Characterization of a ribonuclease III-like protein required for cleavage of the pre-rRNA in the 3'ETS in Arabidopsis.

Ribonuclease III (RNaseIII) is responsible for processing and maturation of RNA precursors into functional rRNA, mRNA and other small RNA. In contrast to bacterial and yeast cells, higher eukaryotes contain at least three classes of RNaseIII, including class IV or dicer-like proteins. Here, we describe the functional characterization of AtRTL2, an Arabidopsis thaliana RNaseIII-like protein that belongs to a small family of genes distinct from the dicer family. We demonstrate that AtRTL2 is required for 3'external transcribed spacer (ETS) cleavage of the pre-rRNA in vivo. AtRTL2 localizes in the nucleus and cytoplasm, a nuclear export signal (NES) in the N-terminal sequence probably controlling AtRTL2 cellular localization. The modeled 3D structure of the RNaseIII domain of AtRTL2 is similar to the bacterial RNaseIII domain, suggesting a comparable catalytic mechanism. However, unlike bacterial RNaseIII, the AtRTL2 protein forms a highly salt-resistant homodimer that is only disrupted on treatment with DTT. These data indicate that AtRTL2 may use a dimeric mechanism to cleave double-stranded RNA, but unlike bacterial or yeast RNase III proteins, AtRTL2 forms homodimers through formation of disulfide bonds, suggesting that redox conditions may operate to regulate the activity of RNaseIII.

Plant thioredoxins and glutaredoxins: identity and putative roles.

Thioredoxins and glutaredoxins are ubiquitous proteins that reduce disulphide bridges of oxidized target proteins in vitro. In contrast to the situations in other organisms, phylogenic analysis has indicated that plant thioredoxins and glutaredoxins are present as multigenic families, and that thioredoxins have several subclasses. Thioredoxins and glutaredoxins are probably involved in similar physiological events - the major challenge is to identify their specific targets and establish the function of these proteins in vivo.

In vivo characterization of a thioredoxin h target protein defines a new peroxiredoxin family.

Disruption of the two thioredoxin genes in yeast dramatically affects cell viability and growth. Expression of Arabidopsis thioredoxin AtTRX3 in the Saccharomyces thioredoxin Delta strain EMY63 restores a wild-type cell cycle, the ability to grow on methionine sulfoxide, and H2O2 tolerance. In order to isolate thioredoxin targets related to these phenotypes, we prepared a C35S (Escherichia coli numbering) thioredoxin mutant to stabilize the intermediate disulfide bridged complex and we added a polyhistidine N-terminal extension in order to purify the complex rapidly. Expression of this mutant thioredoxin in the wild-type yeast induces a reduced tolerance to H2O2, but only limited change in the cell cycle and no change in methionine sulfoxide utilization. Expression in the Delta thioredoxin strain EMY63 allowed us to isolate a complex of the thioredoxin with YLR109, an abundant yeast protein related to PMP20, a peroxisomal protein of Candida. No function has so far been attributed to this protein or to the other numerous homologues described in plants, animals, fungi, and prokaryotes. On the basis of the complementation and of low similarity with peroxiredoxins, we produced YLR109 and one of its Arabidopsis homologues in E. coli to test their peroxiredoxins activity. We demonstrate that both recombinant proteins present a thioredoxin-dependent peroxidase activity in vitro. The possible functions of this new peroxiredoxin family are discussed.

Involvement of a maize proline-rich protein in secondary cell wall formation as deduced from its specific mRNA localization.

A clone encoding a proline-rich protein (ZmPRP) has been obtained from maize root by differential screening of a maturing elongation root cDNA library. The amino acid sequence deduced from the full-length cDNA contains a putative signal peptide and a highly repetitive sequence containing the PEPK motif, indicating that the ZmPRP mRNA may code for a cell wall protein. The PEPK repeat is also found in a previously reported wheat sequence but differs from the repeated sequences found in hydroxyproline-rich glycoproteins (HRGP) and in dicot proline-rich proteins (PRP). In the maize genome, the ZmPRP protein is encoded by a single gene that is expressed in maturing regions of the root, in the hypocotyl and in the pericarp. In these organs, the ZmPRP mRNA accumulates in the xylem and surrounding cells, and in the epidermis. No ZmPRP mRNA was found in the phloem. The pattern of mRNA accumulation is very similar to the one observed for genes coding for proteins involved in lignin biosynthesis and, like most cell wall proteins, ZmPRP synthesis is also induced by wounding. These data support the hypothesis that ZmPRP is a member of a new class of fibrous proteins involved in the secondary cell wall formation in monocot species.

Rice lipid transfer protein (LTP) genes belong to a complex multigene family and are differentially regulated.

Several cDNA clones encoding three different lipid transfer proteins (LTPs) have been isolated from rice (Oryza sativa L.) in order to analyse the complexity, the evolution and the expression of the LTP gene family. The mature proteins deduced from three clones exhibited a molecular mass of 9 kDa, in agreement with the molecular mass of other LTPs from plants. The clones were shown to be homologous in the coding region, while the 3' non-coding regions diverged strongly between the clones. The occurrence of at least three small multigene families encoding these proteins in rice was confirmed by Southern blot analysis. When compared with each other and with LTPs from other plants, the cluster including rice LTPs and other cereal LTPs indicated that these genes duplicated rather recently and independently in the different plant phyla. The expression pattern of each gene family was also investigated. Northern blot experiments demonstrated that they are differentially regulated in the different tissues analysed. Components such as salt, salicylic acid and abscisic acid were shown to modulate Ltp gene expression, depending on tissues and gene classes, suggesting a complex regulation of these genes.

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 beta-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.

The brown midrib3 (bm3) mutation in maize occurs in the gene encoding caffeic acid O-methyltransferase.

The brown midrib mutations are among the earliest described in maize. Plants containing a brown midrib mutation exhibit a reddish brown pigmentation of the leaf midrib starting when there are four to six leaves. These mutations are known to alter lignin composition and digestibility of plants and therefore constitute prime candidates in the breeding of silage maize. Here, we show that two independent brown midrib3 (bm3) mutations have resulted from structural changes in the COMT gene, which encodes the enzyme O-methyltransferase (COMT; EC 2.1.1.6), involved in lignin biosynthesis. Our results indicate that the bm3-1 allele (the reference mutant allele) has arisen from an insertional event producing a COMT mRNA altered in both size and amount. By sequencing a COMT cDNA clone obtained from bm3-1 maize, a retrotransposon with homology to the B5 element has been found to be inserted near the junction of the 3' coding region of the COMT gene intron. The second bm3 allele, bm3-2, has resulted from a deletion of part of the COMT gene. These alterations of the COMT gene were confirmed by DNA gel blot and polymerase chain reaction amplification analyses. These results clearly demonstrate that mutations at the COMT gene give a brown midrib3 phenotype. Thus, the gene genetically recognized as bm3 is the same as the one coding for COMT.

Characterization of a rice gene coding for a lipid transfer protein.

The cloning and sequence analysis of a gene that encodes a lipid transfer protein (LTP) from rice is reported. A genomic DNA library from Oryza sativa was screened using a cDNA encoding a maize LTP. One genomic clone containing the gene (Ltp) was partially sequenced and analyzed. The open reading frame is interrupted by an 89-bp intron. From the results of Southern hybridizations, Ltp appears to be a member of a small multigenic family. Transcripts of the corresponding gene were detected in several tissues including coleoptile, leaf, endosperm, scutellum and root. The transcription start point was determined by primer extension. The deduced amino-acid sequence of the Ltp product is shown to be homologous to LTPs from other crops.

Spatial and temporal expression of a maize lipid transfer protein gene.

We studied the temporal and spatial pattern of lipid transfer protein (LTP) gene expression, as well as the localization of this protein, in maize. Using an LTP gene, we observed an accumulation of LTP mRNA in embryos and endosperms during seed maturation. LTP gene expression was also investigated in young seedlings. After germination, the level of LTP mRNA in the coleoptile increased, with a maximum at 7 days, whereas LTP mRNA levels were low in the scutellum and negligible in roots. The high levels of LTP mRNA found in coleoptiles and embryos were confirmed by in situ hybridization. Moreover, LTP gene expression appeared to be localized in the external cellular layers and around the leaf veins. Using immunogold methods, we also observed that LTP was distributed heterogeneously in the different cells of coleoptiles and leaves. The highest concentrations of LTP were found in the outer epidermis of the coleoptiles as well as the leaf veins. Together, our observations indicate that LTP gene expression is not only organ specific and time specific but also cell specific.

Multiple mRNA coding for phospholipid-transfer protein from Zea mays arise from alternative splicing.

We have isolated a novel cDNA coding for maize phospholipid-transfer protein. The cDNA sequence is similar to the first one obtained by Tchang et al. [J. Biol. Chem. 263 (1988) 16849-16855] differing only by a mslal number of nucleotide substitutions and insertions. One of these insertions is 74 bp long and is flanked by consensus intron splicing sequences. The protein coded by the two cDNA has identical amino acids except in the C terminus. This difference derived from the presence of the 74-bp insert. The possible existence of an alternative splicing mechanism that could introduce heterogeneity in the sequence of these proteins is proposed.