of this sequence as well as a previous AP19 in a plant allows one to propose that specific AP complexes exist in plants in the Golgi complex and in the plasma membrane. The AP17 protein is encoded in maize by a single gene, and its mRNA accumulates in all the organs studied. In the immature embryo, it displays a pattern of expression typical of constitutively expressed genes.

TM20, a Gene Coding for a New Class of Transmembrane Proteins Expressed in the Meristematic Tissues of Maize

In the course of the analysis oflachrima, a recessive, defective kernel, embryo-lethal mutation in Zea mays that alters embryo and endosperm development, a gene coding for a new class of transmembrane proteins was isolated. The mutant was produced by Ac transposon tagging, and a gene located in the insertion region of the transposon was isolated as well as the corresponding cDNA. The predicted protein contains twenty hydrophobic segments that can be grouped in five repeats formed by four segments that fulfill the criteria for membrane spanning domains, and for this reason the gene has been named TM20. The sequences of the domains in each position of each group can be aligned, indicating that TM20 is formed by a four-domain structure duplicated five times. During embryogenesis in wild-type embryos and in the growing plant, TM20 gene expression is associated with meristems.

A new Arabidopsis nucleic-acid-binding protein gene is highly expressed in dividing cells during development

An Arabidopsis thaliana cDNA encoding a new RNA-binding protein (RBP37) was cloned from a silique cDNA library. The predicted amino acid sequence corresponds to a RBP containing two RNA recognition motifs (RRM) and a basic domain. An affinity for nucleic acids was confirmed in binding assays using in vitro synthesised AtRBP37 protein. In situ hybridisation experiments on sections of flowers and siliques showed expression only in growing organs: gynoecium, petals, filaments and during early-embryogenesis expression is located in the embryo proper and the suspensor up to late heart stage. Expression is not detected in the embryo during maturation.This results suggests an expression pattern correlated with dividing cells.

Regulation of the Expression of Genes Coding for Proline-Rich Proteins in Maize

Plant proline-rich proteins can be classified into several families depending on its function or structure (Jose and Puigdomenech, 1993). One of these families are the hydroxyproline-rich glycoproteins (HRGPs) where a large proportion of prolines are hydroxylated post-translationally and contain a variable proportion of glycosylated residues. HRGPs are one of the main extractable components of cell walls from both dicotyledonous (where they are often called extensins) and monocotyledonous species. Another group of genes coding for proline-rich proteins (PRPs) have been better characterized in soybean (Hong et al., 1987). Immunological methods have shown that maize HRGPs (Ludevid et al., 1990) and soybean PRPs (Ye et al., 1991) are located in the cell wall. In general, all these proteins are supposed to have a structural role in the wall (Cassab and Varner, 1988). Other proteins containing domains rich in proline, such as maize HyPRP, have also been described (Jose and Puigdomenech, 1992) but nothing is yet known about their location in the cell.