The WRKY Transcription Factor Family in Citrus: Valuable and Useful Candidate Genes for Citrus Breeding.

WRKY transcription factors belong to a large family of plant transcriptional regulators whose members have been reported to be involved in a wide range of biological roles including plant development, adaptation to environmental constraints and response to several diseases. However, little or poor information is available about WRKY's in Citrus. The recent release of completely assembled genomes sequences of Citrus sinensis and Citrus clementina and the availability of ESTs sequences from other citrus species allowed us to perform a genome survey for Citrus WRKY proteins. In the present study, we identified 100 WRKY members from C. sinensis (51), C. clementina (48) and Citrus unshiu (1), and analyzed their chromosomal distribution, gene structure, gene duplication, syntenic relation and phylogenetic analysis. A phylogenetic tree of 100 Citrus WRKY sequences with their orthologs from Arabidopsis has distinguished seven groups. The CsWRKY genes were distributed across all ten sweet orange chromosomes. A comprehensive approach and an integrative analysis of Citrus WRKY gene expression revealed variable profiles of expression within tissues and stress conditions indicating functional diversification. Thus, candidate Citrus WRKY genes have been proposed as potentially involved in fruit acidification, essential oil biosynthesis and abiotic/biotic stress tolerance. Our results provided essential prerequisites for further WRKY genes cloning and functional analysis with an aim of citrus crop improvement.

Molecular physiology of aquaporins in plants.

In plants, membrane channels of the major intrinsic protein (MIP) super-family exhibit a high diversity with, for instance, 35 homologues in the model species Arabidopsis thaliana. As has been found in other organisms, plant MIPs function as membrane channels permeable to water (aquaporins) and in some cases to small nonelectrolytes. The aim of the present article is to integrate into plant physiology what has been recently learned about the molecular and functional properties of aquaporins in plants. Exhaustive compilation of data in the literature shows that the numerous aquaporin isoforms of plants have specific expression patterns throughout plant development and in response to environmental stimuli. The diversity of aquaporin homologues in plants can also be explained in part by their presence in multiple subcellular compartments. In recent years, there have been numerous reports that describe the activity of water channels in purified membrane vesicles, in isolated organelles or protoplasts, and in intact plant cells or even tissues. Altogether, these data suggest that the transport of water and solutes across plant membranes concerns many facets of plant physiology. Because of the high degree of compartmentation of plant cells, aquaporins may play a critical role in cell osmoregulation. Water uptake in roots represents a typical process in which to investigate the role of aquaporins in transcellular water transport, and the mechanisms and regulations involved are discussed.

Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria.

Cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) catalyses the conversion of cinnamoyl-CoAs into their corresponding cinnamaldehydes, i.e. the first step of the phenylpropanoid pathway specifically dedicated to the monolignol biosynthetic branch. In previous work, we described the isolation and characterisation of the first cDNA encoding CCR in Eucalyptus (Lacombe, E., Hawkins, S., Van Dorsselaere, J., Piquemal, J., Goffner, D., Poeydomenge, O., Boudet, A.M., Grima-Pettenati, J., 1997. Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant Journal 11, 429--441) and shown the role of this enzyme in controlling the carbon flux into lignins (Piquemal, J., Lapierre, C., Myton, K., O'Connell, A., Schuch, W., Grima-Pettenati, J., Boudet, A.M., 1998. Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants. Plant Journal 13, 71--83). Here, we report the characterisation of two functionally and structurally distinct cDNA clones, AtCCR1 and AtCCR2 (81.6% protein sequence identity) in Arabidopsis thaliana. The two recombinant proteins expressed in Escherichia coli are able to use the three cinnamoyl-CoAs tested but with different levels of efficiency. AtCCR1 is five times more efficient with feruloyl-CoA and sinapoyl-CoA than AtCCR2. In addition, the two genes are differentially expressed during development and in response to infection. AtCCR1 is preferentially expressed in tissues undergoing lignification. In contrast, AtCCR2, which is poorly expressed during development, is strongly and transiently induced during the incompatible interaction with Xanthomonas campestris pv. campestris leading to a hypersensitive response. Altogether, these data suggest that AtCCR1 is involved in constitutive lignification whereas AtCCR2 is involved in the biosynthesis of phenolics whose accumulation may lead to resistance.

Two differentially regulated Arabidopsis genes define a new branch of the DFR superfamily.

Two tandem genes were identified on Arabidopsis chromosome II (AtCRL1 and AtCRL2) encoding proteins with homology to members of the dihydroflavonol-4-reductase (DFR) superfamily. The encoded CRL1 and CRL2 proteins share 87% mutual amino acid sequence identity whereas their promoter regions are highly divergent, suggesting differential regulation of the CRL genes. Phylogenetic analysis placed CRL1 and CRL2 in a separate branch of the DFR superfamily. Northern blotting showed strong AtCRL1 induction by abscisic acid (ABA), drought, and heat shock, and high expression level in seeds, thus resembling the expression pattern of late embryogenic abundant ABA-responsive genes. Differential expression of the two genes during plant development was confirmed in plants expressing transcriptional fusions between the two promoters and the Escherichia coli beta-glucuronidase reporter gene. This showed that, whereas high expression of AtCRL1 in mature seeds declines during subsequent vegetative growth, transcriptional activity from the AtCRL2 promoter increases during vegetative growth. Expression of both genes is restricted to vascular tissue. Based upon their homology to proteins involved in lignin synthesis, we propose that AtCRL2 is involved in generating conducting tissue late in development, while AtCRL1 is involved in vascular tissue differentiation and/or synthesis in the germinating embryos.

Immunolocalization of cinnamyl alcohol dehydrogenase 2 (CAD 2) indicates a good correlation with cell-specific activity of CAD 2 promoter in transgenic poplar shoots.

Cinnamyl alcohol dehydrogenase 2 (CAD 2) localization and the cell-specific activity of the eucalyptus CAD 2 promoter were investigated by CAD 2 immunogold localization and promoter beta-glucuronidase (GUS) histochemistry in apical and mature parts of stable transformed poplar (Populus tremula x P. alba) stems. Both CAD 2 protein and GUS activity were found to be confined in the same types of cells in the shoot apices, particularly in the determined meristematic cells in leaf axils and shell zones, procambium and developing tracheids. Within mature stems, CAD 2 and GUS were also identified in cambium and in fully or partially lignified cells derived from it (young xylem, developing phloem fibres, chambered parenchyma cells around phloem). Additionally, GUS activity was found in the scale leaves of apical shoot buds and in the roots (namely in the procambium, cambium, phellogen, young xylem, pericycle) of transformed plants. By employing immunogold cytochemistry, CAD 2 was shown to be localized in the cytoplasm within cambial, ray and young xylem cells in stems, the gold particles being randomly attached to endoplasmic reticulum and Golgi-derived vesicles. These results support a crucial role for CAD 2 in lignification and indicate a new role for this enzyme in branching events within the shoot apex and during lateral root formation.

Cinnamyl Alcohol Dehydrogenase: Identification of New Sites of Promoter Activity in Transgenic Poplar.

Stem sections from poplar that were stably transformed with a eucalypt cinnamyl alcohol dehydrogenase promoter-[beta]-glucuronidase construct were prepared by using either a technique routinely used in herbaceous species or a technique designed to take into account the particular anatomy of woody plants. Although both preparation techniques confirmed the pattern of expression previously observed (C. Feuillet, V. Lauvergeat, C. Deswarte, G. Pilate, A. Boudet and J. Grima-Pettenati [1995] Plant Mol Biol 27: 651-657), the latter technique also allowed the detection of other sites of promoter activity not revealed by the first technique. In situ hybridization confirmed the expression pattern obtained with the second sample preparation technique.

Site-directed mutagenesis of a serine residue in cinnamyl alcohol dehydrogenase, a plant NADPH-dependent dehydrogenase, affects the specificity for the coenzyme.

Using recombinant cinnamyl alcohol dehydrogenase isoform 2 (CAD2, EC 1.1.1.195), an NADPH-dependent aromatic alcohol dehydrogenase involved in lignification in vascular plants, we have investigated the detailed steady-state kinetic mechanism of CAD2 and the role of a serine residue in determining the cofactor specificity of CAD2. Site-directed mutagenesis (S212D) and overexpression of the WT and mutant S212D forms of CAD2 in Escherichia coli, followed by kinetic studies on the purified WT and mutant proteins, confirmed the involvement of S212D in recognizing the phosphate group of NADPH and provided information on the structural requirements for NADPH specificity. From substrate kinetic patterns and product inhibition studies both WT and S212D mutant forms of CAD2 have been shown to follow rapid equilibrium random bireactant kinetics with the value of the interaction factor (alpha) for WT (0.25) being significantly less than that for S212D CAD2 (0.45). The changes in binding energy arising from the mutation on the binding of the 2'-phosphate site of the coenzyme were assessed. A marked degree of physical interaction was detected between the enzymatic binding sites of the coniferyl alcohol substrate and the 2'-phosphate binding region, which are quite distant in the three-dimensional structure. The inhibition by 2',5'-ADP and 5'-AMP was found to be weak for both WT and S212D CAD2. Strong substrate inhibition was detected for CAD2, and its implications for plant physiological studies were assessed.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue- and cell-specific expression of a cinnamyl alcohol dehydrogenase promoter in transgenic poplar plants.

Cinnamyl alcohol dehydrogenase (CAD) which catalyses the synthesis of the cinnamyl alcohols, the immediate precursors of lignins, from the corresponding cinnamaldehydes is considered to be a highly specific marker for lignification. We have isolated and characterized a CAD genomic clone from eucalyptus, a woody species of economic importance. The full-length promoter (EuCAD, 2.5 kb) and a series of 5' deletions were fused to the beta-glucuronidase (GUS) reporter gene. These constructs were tested in a homologous transient expression system of eucalyptus protoplasts which enabled the identification of several regions involved in transcriptional control. In order to study the spatial and developmental regulation of the CAD gene, the chimeric gene fusion (EuCAD-GUS) was then transferred via Agrobacterium tumefaciens-mediated transformation into poplar, an easily transformable woody angiosperm. Quantitative fluorometric assays conducted on eight independent in vitro transformants showed that GUS activity was highest in roots followed thereafter by stems and leaves. Histochemical staining for GUS activity on both in vitro primary transformants and more mature greenhouse-grown plants indicated a specific expression in the vascular tissues of stems, roots, petioles and leaves. At the onset of xylem differentiation, GUS activity was detected in parenchyma cells differentiating between the xylem-conducting elements. After secondary growth has occurred, GUS activity was localized in xylem ray cells and parenchyma cells surrounding the lignified phloem and sclerenchyma fibers. This first characterization of a woody angiosperm CAD promoter provides functional evidence for the role of CAD in lignification and suggests that parenchyma cells expressing CAD may provide lignin precursors to the adjacent lignified elements (vessels and fibres).