Differential expression of a polygalacturonase gene family in Arabidopsis thaliana

By systematic sequencing of a flower bud cDNA library from Arabidopsis thaliana, we have identified four cDNAs encoding polygalacturonase. The corresponding genes, together with seven other A. thaliana genes present in the databases, form a small gene family. Sequence comparisons of the deduced polypeptides within the gene family or with other plant polygalacturonases allow classification of the genes into different clades. Five polygalacturonases, including all those isolated from the flower buds, are closely related to the enzyme in pollen. Of the six remaining polygalacturonases, three are more closely related to the abscission-specific type of enzyme and two others to the fruit polygalacturonase. The last one is more distantly related to the others and might correspond to a new type of polygalacturonase. Expression of the different genes was analysed on Northern blots and by a PCR-based strategy. Results indicate that if, as expected, the cDNAs isolated from the flower bud library are strongly expressed in pollen, other genes are expressed at a low level in young developing tissues, such as in seedlings and roots, suggesting that they could be implicated in the cell wall modifications observed during cell elongation and/or expansion which occur in these tissues.     

Molecular characterization of one of the maize polygalacturonase gene family members which are expressed during late pollen development

A gene exhibiting homology to the polygalacturonases of several species, including tomato and Oenothera, has been shown by RNA dot-blot analysis and in situ hybridization experiments to be expressed post-first microspore mitosis in maize. A 2.87 kbp section of the promoter fused to E. coli β-glucuronidase (uidA) coding sequence conferred the correct spatial and temporal expression in transgenic tobacco plants. However, low levels of expression were detected in other tissues, and in particular in the tissues surrounding the vascular branch points of leaf nodes. The maize polygalacturonase gene is one member of a highly conserved gene family. The lack of detectable expression in sporophytic tissues and the isolation of a number of related cDNAs from maize suggests that all expressed members of this family show the same spatial and temporal regulation.     

Molecular control of early cone development inPinus radiata

Summary A family of genes expressed during early stages of shoot development were isolated fromPinus radiata. A homologue of theLEAFY/FLORICAULA flower meristem-identity genes,NEEDLY (NLY), and three MADS-box genes,PrMADS1, PrMADS2 andPrMADS3 (Pinus radiata MADS-box genes), were expressed at early stages of initiation and differentiation of reproductive (male and female) cone buds, as well as vegetative buds. Expression ofNLY in transgenicArabidopsis thaliana promoted floral fate, demonstrating that it encodes a functional ortholog of theFLORICAUL A/LEAFY genes of angiosperms.Abbreviations DSB dwarf shoot bud - LSTB long-shoot terminal bud - PCB pollen cone bud - SCB seed cone bud - LD long day - SD short day     

β-Glucan synthetase II activity upon callose formation in the flower ofArabidopsis thaliana

Callose formation was observed in the pollens during flower development and pollen tube grown in the pistil ofA. thaliana. The accumulation of callose occurred in the tetrad in the flower bud and pollen tube. Therefore, the activity of β-glucan synthetase II (GS II), which is responsible for synthesizing the callose, was measured in the flowers on the same developmental stages. The enzyme activity was increased by about 10% while the level of callose contents was increased by about 70% in tetrads. Then, callose accumulation was increased during pollen tube growth by about 30% higher than the other stages and enzyme activity was detected, 30% more too. These results suggest that callose plays an important role in the growth of pollen and pollen tube by increasing GS II activity.     

Characterization of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase.

We have isolated and characterized cDNA clones of a gene family (P2) expressed in Oenothera organensis pollen. This family contains approximately six to eight family members and is expressed at high levels only in pollen. The predicted protein sequence from a near full-length cDNA clone shows that the protein products of these genes are at least 38,000 daltons. We identified the protein encoded by one of the cDNAs in this family by using antibodies to beta-galactosidase/pollen cDNA fusion proteins. Immunoblot analysis using these antibodies identifies a family of proteins of approximately 40 kilodaltons that is present in mature pollen, indicating that these mRNAs are not stored solely for translation after pollen germination. These proteins accumulate late in pollen development and are not detectable in other parts of the plant. Although not present in unpollinated or self-pollinated styles, the 40-kilodalton to 45-kilodalton antigens are detectable in extracts from cross-pollinated styles, suggesting that the proteins are present in pollen tubes growing through the style during pollination. The proteins are also present in pollen tubes growing in vitro. Both nucleotide and amino acid sequences are similar to the published sequences for cDNAs encoding the enzyme polygalacturonase, which suggests that the P2 gene family may function in depolymerizing pectin during pollen development, germination, and tube growth. Cross-hybridizing RNAs and immunoreactive proteins were detected in pollen from a wide variety of plant species, which indicates that the P2 family of polygalacturonase-like genes are conserved and may be expressed in the pollen from many angiosperms.     

Isolation and characterization of cDNA clones corresponding to the genes expressed preferentially in floral organs of Arabidopsis thaliana

Seventeen cDNA clones of genes corresponding to mRNAs expressed preferentially in floral organs of Arabidopsis thaliana were obtained by differential screening of a flower bud cDNA library, and classified into five groups (1A, 17A, 1B, 4B and 5B) by cross-hybridization and restriction analysis. Sequence analysis revealed that the 1A-1 and 17A-1 clones encode vegetative storage proteins (VSPs). The VSP mRNAs were detected in a small amount in leaves and increased to a limited level by wounding. Both 1B-1 and 5B-1 clones were homologous to transmembrane protein cDNAs. The protein encoded by 4B-1 clone contained a proline-rich region, but no homologous proteins were found in databases.     

Characterization and localization of short-specific polygalacturonase in distylous Turnera subulata (Turneraceae)

We describe for distylous Turnera subulata a polygalacturonase specific to short-styled plants that is localized to the style transmitting tissue (the tissue through which pollen tubes grow). The polygalacturonase gene is linked to and may be upregulated by the S allele of the distyly locus. Because of its tissue-specific location, the polygalacturonase may be involved in the self-incompatibility response, acting in a complementary or antagonistic manner, or possibly in signalling downstream events. A pollen-specific polygalacturonase was also identified and may be a member of a small multigene family of pollen polygalacturonases. The role, if any, played by the pollen polygalacturonase in distyly, is presently unknown.     

Isolation and characterization of mRNAs accumulated during in-vitro flower bud formation

The development of vegetative and generative buds on thin-layer expiants of tobacco (Nicotiana tabacum L. cv. Samsun) has been studied at the level of translatable mRNA to detect changes in the mRNA population during bud initiation and differentiation, and several quantitative differences were found. By differential screening of a cDNA library obtained from flower-bud-regenerating explants we have isolated a group of six cDNA clones representing genes that are preferentially expressed during in-vitro flower bud formation. Nucleotide sequence analysis of one of these cDNAs, pAP8, showed that the most likely open reading frame has some typical characteristics of, and homology with, extensin-like genes. Northern blot analysis and in-situ hybridization suggest a specific role for these extensin-like genes in flower bud initiation on tobacco pedicel explants.     

Isolation and Characterization of an Anther-Specific Polygalacturonase Gene, BcMF16, in Brassica campestris ssp. chinensis

Many genes in the genic male sterile A/B line (Bajh97-01A/B) of Chinese cabbage pak choi (Brassica campestris L. subsp. chinensis Makino) are expressed differentially, and some play critical roles in the formation of pollen walls. In this study, one of these genes, Brassica campestris Male Fertility 16 (BcMF16), has been isolated and characterized. The BcMF16 gene shares approximately 85% nucleotide sequence homology with two exopolygalacturonase (EC3.2.1.67) genes of Arabidopsis thaliana. Cluster analysis of polygalacturonase peptides indicate that BcMF16 belongs to the pollen polygalacturonase clade. Quantitative real-time PCR analysis has revealed that BcMF16 is specifically expressed in reproductive tissues of the fertile line of genic male sterile A/B line of Chinese cabbage pak choi, and that expression levels dramatically increased during later stages of pollen development. In situ hybridization has demonstrated that BcMF16 is specifically and transiently expressed in both tapetum and pollen following microspore separation at the tetrad stage.     

The presence of a Sar1 gene family in Brassica campestris that suppresses a yeast vesicular transport mutation Sec12-1

Two new members (Bsar1a and Bsar1b) of the Sar1 gene family have been identified from a flower bud cDNA library of Brassica campestris and their functional characteristics were analyzed. The two clones differ from each other at 14 positions of the 193 amino acid residues deduced from their coding region. The amino acid sequences of Bsar1a and Bsar1b are most closely related to the Sar1 family, genes that function early in the process of vesicle budding from the endoplasmic reticulum (ER). The sequences contain all the conserved motifs of the Ras superfamily (G1–G4 motifs) as well as the distinctive structural feature near the C-terminus that is Sar1 specific. Our phylogenetic analysis confirmed that these two clones can indeed be considered members of the Sar1 family and that they have a close relationship to the ARF family. The Bsar1 proteins, expressed in Escherichia coli, cross-reacted with a polyclonal antibody prepared against Saccharomyces cerevisiae Sar1 protein. It also exhibited GTP-binding activity. Genomic Southern blot analysis, using the 3'-gene-specific regions of the Bsar1 cDNAs as probes, revealed that the two cDNA clones are members of a B. campestris Sar1 family that consists of 2 to 3 genes. RNA blot analysis, using the same gene-specific probes, showed that both genes are expressed with similar patterns in most tissues of the plant, including leaf, stem, root, and flower buds. Furthermore, when we placed the two Bsar1 genes under the control of the yeast pGK1 promoter into the temperature-sensitive mutant yeast strain S. cerevisiae Sec12-1, they suppressed the mutation which consists of a defect in vesicle transport. The amino acid sequence similarity, the GTP-binding activity, and the functional suppression of the yeast mutation suggest that the Bsar1 proteins are functional homologues of the Sar1 protein in S. cerevisiae and that they may perform similar biological functions.     

The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes.

Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases I and II (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lambda EMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergillus nidulans transformants on the basis of activity measurements and Western-blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A. nidulans transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A. nidulans transformed with the subcloned pgaC gene. This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intron/exon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N-terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A. niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to represent a binding site for a regulatory protein as it shows a high similarity to the yeast CYC1 upstream activation site recognized by the HAP2/3/4 activation complex.     

Structure and expression of three src2 homologues and a novel subfamily of flavoprotein monooxygenase genes revealed by the analysis of a 25 kb fragment from Arabidopsis thaliana chromosome IV

Biological and computer-assisted analyses of a 25 kb fragment from Arabidopsis thaliana chromosome IV led to the characterization of two multigene families and three novel orphan genes, not previously described. The first gene family named AtMO1-4 encodes monooxygenases, related to the prokaryotic salicylate hydroxylases. The second gene family contains three members, two on the analysed 25 kb fragment and one on chromosome I. The latter three genes lack introns and are homologous to the previously studied Glycine max src2 gene which is overexpressed at low temperature. Gene expression and primary structure of the deduced proteins are described and compared. Three genes of unknown function, showing tissue specific expressions, are characterized on the 25 kb fragment. Full length or partial cognate cDNAs have been sequenced for all the genes studied.     

Expression of a cytochrome P450 gene family in maize

Maize seedlings, like seedlings of many other plants, are rich in cytochrome P450 (P450) enzyme activity. Four P450 genes (CYPzm1–4), isolated from a seedling-specific cDNA library, are characterised by a transient and seedling-specific expression pattern. The maximum steady state mRNA levels are reached at 3 days in root and at 7 days in shoot tissue, respectively. All four genes belong to one gene family and are closely related to the CYP71 family of plant P450 genes, which includes the enzymes of the ripening avocado fruit (CYP71A1) and eggplant hypocotyls (CYP71A2, A3, A4). The expression of these related P450 genes in monocot and dicot plants indicates that these enzymes play a significant role in plants; however, the in vivo enzyme functions are unknown. The divergence of the four members of the maize gene family is sufficiently high to account for different substrate and/or reaction specificity. Although the general expression pattern of the four genes is identical, the maximum steady-state mRNA levels vary in different maize lines. In situ hybridisation reveals the highest mRNA levels in the coleoptile, the first developed leaflets, the ground tissue of the nodular complex, and in the cortex and pith of the region of cell division in the root. The mapping of the maize CYPzm genes shows that, as in animals, P450 genes of the same family can be clustered. The presence of the CYPzm gene cluster in maize argues for generation of distinct plant P450 gene families by gene duplication.     

Gene structure and molecular analysis of the laccase-like multicopper oxidase (LMCO) gene family in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Completed genome sequences have made it clear that multicopper oxidases related to laccase are widely distributed as multigene families in higher plants. Laccase-like multicopper oxidase (LMCO) sequences culled from GenBank and the Arabidopsis thaliana genome, as well as those from several newly cloned genes, were used to construct a gene phylogeny that clearly divided plant LMCOs into six distinct classes, at least three of which predate the evolutionary divergence of angiosperms and gymnosperms. Alignments of the predicted amino acid sequences highlighted regions of variable sequence flanked by the highly conserved copper-binding domains that characterize members of this enzyme family. All of the predicted proteins contained apparent signal sequences. The expression of 13 of the 17 LMCO genes in A. thaliana was assessed in different tissues at various stages of development using RT-PCR. A diversity of expression patterns was demonstrated with some genes being expressed in a constitutive fashion, while others were only expressed in specific tissues at a particular stage of development. Only a few of the LMCO genes were expressed in a pattern that could be considered consistent with a major role for these enzymes in lignin deposition. These results are discussed in the context of other potential physiological functions for plant LMCOs, such as iron metabolism and wound healing.     

A polygalacturonase inhibitory protein gene (<Emphasis Type="Italic">BcMF19</Emphasis>) expressed during pollen development in Chinese cabbage-pak-choi

The level of polygalacturonase inhibitory protein (PGIP) genes involved in pollen development remains unclear. Characterization of the different PGIP genes that are expressed in pollen is necessary in understanding the similarities and differences of functions between the members of this gene family, as well as the underlying mechanism of pollen development. A gene-encoding putative PGIP, BcMF19 was successfully cloned on a cDNA-amplified fragment length polymorphism fragment after it was found to be up-regulated in the fertile flower buds of Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) genic male sterile AB line (Bajh97-01A/B). The amino acid sequence of BcMF19 possessed the basic feature of PGIPs, containing an N-terminal signal peptide, several potential N-glycosylation sites, two disulfide bridges flanking both the N- and C-terminal regions, and 10 leucine-rich repeat (LRR) consensus sequences. Real-time RT-PCR verified the higher expression of BcMF19 in the fertile flower buds compared to the sterile flower buds. In situ hybridization showed that BcMF19 was exclusively expressed in the tapetal cells and microspores during anther development. These results indicate that BcMF19 is a novel PGIP gene that might be involved in pollen or tapetum development.     

Two amidophosphoribosyltransferase genes of Arabidopsis thaliana expressed in different organs

Amidophosphoribosyltransferase (ATase: EC 2.4.2.14) is a key enzyme in the pathway of purine nucleotide biosynthesis. We have identified several cDNA clones whose amino acid sequences exhibit similarity with the known ATases in a cDNA library of young floral buds of Arabidopsis thaliana. The cDNA clones are derived from two genes homologous with each other. These cDNAs represent the first plant representatives of ATase gene. Structural comparison with ATases of other organisms has revealed that the two genes encode [4Fe-4S] cluster-dependent ATases. Northern blot analysis showed that expression level of the genes is different in three organs; one gene is expressed in flowers and roots, while the other gene is mainly expressed in leaves.     

The relatively large beta-tubulin gene family of Arabidopsis contains a member with an unusual transcribed 5′ noncoding sequence

We have characterized the beta-tubulin gene family of Arabidopsis thaliana. Five distinct genes were cloned and analyzed by restriction enzyme mapping and cross-hybridization studies. Three of the genes appear to be dispersed, whereas two others are linked within 1.5 kb of one another. The two linked genes are closely related and appear to have resulted from a fairly recent duplication. The three dispersed genes do not cross-hybridize to one another or to the two linked genes under highly stringent hybridization conditions, suggesting that they arose from more ancient duplications. From Southern analysis we estimate that there are a total of between six and ten beta-tubulin genes in Arabidopsis. Additional analyses indicate that the gene family is equal in size or larger than those in other plants, but significantly smaller than those in related Brassica species. Sequence determination of one of the Arabidopsis genes revealed a highly unusual transcribed leader sequence. The leader contains two fairly long tracks of adenines. One is located toward the 5 end of the mRNA and the other is just before the initiation codon. A track of uridines is located between the adenine tracks. This leader can form two different secondary structures that may have regulatory significance.