The poplar glycosyltransferase GT47C is functionally conserved with Arabidopsis Fragile fiber8

Xylan is the major hemicellulose in dicot wood. Unraveling genes involved in the biosynthesis of xylan will be of importance in understanding the process of wood formation. In this report, we investigated the possible role of poplar GT47C, a glycosyltransferase belonging to family GT47, in the biosynthesis of xylan. PoGT47C from the hybrid poplar Populus alba x tremula exhibits 84% sequence similarity to Fragile fiber8 (FRA8), which is involved in the biosynthesis of glucuronoxylan in Arabidopsis. Phylogenetic analysis of glycosyltransferase family GT47 in the Populus trichocarpa genome revealed that GT47C is the only close homolog of FRA8. In situ hybridization showed that the PoGT47C gene was expressed in developing primary xylem, secondary xylem and phloem fibers of stems, and in developing secondary xylem of roots. Sequence analysis suggests that PoGT47C is a type II membrane protein, and study of the subcellular localization demonstrated that fluorescent protein-tagged PoGT47C was located in the Golgi. Immunolocalization with a xylan monoclonal antibody LM10 revealed a nearly complete loss of xylan signals in the secondary walls of fibers and vessels in the Arabidopsis fra8 mutant. Expression of PoGT47C in the fra8 mutant restored the secondary wall thickness and xylan content to the wild-type level. Together, these results suggest that PoGT47C is functionally conserved with FRA8 and it is probably involved in xylan synthesis during wood formation.     

Unraveling the functions of glycosyltransferase family 47 in plants

The function of glycosyltransferases (GTs) from family GT47 was first identified in animal exostosins as β-glucuronyltransferase involved in the synthesis of heparan sulfate. Two recent papers report the functions of two plant members in this family as a pectin β-glucuronyltransferase and a xyloglucan β-galactosyltransferase. These findings greatly extend our understanding of the biological functions of family GT47 and also represent an important leap toward the molecular dissection of cell wall biosynthesis.     

Identification and molecular characterization of a Brachypodium distachyon GIGANTEA gene: functional conservation in monocot and dicot plants

Developmental phase change and flowering transition are emerging as potential targets for biomass agriculture in recent years. The GIGANTEA (GI) gene is one of the central regulators that direct flowering promotion and phase transition. In this work, we isolated a GI gene orthologue from the small annual grass Brachypodium distachyon inbred line Bd21 (Brachypodium), which is perceived as a potential model monocot for studies on bioenergy grass species. A partial GI gene sequence was identified from a Brachypodium expressed sequence tag library, and a full-size gene (BdGI) was amplified from a Brachypodium cDNA library using specific primer sets designed through analysis of monocot GI gene sequences. The BdGI gene was up-regulated by light and cold. A circadian rhythm set by light–dark transition also regulated the expression of the BdGI gene. The deduced amino acid sequence of the BdGI protein shares higher than 70% of sequence identity with the GI proteins in monocots and Arabidopsis. In addition, the BdGI protein is constitutively targeted to the nucleus and physically interacts with the ZEITLUPE (ZTL) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) proteins, like the Arabidopsis GI protein. Interestingly, heterologous expression of the BdGI gene in a GI-deficient Arabidopsis mutant rescued efficiently the late flowering phenotype. Together, our data indicate that the role of the GI gene in flowering induction is conserved in Arabidopsis and Brachypodium. It is envisioned that the GI genes of bioenergy grasses as well as Brachypodium could be manipulated to improve biomass by engineering developmental timing of phase transitions.     

Strong expression of the rice catalase gene CatB promoter in protoplasts and roots of both a monocot and dicots.

The rice (Oryza sativa L.) catalase (EC 1.11.1.6) gene CatB is expressed in roots and cultured cells. We examined the promoter activity of its 5'-flanking region in a monocot and in two dicots. Transient expression assays in rice Oc and tobacco BY-2 suspension cell protoplasts showed that CatB's 5'-flanking DNA fragments (nucleotides -1066 to +298) had about 20 and 3-4 times as much promoter activity, respectively, as the CaMV 35S promoter. Serial deletion analyses of the CatB promoter region revealed that the shortest fragment (-56 to +298) still had about 10 times as much promoter activity as the CaMV 35S promoter in rice protoplasts. In tobacco protoplasts, the activity of the fragment (-56 to +298) was about half of the CaMV 35S promoter. Transgenic rice and Arabidopsis plants carrying GUS genes driven by the 5'-truncated CatB promoters were generated and their GUS activity was examined. The region ranging from -329 to +298 showed preferential expression in the roots of rice and Arabidopsis, and in the shoot apical meristems of Arabidopsis. In situ hybridization revealed that CatB was highly expressed in branch root primordia and root apices of rice. Fusion of the GUS gene to the region (-329 to +298) conferred strong expression in these same areas, indicating that the presence of this region was sufficient to express CatB specifically in the roots. There may be new regulatory element(s) in this region, because it contained no previously known cis-regulatory elements specific for gene expression in roots.     

Functional conservation of MADS-box factors controlling floral organ identity in rice and Arabidopsis

Studies on MADS-box genes in Arabidopsis and other higher eudicotyledonous flowering plants have shown that they are key regulators of flower development. Since Arabidopsis and monocotyledonous rice are distantly related plant species it is interesting to investigate whether the floral organ identity factors have been conserved in their functions, and if not, to understand the differences. Arabidopsis and rice are very suitable for these studies since they are both regarded as models for plant functional genomics. Both their genomes are sequenced and tools are available for the analysis of gene function. These developments have accelerated experiments and increased our knowledge on rice gene function. Therefore it is the right moment to perform a comparative analysis on MADS-box factors controlling floral organ identity as reported in this review.     

Leveraging the rice genome sequence for monocot comparative and translational genomics

Common genome anchor points across many taxa greatly facilitate translational and comparative genomics and will improve our understanding of the Tree of Life. To add to the repertoire of genomic tools applicable to the study of monocotyledonous plants in general, we aligned Allium and Musa ESTs to Oryza BAC sequences and identified candidate Allium-Oryza and Musa-Oryza conserved intron-scanning primers (CISPs). A random sampling of 96 CISP primer pairs, representing loci from 11 of the 12 chromosomes in rice, were tested on seven members of the order Poales and on representatives of the Arecales, Asparagales, and Zingiberales monocot orders. The single-copy amplification success rates of Allium (31.3%), Cynodon (31.4%), Hordeum (30.2%), Musa (37.5%), Oryza (61.5%), Pennisetum (33.3%), Sorghum (47.9%), Zea (33.3%), Triticum (30.2%), and representatives of the palm family (32.3%) suggest that subsets of these primers will provide DNA markers suitable for comparative and translational genomics in orphan crops, as well as for applications in conservation biology, ecology, invasion biology, population biology, systematic biology, and related fields. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. H. C. Lohithaswa and F. A. Feltus contributed equally to this work.     

Functional conservation of the Drosophila hybrid incompatibility gene Lhr

Background  

Hybrid incompatibilities such as sterility and lethality are commonly modeled as being caused by interactions between two genes, each of which has diverged separately in one of the hybridizing lineages. The gene Lethal hybrid rescue (Lhr) encodes a rapidly evolving heterochromatin protein that causes lethality of hybrid males in crosses between Drosophila melanogaster females and D. simulans males. Previous genetic analyses showed that hybrid lethality is caused by D. simulans Lhr but not by D. melanogaster Lhr, confirming a critical prediction of asymmetry in the evolution of a hybrid incompatibility gene.     

Functional conservation of the Drosophila gooseberry gene and its evolutionary alleles

The Drosophila Pax gene gooseberry (gsb) is required for development of the larval cuticle and CNS, survival to adulthood, and male fertility. These functions can be rescued in gsb mutants by two gsb evolutionary alleles, gsb-Prd and gsb-Pax3, which express the Drosophila Paired and mouse Pax3 proteins under the control of gooseberry cis-regulatory region. Therefore, both Paired and Pax3 proteins have conserved all the Gsb functions that are required for survival of embryos to fertile adults, despite the divergent primary sequences in their C-terminal halves. As gsb-Prd and gsb-Pax3 uncover a gsb function involved in male fertility, construction of evolutionary alleles may provide a powerful strategy to dissect hitherto unknown gene functions. Our results provide further evidence for the essential role of cis-regulatory regions in the functional diversification of duplicated genes during evolution.     

Xyloglucan exoglycosidases in the monocot model Brachypodium distachyon and the conservation of xyloglucan disassembly in angiosperms

Plant Molecular Biology - Brachypodium distachyon has a full set of exoglycosidases active on xyloglucan, including α-xylosidase, β-galactosidase, soluble and membrane-bound...     

Expression and characterization of monocot rice cytosolic CuZnSOD protein in dicot Arabidopsis

Cytosolic CuZnSOD removes deleterious superoxides from plant cells. In order to understand its function better, we sought to express a monocot CuZnSODgene in transgenic Arabidopsis. We constructed a transgene usingthe CaMV 35S promoter to express a rice cytosolic CuZnSOD gene in Arabidopsis and generated over 200 transformants. A 16kD polypeptide, the same size as the native rice CuZnSOD polypeptide, was detected inthe transgenic Arabidopsis. Interestingly, two forms of riceCuZnSOD, rSODI and rSODII, having the same dimeric size, were detectedin the transgenic plants. rSODII protein was relatively abundant but hadlow specific activity. In contrast, rSODI protein was relatively rareand had high specific activity. Inter-conversion of rSODI and rSODIIcould be achieved by the addition and removal of copper ions into the purifiedrecombinant SOD and to the leaf extract of transgenic plants. Ouranalysis indicates that rSODI most likely corresponds to native riceCuZnSOD that has incorporated the Cu and Zn ions required for fullactivity, whereas the less active rSODII form may not have properlyincorporated the necessary copper ions.     

Functional conservation of wheat and rice Mlo orthologs in defense modulation to the powdery mildew fungus

Homologs of barley Mlo are found in syntenic positions in all three genomes of hexaploid bread wheat, Triticum aestivum, and in rice, Oryza sativa. Candidate wheat orthologs, designated TaMlo-A1, TaMlo-B1, and TaMlo-D1, encode three distinct but highly related proteins that are 88% identical to barley MLO and appear to originate from the three diploid ancestral genomes of wheat. TaMlo-B1 and the rice ortholog, OsMlo2, are able to complement powdery mildew-resistant barley mlo mutants at the single-cell level. Overexpression of TaMlo-B1 or barley Mlo leads to super-susceptibility to the appropriate powdery mildew formae speciales in both wild-type barley and wheat. Surprisingly, overexpression of either Mlo or TaMlo-B1 also mediates enhanced fungal development to tested inappropriate formae speciales. These results underline a regulatory role for MLO and its wheat and rice orthologs in a basal defense mechanism that can interfere with forma specialis resistance to powdery mildews.     

Functional investigation of a gene encoding pteridine glycosyltransferase for cyanopterin synthesis in Synechocystis sp. PCC 6803

A gene (slr1166) putatively encoding pteridine glycosyltransferase was disrupted with a kanamycin resistance cassette in Synechocystis sp. PCC 6803, which produces cyanopterin. The deduced polypeptide from slr1166 consisted of 354 amino acid residues sharing 45% sequence identity with UDP-glucose:tetrahydrobiopterin alpha-glucosyltransferase (BGluT) isolated previously from Synechococcus sp. PCC 7942. The knockout mutant was unable to produce cyanopterin but only 6-hydroxymethylpterin-beta-galactoside, verifying that slr1166 encodes a pteridine glycosyltransferase, which is responsible for transfer of the second sugar glucuronic acid in cyanopterin synthesis. The mutant was affected in its intracellular pteridine content and growth rate, which were 74% and 80%, respectively, of wild type, demonstrating that the second sugar residue is still required for quantitative maintenance of cyanopterin. This supports the previous suggestion that glycosylation may contribute to high cellular concentration of pteridine compounds.     

A bacterial glycosyltransferase gene toolbox: Generation and applications

The bioactivity of many natural products produced by microorganisms can be attributed to their sugar substituents. These substituents are transferred as nucleotide-activated sugars to an aglycon by glycosyltransferases. Engineering these enzymes can broaden their substrate specificity and can therefore have an impact on the bioactivity of the secondary metabolites.In this review we present the generation of a glycosyltransferase gene toolbox which contains more than 70 bacterial glycosyltransferases to date. Investigations of the function, specificity and structure of these glycosyltransferases help to understand the great potential of these enzymes for natural product biosynthesis.