mRNA cap binding proteins: effects on abscisic acid signal transduction, mRNA processing, and microarray analyses.

The plant hormone abscisic acid (ABA) intricately regulates a multitude of processes during plant growth and development. Recent studies have established a connection between genes participating in various steps of cellular RNA metabolism and the ABA signal transduction machinery. In this chapter we focus on the plant nuclear mRNA cap binding proteins, CBP20 and CBP80. We summarize and report recent findings on their effects on cellular signal transduction networks and mRNA processing events. ABA hypersensitive 1 (abh1) harbors a gene disruption in the Arabidopsis CBP80 gene. Loss-of-function mutation of ABH1 can also result in an early flowering phenotype in the Arabidopsis accession C24. abh1 revealed noncoding cis-natural antisense transcripts (cis-NATs) at the CONSTANS locus in wild-type plants with elevated cis-NAT expression in the mutant. abh1 also revealed an influence on the splicing of the MADS box transcription factor Flowering Locus C pre-mRNA, which may result in the regulation of flowering time. Furthermore, new experiments analyzing complementation of cpb20 with site-directed cpb20 mutants provide evidence that the CAP binding activity of CBP20 is essential for the observed cbp-associated phenotypes. In conclusion, mutants in genes participating in RNA processing provide excellent tools to uncover novel molecular mechanisms for the regulation of RNA metabolism and of signal transduction networks in wild-type plants.

Genome-wide transcriptome profiling of the early cadmium response of Arabidopsis roots and shoots.

Transcriptional regulation in response to cadmium treatment was investigated in both roots and leaves of Arabidopsis, using the whole genome CATMA microarray containing at least 24,576 independent probe sets. Arabidopsis plants were hydroponically treated with low (5 microM) or high (50 microM) cadmium concentrations during 2, 6, and 30 hours. At each time point, Cd level was determined using ICP-AES showing that both plant tissues are able to accumulate the heavy metal. RT-PCR of eight randomly selected genes confirmed the reliability of our microarray results. Analyses of response profiles demonstrate the existence of a regulatory network that differentially modulates gene expression in a tissue- and kinetic-specific manner in response to cadmium. One of the main response observed in roots was the induction of genes involved in sulfur assimilation-reduction and glutathione (GSH) metabolism. In addition, HPLC analysis of GSH and phytochelatin (PC) content shows a transient decrease of GSH after 2 and 6 h of metal treatment in roots correlated with an increase of PC contents. Altogether, our results suggest that to cope with cadmium, plants activate the sulfur assimilation pathway by increasing transcription of related genes to provide an enhanced supply of GSH for PC biosynthesis. Interestingly, in leaves an early induction of several genes encoding enzymes involved in the biosynthesis of phenylpropanoids was observed. Finally, our results provide new insights to understand the molecular mechanisms involved in transcriptional regulation in response to cadmium exposure in plants.

An mRNA cap binding protein, ABH1, modulates early abscisic acid signal transduction in Arabidopsis.

The plant hormone abscisic acid (ABA) regulates important stress and developmental responses. We have isolated a recessive ABA hypersensitive mutant, abh1, that shows hormone specificity to ABA. ABH1 encodes the Arabidopsis homolog of a nuclear mRNA cap binding protein and functions in a heterodimeric complex to bind the mRNA cap structure. DNA chip analyses show that only a few transcripts are down-regulated in abh1, several of which are implicated in ABA signaling. Consistent with these results, abh1 plants show ABA-hypersensitive stomatal closing and reduced wilting during drought. Interestingly, ABA-hypersensitive cytosolic calcium increases in abh1 guard cells demonstrate amplification of early ABA signaling. Thus, ABH1 represents a modulator of ABA signaling proposed to function by transcript alteration of early ABA signaling elements.

Effects of targeted replacement of the tomatinase gene on the interaction of Septoria lycopersici with tomato plants.

Many plants produce constitutive antifungal molecules belonging to the saponin family of secondary metabolites, which have been implicated in plant defense. Successful pathogens of these plants must presumably have some means of combating the chemical defenses of their hosts. In the oat root pathogen Gaeumannomyces graminis, the saponin-detoxifying enzyme avenacinase has been shown to be essential for pathogenicity. A number of other phytopathogenic fungi also produce saponin-degrading enzymes, although the significance of these for saponin resistance and pathogenicity has not yet been established. The tomato leaf spot pathogen Septoria lycopersici secretes the enzyme tomatinase, which degrades the tomato steroidal glycoalkaloid alpha-tomatine. Here we report the isolation and characterization of tomatinase-deficient mutants of S. lycopersici following targeted gene disruption. Tomatinase-minus mutants were more sensitive to alpha-tomatine than the wild-type strain. They could, however, still grow in the presence of 1 mM alpha-tomatine, suggesting that nondegradative mechanisms of tolerance are also important. There were no obvious effects of loss of tomatinase on macroscopic lesion formation on tomato leaves, but trypan blue staining of infected tissue during the early stages of infection revealed more dying mesophyll cells in leaves that had been inoculated with tomatinase-minus mutants. Expression of a defense-related basic beta-1,3 glucanase gene was also enhanced in these leaves. These differences in plant response may be associated with subtle differences in the growth of the wild-type and mutant strains during infection. Alternatively, tomatinase may be involved in suppression of plant defense mechanisms.

Entry of Xanthomonas campestris pv. campestris into hydathodes of Arabidopsis thaliana leaves: a system for studying early infection events in bacterial pathogenesis.

Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen of cruciferous plants that normally gains entry to plants via hydathodes. In order to study the basis of the preference for this protal of entry we have developed an Arabidopsis thaliana model with attached or detached leaves partially immersed in a bacterial suspension. Entry of bacteria into leaves, assessed by resistance to surface sterilization, could be detected after 1 h. Dissection of leaves and histochemical staining for beta-glucuronidase produced by the bacteria indicated that they were located in hydathodes. In contrast, similar experiments with the leaf-spotting pathogen X. campestris pv. armoraciae gave patterns of localized staining dispersed over the leaf area, indicative of entry through stomata. A survey of 41 A. thaliana accessions showed that they fell into three classes distinguishable by total numbers of Xcc that entered under standard conditions and by preference for hydathode colonization. Previously isolated Xcc mutants affected in pathogenicity were tested for hydathode colonization: an hrp mutant behaved indistinguishably from the wild type, and rpf regulatory mutants gave 10-fold reduced colonization, whereas with rfaX mutants with altered lipopolysaccharide, few if any viable bacteria were recoverable from hydathodes. This fact, together with the rapid induction of superoxide dismutase in the bacteria located in hydathodes, suggests that an early defense reaction is mounted in the hydathode.

Induction by (alpha)-L-Arabinose and (alpha)-L-Rhamnose of Endopolygalacturonase Gene Expression in Colletotrichum lindemuthianum.

The production of endopolygalacturonase (endoPG) by Colletotrichum lindemuthianum, a fungal pathogen causing anthracnose on bean seedlings, was enhanced when the fungus was grown in liquid medium with L-arabinose or L-rhamnose as the sole carbon source. These two neutral sugars are present in plant cell wall pectic polysaccharides. The endolytic nature of the enzyme was demonstrated by its specific interaction with the polygalacturonase-inhibiting protein of the host plant as well as by sugar analysis of the products released from its action on oligogalacturonides. Additional characterization of the protein was achieved with an antiserum raised against the pure endoPG of the fungus. Induction by arabinose and rhamnose was more prolonged and led to a level of enzyme activity at least five times higher than that on pectin. Northern blot experiments showed that this effect was correlated to the induction of a 1.6-kb transcript. A dose-response study indicated that the endoPG transcript level was already increased at a concentration of each sugar as low as 2.75 mM in the medium and was maximum at 55 mM arabinose and 28 mM rhamnose. Glucose, the main plant cell wall sugar residue which is also present in the apoplast, prevented endoPG gene expression, partially when added to pectin at concentrations ranging from 5 to 110 mM and totally when added at 55 mM to arabinose. Inhibition by glucose of the rhamnose-induced endoPG was correlated to nonuptake of rhamnose. This is the first report that arabinose and rhamnose stimulate endoPG gene expression in a fungus. The possible involvement of these various sugars on endoPG gene expression during pathogenesis is discussed.