Synergistic interaction of the two paralogous Arabidopsis genes LRX1 and LRX2 in cell wall formation during root hair development.

LRR-extensins (LRX) form a family of structural cell wall proteins containing a receptor-like domain. The functional analysis of Arabidopsis LRX1 has shown that it is involved in cell morphogenesis of root hairs. In this work, we have studied LRX2, a paralog of LRX1. LRX2 expression is mainly found in roots and is responsive to factors promoting or repressing root hair formation. The function of LRX1 and LRX2 was tested by the expression of a truncated LRX2 and different LRX1/LRX2 chimaeric proteins. Using complementation of the lrx1 phenotype as the parameter for protein function, our experiments indicate that LRX1 and LRX2 are functionally similar but show differences in their activity. Genetic analysis revealed that single lrx2 mutants do not show any defect in root hair morphogenesis, but synergistically interact with the lrx1 mutation. lrx1/lrx2 double mutants have a significantly enhanced lrx1 phenotype, resulting in frequent rupture of the root hairs soon after their initiation. Analysis of the root hair cell wall ultrastructure by transmission electron microscopy (TEM) revealed the formation of osmophilic aggregates within the wall, as well as local disintegration of the wall structure in the double mutant, but not in wild-type plants. Our results indicate that LRX1 and LRX2 have overlapping functions in root hair formation, and that they likely regulate cell morphogenesis by promoting proper development of the cell wall.

Glycine-rich proteins as structural components of plant cell walls.

Glycine-rich proteins (GRPs) have been found in the cell walls of many higher plants and form a third group of structural protein components of the wall in addition to extensins and proline-rich proteins. The primary sequences of GRPs contain more than 60% glycine. GRPs are localized mainly in the vascular tissue of the plant, and their coding genes provide an excellent system to analyze the molecular basis of vascular-specific gene expression. In French bean, the major cell wall GRP has been localized at the ultrastructural level in the modified primary cell wall of protoxylem. Immunological studies showed that it forms a major part of these highly extensible and specialized cell walls. Specific digestion of GRP1.8 from bean by collagenase suggests that it shares structural similarities with collagen. The protein is synthesized by living protoxylem cells as well as xylem parenchyma cells. After cell death, GRPs are exported from neighboring xylem parenchyma cells to the protoxylem wall, a rare example of protein transport between cells in plants. We propose that GRPs are part of a repair system of the plant during the stretching phase of protoxylem.

The chimeric leucine-rich repeat/extensin cell wall protein LRX1 is required for root hair morphogenesis in Arabidopsis thaliana.

In plants, the cell wall is a major determinant of cell morphogenesis. Cell enlargement depends on the tightly regulated expansion of the wall, which surrounds each cell. However, the qualitative and quantitative mechanisms controlling cell wall enlargement are still poorly understood. Here, we report the molecular and functional characterization of LRX1, a new Arabidopsis gene that encodes a chimeric leucine-rich repeat/extensin protein. LRX1 is expressed in root hair cells and the protein is specifically localized in the wall of the hair proper, where it becomes insolubilized during development. lrx1-null mutants, isolated by a reverse-genetic approach, develop root hairs that frequently abort, swell, or branch. Complementation and overexpression experiments using modified LRX1 proteins indicate that the interaction with the cell wall is important for LRX1 function. These results suggest that LRX1 is an extracellular component of a mechanism regulating root hair morphogenesis and elongation by controlling either polarized growth or cell wall formation and assembly.

Hydrophobic interactions of the structural protein GRP1.8 in the cell wall of protoxylem elements.

The glycine-rich structural protein GRP1.8 of French bean (Phaseolus vulgaris) is specifically localized in the modified primary cell walls of protoxylem elements. Continuous deposition of GRP1.8 into the cell walls during elongation growth of the plant suggests that GRP1.8 is part of a repair mechanism to strengthen the protoxylem. In this work, a reporter-protein system was developed to study the interaction of GRP1.8 with the extracellular matrix. Fusion proteins of a highly soluble chitinase with different domains of GRP1.8 were expressed in the vascular tissue of tobacco (Nicotiana tabacum), and the chemical nature of the interaction of these fusion proteins in the cell wall compartment was analyzed. In contrast with chitinase that required only low-salt conditions for complete extraction, the different chitinase/GRP1.8 fusion proteins were completely extracted only by a nonionic or ionic detergent, indicating hydrophobic interactions of GRP1.8. The same interactions were found with the endogenous GRP1.8 in bean hypocotyls. In addition, in vitro experiments indicate that oxidative cross-linking of tyrosines might account for the insolubilization of GRP1.8 observed in later stages of protoxylem development. Our data suggest that GRP1.8 forms a hydrophobic protein-layer in the cell wall of protoxylem vessels.

The FAR1 locus encodes a novel nuclear protein specific to phytochrome A signaling.

The phytochrome family of photoreceptors has a well-defined role in regulating gene expression in response to informational light signals. Little is known, however, of the early steps of phytochrome signal transduction. Here we describe a new Arabidopsis mutant, far1 (far-red-impaired response), which has reduced responsiveness to continuous far-red light, but responds normally to other light wavelengths. This phenotype implies a specific requirement for FAR1 in phyA signal transduction. The far1 locus maps to the south arm of chromosome 4, and is not allelic to photomorphogenic loci identified previously. All five far1 alleles isolated have single nucleotide substitutions that introduce stop codons in a single ORF. The FAR1 gene encodes a protein with no significant sequence similarity to any proteins of known function. The FAR1 protein contains a predicted nuclear localization signal and is targeted to the nucleus in transient transfection assays. This result supports an emerging view that early steps in phytochrome signaling may be centered in the nucleus. The FAR1 gene defines a new multigene family, which consists of at least four genes in Arabidopsis. This observation raises the possibility of redundancy in the phyA-signaling pathway, which could account for the incomplete block of phyA signaling observed in the far1 mutant.

Involvement of an ABC transporter in a developmental pathway regulating hypocotyl cell elongation in the light

In the dark, plant seedlings follow the skotomorphogenetic developmental program, which results in hypocotyl cell elongation. When the seedlings are exposed to light, a switch to photomorphogenetic development occurs, and hypocotyl cell elongation is inhibited. We have manipulated the expression of the AtPGP1 (for Arabidopsis thaliana P glycoprotein1) gene in transgenic Arabidopsis plants by using sense and antisense constructs. We show that within a certain light fluence rate window, overexpression of the AtPGP1 gene under the control of the cauliflower mosaic virus 35S promoter causes plants to develop longer hypocotyls, whereas expression of the gene in antisense orientation results in hypocotyls shorter than those occurring in the wild type. In the dark, hypocotyls of transgenic and wild-type plants are indistinguishable. Because the AtPGP1 gene encodes a member of the superfamily of ATP binding cassette-containing (ABC) transporters, these results imply that a transport process is involved in a hypocotyl cell elongation pathway active in the light. The AtPGP1 transporter is localized in the plasmalemma, as indicated by immunohistochemical techniques and biochemical membrane separation methods. Analysis of the AtPGP1 expression pattern by using reporter gene constructs and in situ hybridization shows that in wild-type seedlings, AtPGP1 is expressed in both the root and shoot apices.

Specific interaction of the tomato bZIP transcription factor VSF-1 with a non-palindromic DNA sequence that controls vascular gene expression.

The grp1.8 gene of French bean (Phaseolus vulgaris) is specifically expressed in vascular tissue and encodes a glycine-rich structural protein (GRP1.8) of the cell wall. Earlier promoter analysis had shown that a 28 bp fragment of the grp1.8 promoter (vs-1) confers vascular expression to heterologous minimal promoters and is bound by the tomato bZIP transcription factor VSF-1. Here, we analysed the interaction of VSF-1 with fragments of the vs-1 element and studied the molecular basis of specific binding both in the DNA sequence of the promoter element as well as in the protein. The minimal binding site of VSF-1 is a 9 bp, non-palindromic sequence with two non-identical half-sites and a central nucleotide which separates them. The amino acid sequence of the VSF-1 DNA-binding basic domain has a Lys at position -10 instead of a conserved Arg found in the other bZIP factors isolated so far. This lysine was found to be required for specific recognition of the non-palindromic binding site: a mutant VSF-1 with a Lys-to-Arg substitution at position -10 bound with higher affinity to a palindromic sequence than the wild-type protein. The minimal binding site of VSF-1 was sufficient and necessary to confer vascular-specific expression to a heterologous promoter in vivo. The vsf-1 promoter also showed vascular-specific expression in transgenic tobacco. The close similarity of these expression patterns suggests that VSF-1 is specifically involved in vascular expression of the grp1.8 gene in plants.

In vitro binding of the tomato bZIP transcriptional activator VSF-1 to a regulatory element that controls xylem-specific gene expression.

The bean grp1.8 gene is specifically expressed in vascular tissue. Monomers and multimers of a 28 bp regulatory element of the grp1.8 promoter (vs-1) specifically activated both the -82 CaMV 35S and the -76/grp1.8 minimal promoters in vascular tissue of transgenic tobacco plants. vs-1 partially overlaps with a negative regulatory element in the grp1.8 promoter that is necessary for restriction of gene expression to vascular tissue. Nuclear extracts from tobacco and tomato cells contain a factor that binds to vs-1 in vitro. To study the molecular basis of xylem-specific expression mediated by the vs-1 promoter element, a gene was isolated from tomato encoding a protein that binds to vs-1 in vitro. This protein, designated VSF-1, contains a bZIP motif close to the C-terminus. Mutated vs-1 elements were no longer bound by VSF-1 and also failed to activate the minimal -82 CaMV 35S promoter in vivo. Transient expression of VSF-1 in protoplasts stimulated vs-1 dependent activation of the -76/grp1.8 minimal promoter. Binding studies and use of a polyclonal antiserum against VSF-1 provided further evidence that vs-1 is a potential in vivo target site, as VSF-1 was a part of the observed complex formed between vs-1 and nuclear protein extract. vs-1 does not contain the 5'-ACGT-3' core sequence that is part of known plant bZIP protein binding sites or another palindromic sequence. Based on the unusual binding specificity and a characteristic amino acid sequence in the bZIP domain we propose that VSF-1 and the partially homologous PosF21, a bZIP protein from Arabidopsis, belong to a new family of plant bZIP proteins.