Rachel Baltz

Downregulation of a Pathogen-Responsive Tobacco UDP-Glc:Phenylpropanoid Glucosyltransferase Reduces Scopoletin Glucoside Accumulation, Enhances Oxidative Stress, and Weakens Virus Resistance

Plant UDP-Glc:phenylpropanoid glucosyltransferases (UGTs) catalyze the transfer of Glc from UDP-Glc to numerous substrates and regulate the activity of compounds that play important roles in plant defense against pathogens. We previously characterized two tobacco salicylic acid– and pathogen-inducible UGTs (TOGTs) that act very efficiently on the hydroxycoumarin scopoletin and on hydroxycinnamic acids. To identify the physiological roles of these UGTs in plant defense, we generated TOGT-depleted tobacco plants by antisense expression. After inoculation with Tobacco mosaic virus (TMV), TOGT-inhibited plants exhibited a significant decrease in the glucoside form of scopoletin (scopolin) and a decrease in scopoletin UGT activity. Unexpectedly, free scopoletin levels also were reduced in TOGT antisense lines. Scopolin and scopoletin reduction in TOGT-depleted lines resulted in a strong decrease of the blue fluorescence in cells surrounding TMV lesions and was associated with weakened resistance to infection with TMV. Consistent with the proposed role of scopoletin as a reactive oxygen intermediate (ROI) scavenger, TMV also triggered a more sustained ROI accumulation in TOGT-downregulated lines. Our results demonstrate the involvement of TOGT in scopoletin glucosylation in planta and provide evidence of the crucial role of a UGT in plant defense responses. We propose that TOGT-mediated glucosylation is required for scopoletin accumulation in cells surrounding TMV lesions, where this compound could both exert a direct antiviral effect and participate in ROI buffering.

The Arabidopsis DELLA RGA-LIKE3 Is a Direct Target of MYC2 and Modulates Jasmonate Signaling Responses

The DELLA proteins function as negative regulators of the gibberellin (GA) signaling pathway. This article reports that RGL3 is a distinct DELLA protein that acts as an integrating factor that links GA and jasmonate signaling to enable adaptive regulation of plant resistance to pathogens. Gibberellins (GAs) are plant hormones involved in the regulation of plant growth in response to endogenous and environmental signals. GA promotes growth by stimulating the degradation of nuclear growth–repressing DELLA proteins. In Arabidopsis thaliana, DELLAs consist of a small family of five proteins that display distinct but also overlapping functions in repressing GA responses. This study reveals that DELLA RGA-LIKE3 (RGL3) protein is essential to fully enhance the jasmonate (JA)-mediated responses. We show that JA rapidly induces RGL3 expression in a CORONATINE INSENSITIVE1 (COI1)– and JASMONATE INSENSITIVE1 (JIN1/MYC2)–dependent manner. In addition, we demonstrate that MYC2 binds directly to RGL3 promoter. Furthermore, we show that RGL3 (like the other DELLAs) interacts with JA ZIM-domain (JAZ) proteins, key repressors of JA signaling. These findings suggest that JA/MYC2-dependent accumulation of RGL3 represses JAZ activity, which in turn enhances the expression of JA-responsive genes. Accordingly, we show that induction of primary JA-responsive genes is reduced in the rgl3-5 mutant and enhanced in transgenic lines overexpressing RGL3. Hence, RGL3 positively regulates JA-mediated resistance to the necrotroph Botrytis cinerea and susceptibility to the hemibiotroph Pseudomonas syringae. We propose that JA-mediated induction of RGL3 expression is of adaptive significance and might represent a recent functional diversification of the DELLAs.

Two tobacco genes induced by infection, elicitor and salicylic acid encode glucosyltransferases acting on phenylpropanoids and benzoic acid derivatives, including salicylic acid

Two tobacco genes (TOGT) with homology to glucosyltransferase genes known to be induced by salicylic acid (SA) also responded rapidly to a fungal elicitor or to an avirulent pathogen. SA, although an efficient inducer, was shown not to be essential in the signal transduction pathway regulating TOGT gene expression during the resistance response. Recombinant TOGT proteins produced in Escherichia coli exhibited low, but significant, glucosyltransferase activity towards SA, but very high activity towards hydroxycoumarins and hydroxycinnamic acids, with glucose esters being the predominant products. These results point to a possible important function in defense of these glucosyltransferases in conjugating aromatic metabolites prior to their transport and cross‐linking to the cell wall.

A LIM motif is present in a pollen-specific protein.

We have recently described a sunflower cDNA sequence coding for a pollenspecific protein (SF3) with putative zinc finger domains (Baltz et al., 1992). In a more recent analysis we have found that these domains correspond to the conserved LIM motif identified so far only in a family of metal binding, cysteine-rich proteins from animais. This motif, n~55 amino acids long, is characterized by a unique organization of cysteine and histidine residues into two adjacent putative zinc fingers. LIM motif-containing proteins include developmental regulators such as the rat insulin gene enhancer binding protein ISL-1 (Karlsson et al., 1990), the Caenorhabdifis elegans proteins LIN-11 (Freyd et al., 1990) and MEC-3 (Way and Chalfie, 1988), the Drosophila APTEROUS protein (Cohen et al., 1992), the XenopusXLIM-1 protein (Tairaet al., 1992), and the mammalian oncoproteins TTG-1 and TTG-2 (also known as RHOMQ) of the rhombotin family (McGuire et ai., 1989; Boehm et al., 1990, 1991; Royer-Pokora et al., 1991). The mammalian cysteine-rich proteins CRlP (Birkenmeyer and Gordon, 1986), hCRP (Liebhaber et al., 1990; Wang et al., 1992), and ESP-l (Nalik et al., 1989), all of which are of yet unknown function, also contain LIM motifs. LIM motifs are found either alone (in CRIP, TTG-1, TTGP, ESP-1, and hCRP) or in association with a homeodomain (in MEC-3, ISL-1, LIM-11, XLIM-1, and APTEROUS). Figure 1 shows an alignment of the LIM motifs of the pollen-specific protein SF3 with those of the animal LIM proteins. ConSeNed residues are shown in bold type. Aclose examination of a number of semiconserved positions (see boxed residues) shows evidence for the existence of two subfamilies of LIM proteins: subfamily A, which includes SF3, hCRP, CRlP and ESP-1, and subfamily B, which comprises the seven other proteins. The most frequently occurring metalchelating residues in the potential zinc fingers are cysteines and histidines. However, in the majority of the LIM proteins, aspartate (D) is the last residue in the second finger (position 57). This is not necessarily surprising because aspartate has been identified as a metal-chelating residue in zinc-containing enzymes (Vallee and Auld, 1990). As potential zinc finger domains, the LIM motifs could be directly involved in DNA binding, although a possible role in protein-protein interactions has been

Molecular and expression analysis of a LIM protein gene family from flowering plants.

Abstract. LIM-domain proteins participate in important cellular processes in eukaryotes, including gene transcription and actin cytoskeleton organization. They are predominantly found in animals, but have also been identified in yeast and plants. Following the characterization of a LIM-domain protein in sunflower pollen, we carried out an extensive search for these proteins in flowering plants. We have isolated and studied cDNAs and/or genomic sequences for two novel LIM-domain proteins from sunflower, three from tobacco, and one from Arabidopsis. The plant proteins are structurally related to the cytoskeleton-associated CRP class of LIM proteins in animals, but show several distinctive features, including a second, atypical, LIM domain. We have performed comparative expression studies of these genes, as well as of one other gene from tobacco and two additional Arabidopsis genes whose sequences are available from databases. These studies, carried out by RT-PCR in the presence of gene-specific primers, showed that, in sunflower and tobacco, pollen grains and sporophytic tissues express different sets of LIM proteins. With the exception of one Arabidopsis gene – which has two introns – all the genes analyzed contain four introns at conserved positions, indicating that the ancestral gene from which the various copies evolved in higher plants already had this split structure.

AN EARLY SALICYLIC ACID-, PATHOGEN- AND ELICITOR-INDUCIBLE TOBACCO GLUCOSYLTRANSFERASE : ROLE IN COMPARTMENTALIZATION OF PHENOLICS AND H2O2 METABOLISM

Treatment of tobacco cell suspension cultures with a fungal elicitor of defense responses resulted in an early accumulation of the phenylpropanoid glucosyltransferase TOGT, along with the rapid synthesis and secretion of scopolin, the glucoside of scopoletin. Elicitor‐triggered extracellular accumulation of the aglycone scopoletin and of free caffeic and ferulic acids could only be revealed in the presence of diphenylene iodonium, an inhibitor of extracellular H2O2 production. Our results strongly support a role for TOGT in the elicitor‐stimulated production of transportable phenylpropanoid glucosides, followed by the release of free antioxidant phenolics into the extracellular medium and subsequent H2O2 scavenging.

Differential localization of the LIM domain protein PLIM-1 in microspores and mature pollen grains from sunflower

Abstract PLIM-1 is a LIM domain protein specifically expressed in pollen grains. Using two PLIM-1-specific monoclonal antibodies we studied its expression and intracellular location at various developmental stages of sunflower (Helianthus annuus L.) pollen. Our studies show that the protein appears at the microspore stage in a limited number of cytoplasmic bodies, becomes undetectable in bicellular pollen, and reappears in tricellular pollen grains in cortical patches particularly concentrated in the F-actin-enriched germination cones of the vegetative cell. The developmental stage-dependent, different location of the protein suggests a dual function during pollen development. While this function in microspore development remains obscure, the high concentration of PLIM-1 in the germination cones of mature pollen suggests that it participates in the germination process as well as in pollen tube growth.

The pollen-specific LIM protein PLIM-1 from sunflower binds nucleic acids in vitro

The protein PLIM-1 (formerly SF3) from sunflower is expressed exclusively in mature, free pollen. It contains two LIM domains associated with an acidic C-terminus comprising six copies of the pentapeptide motif (A,T,S) (E,D) TQN. We have expressed the pollen protein as well as some of its mutant forms inEscherichia coli and have used the bacterially produced proteins to study interactions with nucleic acids. Our studies show that the protein binds DNA and RNA in vitro to form large complexes, while mutant polypeptides containing either a single LIM domain or a destabilized first or second LIM domain do not. Although these data suggest that the biological function of PLIM-1 involves interactions with nucleic acids, its role in pollen development remains unclear.

Anther- and Pollen-Specific Gene Expression in Sunflower

Among the living organisms plants have a unique life cycle characterized by an alternation of diploid and haploid generations. The role of the diploid sporophytic generation is to produce, following meiosis, haploid spores that develop into haploid male or female gametophytes. Specific cells of the gametophytes then differentiate into male or female gametes which fuse to yield a diploid embryo that finally grows into mature sporophyte. In flowering plants, the diploid, sporophytic generation is the dominant phase, whereas the haploid gametophyte is microscopic and consists of a very reduced number of cells: two or three in the case of the male gametophyte and seven (one of which being binucleate) in the case of the female gametophyte. The haploid male gametophytes, or pollen grains, are produced in the anthers, while the female gametophytes, or embryo sacs, develop in the ovaries (anthers and ovaries are diploid, sporophytic structures). In higher plants, the development of the male and female gametophytes is therefore closely associated with the development of sporophytic tissues. This contrasts with the situation in lower plants where the gametophytes can develop and live as independent free organisms.