Molecular Plant - current issue

Editorial

Cosgrove, D. J., Fincher, G., Hofte, H. — Tue, 22 Sep 2009 14:14:16 PDT

Plant Cell Wall Matrix Polysaccharide Biosynthesis

Sandhu, A. P. S., Randhawa, G. S., Dhugga, K. S. — Tue, 22 Sep 2009 14:14:16 PDT

The wall of an expanding plant cell consists primarily of cellulose microfibrils embedded in a matrix of hemicellulosic and pectic polysaccharides along with small amounts of structural and enzymatic proteins. Matrix polysaccharides are synthesized in the Golgi and exported to the cell wall by exocytosis, where they intercalate among cellulose microfibrils, which are made at the plasma membrane and directly deposited into the cell wall. Involvement of Golgi glucan synthesis in auxin-induced cell expansion has long been recognized; however, only recently have the genes corresponding to glucan synthases been identified. Biochemical purification was unsuccessful because of the labile nature and very low abundance of these enzymes. Mutational genetics also proved fruitless. Expression of candidate genes identified through gene expression profiling or comparative genomics in heterologous systems followed by functional characterization has been relatively successful. Several genes from the cellulose synthase-like (Csl) family have been found to be involved in the synthesis of various hemicellulosic glycans. The usefulness of this approach, however, is limited to those enzymes that probably do not form complexes consisting of unrelated proteins. Nonconventional approaches will continue to incrementally unravel the mechanisms of Golgi polysaccharide biosynthesis.

Homogalacturonan Methyl-Esterification and Plant Development

Wolf, S., Mouille, G., Pelloux, J. — Tue, 22 Sep 2009 14:14:16 PDT

The ability of a plant cell to expand is largely defined by the physical constraints imposed by its cell wall. Accordingly, cell wall properties have to be regulated during development. The pectic polysaccharide homogalacturonan is a major component of the plant primary walls. Biosynthesis and in muro modification of homogalacturonan have recently emerged as key determinants of plant development, controlling cell adhesion, organ development, and phyllotactic patterning. This review will focus on recent findings regarding impact of homogalacturonan content and methyl-esterification status of this polymer on plant life. De-methyl-esterification of homogalacturonan occurs through the action of the ubiquitous enzyme ‘pectin methyl-esterase’. We here describe various strategies developed by the plant to finely tune the methyl-esterification status of homogalacturonan along key events of the plant lifecycle.

Feruloylation in Grasses: Current and Future Perspectives

de O. Buanafina, M. M. — Tue, 22 Sep 2009 14:14:16 PDT

In the cell walls of forage grasses, ferulic acid is esterified to arabinoxylans and participates with lignin monomers in oxidative coupling pathways to generate ferulate–polysaccharide–lignin complexes that cross-link the cell wall. The accumulation of ferulates and the cross-linking of arabinoxylans via diferulate esters are hypothesized to function in various processes in plants. The specific roles of arabinoxylan feruloylation as well as the nature, cellular localization, and substrate for arabinoxylans feruloylation of cell walls are reviewed. The various approaches that have been used for assessing the specific roles of feruloylation are described and assessed. I argue that, until recently, the specific role of feruloylation in these various processes has been established largely by indirect experiments and, although these studies reached similar conclusions about the potential importance of wall feruloylation, they suffer from a common problem: namely they depend on correlations between two processes and do not stem from a detailed understanding of the mechanisms of feruloylation. I also argue that the nature of arabinoxylan feruloylation remains uncertain.

(1,3;1,4)-{beta}-D-Glucans in Cell Walls of the Poaceae, Lower Plants, and Fungi: A Tale of Two Linkages

Burton, R. A., Fincher, G. B. — Tue, 22 Sep 2009 14:14:16 PDT

(1,3;1,4)-β-D-Glucans consist of unbranched and unsubstituted chains of (1,3)- and (1,4)-β-glucosyl residues, in which the ratio of (1,4)-β-D-glucosyl residues to (1,3)-β-D-glucosyl residues appears to influence not only the physicochemical properties of the polysaccharide and therefore its functional properties in cell walls, but also its adoption by different plant species during evolution. The (1,3;1,4)-β-D-glucans are widely distributed as non-cellulosic matrix phase polysaccharides in cell walls of the Poaceae, which evolved relatively recently and consist of the grasses and commercially important cereal species, but they are less commonly found in lower vascular plants, such as the horsetails, in algae and in fungi. The (1,3;1,4)-β-D-glucans have often been considered to be components mainly of primary cell walls, but recent observations indicate that they can also be located in secondary walls of certain tissues. Enzymes involved in the depolymerisation of (1,3;1,4)-β-D-glucans have been well characterized. In contrast, initial difficulties in purifying the enzymes responsible for (1,3;1,4)-β-D-glucan biosynthesis slowed progress in the identification of the genes that encode (1,3;1,4)-β-D-glucan synthases, but emerging comparative genomics and associated techniques have allowed at least some of the genes that contribute to (1,3;1,4)-β-D-glucan synthesis in the Poaceae to be identified. Whether similar genes and enzymes also mediate (1,3;1,4)-β-D-glucan biosynthesis in lower plants and fungi is not yet known. Here, we compare the different fine structures of (1,3;1,4)-β-D-glucans across the plant kingdom, present current information on the genes that have been implicated recently in their biosynthesis, and consider aspects of the cell biology of (1,3;1,4)-β-D-glucan biosynthesis in the Poaceae.

Feruloylated Arabinoxylans Are Oxidatively Cross-Linked by Extracellular Maize Peroxidase but Not by Horseradish Peroxidase

Burr, S. J., Fry, S. C. — Tue, 22 Sep 2009 14:14:16 PDT

Covalent cross-linking of soluble extracellular arabinoxylans in living maize cultures, which models the cross-linking of wall-bound arabinoxylans, is due to oxidation of feruloyl esters to oligoferuloyl esters and ethers. The oxidizing system responsible could be H₂O₂/peroxidase, O₂/laccase, or reactive oxygen species acting non-enzymically. To distinguish these possibilities, we studied arabinoxylan cross-linking in vivo and in vitro. In living cultures, exogenous, soluble, extracellular, feruloylated [pentosyl-³H]arabinoxylans underwent cross-linking, beginning abruptly 8 d after sub-culture. Cross-linking was suppressed by iodide, an H₂O₂ scavenger, indicating dependence on endogenous H₂O₂. However, exogenous H₂O₂ did not cause precocious cross-linking, despite the constant presence of endogenous peroxidases, suggesting that younger cultures contained natural cross-linking inhibitors. Dialysed culture-filtrates cross-linked [³H]arabinoxylans in vitro only if H₂O₂ was also added, indicating a peroxidase requirement. This cross-linking was highly ionic-strength-dependent. The peroxidases responsible were heat-labile, although relatively heat-stable peroxidases (assayed on o-dianisidine) were also present. Surprisingly, added horseradish peroxidase, even after heat-denaturation, blocked the arabinoxylan-cross-linking action of maize peroxidases, suggesting that the horseradish protein was a competing substrate for [³H]arabinoxylan coupling. In conclusion, we show for the first time that cross-linking of extracellular arabinoxylan in living maize cultures is an action of apoplastic peroxidases, some of whose unusual properties we report.

Xyloglucan for Generating Tensile Stress to Bend Tree Stem

Tue, 22 Sep 2009 14:14:16 PDT

In response to environmental variation, angiosperm trees bend their stems by forming tension wood, which consists of a cellulose-rich G (gelatinous)-layer in the walls of fiber cells and generates abnormal tensile stress in the secondary xylem. We produced transgenic poplar plants overexpressing several endoglycanases to reduce each specific polysaccharide in the cell wall, as the secondary xylem consists of primary and secondary wall layers. When placed horizontally, the basal regions of stems of transgenic poplars overexpressing xyloglucanase alone could not bend upward due to low strain in the tension side of the xylem. In the wild-type plants, xyloglucan was found in the inner surface of G-layers during multiple layering. In situ xyloglucan endotransglucosylase (XET) activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, began at the inner surface layers S1 and S2 and was retained throughout G-layer development, while the incorporation of xyloglucan heptasaccharide (XXXG) for wall loosening occurred in the primary wall of the expanding zone. We propose that the xyloglucan network is reinforced by XET to form a further connection between wall-bound and secreted xyloglucans in order to withstand the tensile stress created within the cellulose G-layer microfibrils.

Loosening Xyloglucan Accelerates the Enzymatic Degradation of Cellulose in Wood

Tue, 22 Sep 2009 14:14:16 PDT

In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrils was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood.

Cell Wall Microstructure Analysis Implicates Hemicellulose Polysaccharides in Cell Adhesion in Tomato Fruit Pericarp Parenchyma

Ordaz-Ortiz, J. J., Marcus, S. E., Paul Knox, J. — Tue, 22 Sep 2009 14:14:16 PDT

Methods developed to isolate intact cells from both unripe and ripe tomato fruit pericarp parenchyma have allowed the cell biological analysis of polysaccharide epitopes at the surface of separated cells. The LM7 pectic homogalacturonan epitope is a marker of the junctions of adhesion planes and intercellular spaces in parenchyma systems. The LM7 epitope persistently marked the former edge of adhesion planes at the surface of cells separated from unripe and ripened tomato fruit and also from fruits with the Cnr mutation. The LM11 xylan epitope was associated, in sections, with cell walls lining intercellular space but the epitope was not detected at the surface of isolated cells, being lost during cell isolation. The LM15 xyloglucan epitope was present at the surface of cells isolated from unripe fruit in a pattern reflecting the former edge of cell adhesion planes/intercellular space but with gaps and apparent breaks. An equivalent pattern of LM15 epitope occurrence was revealed at the surface of cells isolated by pectate lyase action but was not present in cells isolated from ripe fruit or from Cnr fruit. In contrast to wild-type cells, the LM5 galactan and LM21 mannan epitopes occurred predominantly in positions reflecting intercellular space in Cnr, suggesting a concerted alteration in cell wall microstructure in response to this mutation. Galactanase and mannanase, along with pectic homogalacturonan-degrading enzymes, were capable of releasing cells from unripe fruit parenchyma. These observations indicate that hemicellulose polymers are present in architectural contexts reflecting cell adhesion and that several cell wall polysaccharide classes are likely to contribute to cell adhesion/cell separation in tomato fruit pericarp parenchyma.

Microanalysis of Plant Cell Wall Polysaccharides

Obel, N., Erben, V., Schwarz, T., Kuhnel, S., Fodor, A., Pauly, M. — Tue, 22 Sep 2009 14:14:16 PDT

Oligosaccharide Mass Profiling (OLIMP) allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry (MALDI–TOF MS). The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI–TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first isolating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apoplastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.

Identification of Lignin and Polysaccharide Modifications in Populus Wood by Chemometric Analysis of 2D NMR Spectra from Dissolved Cell Walls

Tue, 22 Sep 2009 14:14:16 PDT

2D ¹³C–¹H HSQC NMR spectroscopy of acetylated cell walls in solution gives a detailed fingerprint that can be used to assess the chemical composition of the complete wall without extensive degradation. We demonstrate how multivariate analysis of such spectra can be used to visualize cell wall changes between sample types as high-resolution 2D NMR loading spectra. Changes in composition and structure for both lignin and polysaccharides can subsequently be interpreted on a molecular level. The multivariate approach alleviates problems associated with peak picking of overlapping peaks, and it allows the deduction of the relative importance of each peak for sample discrimination. As a first proof of concept, we compare Populus tension wood to normal wood. All well established differences in cellulose, hemicellulose, and lignin compositions between these wood types were readily detected, confirming the reliability of the multivariate approach. In a second example, wood from transgenic Populus modified in their degree of pectin methylesterification was compared to that of wild-type trees. We show that differences in both lignin and polysaccharide composition that are difficult to detect with traditional spectral analysis and that could not be a priori predicted were revealed by the multivariate approach. 2D NMR of dissolved cell wall samples combined with multivariate analysis constitutes a novel approach in cell wall analysis and provides a new tool that will benefit cell wall research.

Xyloglucans of Monocotyledons Have Diverse Structures

Hsieh, Y. S.Y., Harris, P. J. — Tue, 22 Sep 2009 14:14:16 PDT

Except in the Poaceae, little is known about the structures of the xyloglucans in the primary walls of monocotyledons. Xyloglucan structures in a range of monocotyledon species were examined. Wall preparations were isolated, extracted with 6 M sodium hydroxide, and the extracts treated with a xyloglucan-specific endo-(1 -> 4)-β-glucanase preparation. The oligosaccharides released were analyzed by high-performance anion-exchange chromatography and by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. Oligosaccharide profiles of the non-commelinid monocotyledons were similar to those of most eudicotyledons, indicating the xyloglucans were fucogalactoxyloglucans, with a XXXG a core motif and the fucosylated units XXFG and XLFG. An exception was Lemna minor (Araceae), which yielded no fucosylated oligosaccharides and had both XXXG and XXG_n core motifs. Except for the Arecales (palms) and the Dasypogonaceae, which had fucogalactoxyloglucans, the xyloglucans of the commelinid monocotyledons were structurally different. The Zingiberales and Commelinales had xyloglucans with both XXG_n and XXXG core motifs; small proportions of XXFG units, but no XLFG units, were present. In the Poales, the Poaceae had xyloglucans with a XXG_n core motif and no fucosylated units. In the other Poales families, some had both XXXG and XXG_n core motifs, others had only XXXG; XXFG units were present, but XLFG units were not.

Arabinan Metabolism during Seed Development and Germination in Arabidopsis

Tue, 22 Sep 2009 14:14:16 PDT

Arabinans are found in the pectic network of many cell walls, where, along with galactan, they are present as side chains of Rhamnogalacturonan l. Whilst arabinans have been reported to be abundant polymers in the cell walls of seeds from a range of plant species, their proposed role as a storage reserve has not been thoroughly investigated. In the cell walls of Arabidopsis seeds, arabinose accounts for approximately 40% of the monosaccharide composition of non-cellulosic polysaccharides of embryos. Arabinose levels decline to ~15% during seedling establishment, indicating that cell wall arabinans may be mobilized during germination. Immunolocalization of arabinan in embryos, seeds, and seedlings reveals that arabinans accumulate in developing and mature embryos, but disappear during germination and seedling establishment. Experiments using ¹⁴C-arabinose show that it is readily incorporated and metabolized in growing seedlings, indicating an active catabolic pathway for this sugar. We found that depleting arabinans in seeds using a fungal arabinanase causes delayed seedling growth, lending support to the hypothesis that these polymers may help fuel early seedling growth.

Plant Cell Wall Proteomics: Mass Spectrometry Data, a Trove for Research on Protein Structure/Function Relationships

Tue, 22 Sep 2009 14:14:16 PDT

Proteomics allows the large-scale study of protein expression either in whole organisms or in purified organelles. In particular, mass spectrometry (MS) analysis of gel-separated proteins produces data not only for protein identification, but for protein structure, location, and processing as well. An in-depth analysis was performed on MS data from etiolated hypocotyl cell wall proteomics of Arabidopsis thaliana. These analyses show that highly homologous members of multigene families can be differentiated. Two lectins presenting 93% amino acid identity were identified using peptide mass fingerprinting. Although the identification of structural proteins such as extensins or hydroxyproline/proline-rich proteins (H/PRPs) is arduous, different types of MS spectra were exploited to identify and characterize an H/PRP. Maturation events in a couple of cell wall proteins (CWPs) were analyzed using site mapping. N-glycosylation of CWPs as well as the hydroxylation or oxidation of amino acids were also explored, adding information to improve our understanding of CWP structure/function relationships. A bioinformatic tool was developed to locate by means of MS the N-terminus of mature secreted proteins and N-glycosylation.

Pectin May Hinder the Unfolding of Xyloglucan Chains during Cell Deformation: Implications of the Mechanical Performance of Arabidopsis Hypocotyls with Pectin Alterations

Tue, 22 Sep 2009 14:14:16 PDT

Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the soft matrix phase and on the matrix deformation itself. Using specific Arabidopsis thaliana mutants, we studied the mechanical role of the matrix assembly in primary cell walls of hypocotyls with altered xyloglucan and pectin composition. Standard microtensile tests and cyclic loading protocols were performed on mur1 hypocotyls with affected RGII borate diester cross-links and a hindered xyloglucan fucosylation as well as qua2 exhibiting 50% less homogalacturonan in comparison to wild-type. As a control, wild-type plants (Col-0) and mur2 exhibiting a specific xyloglucan fucosylation and no differences in the pectin network were utilized. In the standard tensile tests, the ultimate stress levels (~tensile strength) of the hypocotyls of the mutants with pectin alterations (mur1, qua2) were rather unaffected, whereas their tensile stiffness was noticeably reduced in comparison to Col-0. The cyclic loading tests indicated a stiffening of all hypocotyls after the first cycle and a plastic deformation during the first straining, the degree of which, however, was much higher for mur1 and qua2 hypocotyls. Based on the mechanical data and current cell wall models, it is assumed that folded xyloglucan chains between cellulose fibrils may tend to unfold during straining of the hypocotyls. This response is probably hindered by geometrical constraints due to pectin rigidity.

Arabidopsis thaliana T-DNA Mutants Implicate GAUT Genes in the Biosynthesis of Pectin and Xylan in Cell Walls and Seed Testa

Caffall, K. H., Pattathil, S., Phillips, S. E., Hahn, M. G., Mohnen, D. — Tue, 22 Sep 2009 14:14:16 PDT

Galacturonosyltransferase 1 (GAUT1) is an 1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5'-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan (Sterling et al., 2006). The 25-member Arabidopsis thaliana GAUT1-related gene family encodes 15 GAUT and 10 GAUT-like (GATL) proteins with, respectively, 56–84 and 42–53% amino acid sequence similarity to GAUT1. Previous phylogenetic analyses of AtGAUTs indicated three clades: A through C. A comparative phylogenetic analysis of the Arabidopsis, poplar and rice GAUT families has sub-classified the GAUTs into seven clades: clade A-1 (GAUTs 1 to 3); A-2 (GAUT4); A-3 (GAUTs 5 and 6); A-4 (GAUT7); B-1 (GAUTs 8 and 9); B-2 (GAUTs 10 and 11); and clade C (GAUTs 12 to 15). The Arabidopsis GAUTs have a distribution comparable to the poplar orthologs, with the exception of GAUT2, which is absent in poplar. Rice, however, has no orthologs of GAUTs 2 and 12 and has multiple apparent orthologs of GAUTs 1, 4, and 7 compared with either Arabidopsis or poplar. The cell wall glycosyl residue compositions of 26 homozygous T-DNA insertion mutants for 13 of 15 Arabidopsis GAUT genes reveal significantly and reproducibly different cell walls in specific tissues of gaut mutants 6, 8, 9, 10, 11, 12, 13, and 14 from that of wild-type Arabidopsis walls. Pectin and xylan polysaccharides are affected by the loss of GAUT function, as demonstrated by the altered galacturonic acid, xylose, rhamnose, galactose, and arabinose composition of distinct gaut mutant walls. The wall glycosyl residue compositional phenotypes observed among the gaut mutants suggest that at least six different biosynthetic linkages in pectins and/or xylans are affected by the lesions in these GAUT genes. Evidence is also presented to support a role for GAUT11 in seed mucilage expansion and in seed wall and mucilage composition.

Transcriptional Wiring of Cell Wall-Related Genes in Arabidopsis

Mutwil, M., Ruprecht, C., Giorgi, F. M., Bringmann, M., Usadel, B., Persson, S. — Tue, 22 Sep 2009 14:14:16 PDT

Transcriptional coordination, or co-expression, of genes may signify functional relatedness of the corresponding proteins. For example, several genes involved in secondary cell wall cellulose biosynthesis are co-expressed with genes engaged in the synthesis of xylan, which is a major component of the secondary cell wall. To extend these types of analyses, we investigated the co-expression relationships of all Carbohydrate-Active enZYmes (CAZy)-related genes for Arabidopsis thaliana. Thus, the intention was to transcriptionally link different cell wall-related processes to each other, and also to other biological functions. To facilitate easy manual inspection, we have displayed these interactions as networks and matrices, and created a web-based interface (http://aranet.mpimp-golm.mpg.de/corecarb) containing downloadable files for all the transcriptional associations.

The CELLULOSE-SYNTHASE LIKE C (CSLC) Family of Barley Includes Members that Are Integral Membrane Proteins Targeted to the Plasma Membrane

Tue, 22 Sep 2009 14:14:16 PDT

The CELLULOSE SYNTHASE-LIKE C (CSLC) family is an ancient lineage within the CELLULOSE SYNTHASE/CELLULOSE SYNTHASE-LIKE (CESA/CSL) polysaccharide synthase superfamily that is thought to have arisen before the divergence of mosses and vascular plants. As studies in the flowering plant Arabidopsis have suggested synthesis of the (1,4)-β-glucan backbone of xyloglucan (XyG), a wall polysaccharide that tethers adjacent cellulose microfibrils to each other, as a probable function for the CSLCs, CSLC function was investigated in barley (Hordeum vulgare L.), a species with low amounts of XyG in its walls. Four barley CSLC genes were identified (designated HvCSLC1–4). Phylogenetic analysis reveals three well supported clades of CSLCs in flowering plants, with barley having representatives in two of these clades. The four barley CSLCs were expressed in various tissues, with in situ PCR detecting transcripts in all cell types of the coleoptile and root, including cells with primary and secondary cell walls. Co-expression analysis showed that HvCSLC3 was coordinately expressed with putative XyG xylosyltransferase genes. Both immuno-EM and membrane fractionation showed that HvCSLC2 was located in the plasma membrane of barley suspension-cultured cells and was not in internal membranes such as endoplasmic reticulum or Golgi apparatus. Based on our current knowledge of the sub-cellular locations of polysaccharide synthesis, we conclude that the CSLC family probably contains more than one type of polysaccharide synthase.

Two Poplar Glycosyltransferase Genes, PdGATL1.1 and PdGATL1.2, Are Functional Orthologs to PARVUS/AtGATL1 in Arabidopsis

Kong, Y., Zhou, G., Avci, U., Gu, X., Jones, C., Yin, Y., Xu, Y., Hahn, M. G. — Tue, 22 Sep 2009 14:14:16 PDT

Several genes in Arabidopsis, including PARVUS/AtGATL1, have been implicated in xylan synthesis. However, the biosynthesis of xylan in woody plants, where this polysaccharide is a major component of wood, is poorly understood. Here, we characterize two Populus genes, PdGATL1.1 and PdGATL1.2, the closest orthologs to the Arabidopsis PARVUS/GATL1 gene, with respect to their gene expression in poplar, their sub-cellular localization, and their ability to complement the parvus mutation in Arabidopsis. Overexpression of the two poplar genes in the parvus mutant rescued most of the defects caused by the parvus mutation, including morphological changes, collapsed xylem, and altered cell wall monosaccharide composition. Quantitative RT–PCR showed that PdGATL1.1 is expressed most strongly in developing xylem of poplar. In contrast, PdGATL1.2 is expressed much more uniformly in leaf, shoot tip, cortex, phloem, and xylem, and the transcript level of PdGATL1.2 is much lower than that of PdGATL1.1 in all tissues examined. Sub-cellular localization experiments showed that these two proteins are localized to both ER and Golgi in comparison with marker proteins resident to these sub-cellular compartments. Our data indicate that PdGATL1.1 and PdGATL1.2 are functional orthologs of PARVUS/GATL1 and can play a role in xylan synthesis, but may also have role(s) in the synthesis of other wall polymers.

New GATEWAY vectors for High Throughput Analyses of Protein-Protein Interactions by Bimolecular Fluorescence Complementation

Gehl, C., Waadt, R., Kudla, J., Mendel, R.-R., Hansch, R. — Tue, 22 Sep 2009 14:14:16 PDT

Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation (BiFC) is a suitable technique to investigate the formation of protein complexes and the localization of protein–protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N- or C-terminal sub-fragment of S(CFP)3A or Venus, respectively, and enable the performance of multicolor BiFC (mcBiFC). We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.

Rice-Specific Mitochondrial Iron-Regulated Gene (MIR) Plays an Important Role in Iron Homeostasis

Tue, 22 Sep 2009 14:14:16 PDT

Mitochondria utilize iron (Fe), but the proteins involved in mitochondrial Fe regulation are not characterized in plants. We cloned and characterized a mitochondrial iron-regulated (MIR) gene in rice involved in Fe homeostasis. MIR, when expressed in tobacco BY-2 cells, was localized to the mitochondria. MIR transcripts were greatly increased in response to Fe deficiency in roots and shoot tissue. MIR is not homologous to any known protein, as homologs were not found in the rice or Arabidopsis genome databases, or in the EST database for other organisms. Growth in the MIR T-DNA knockout rice mutant (mir) was significantly impaired compared to wild-type (WT) plants when grown under Fe-deficient or -sufficient conditions. Furthermore, mir plants accumulated more than twice the amount of Fe in shoot and root tissue compared to WT plants when grown under either Fe-sufficient or -deficient conditions. Despite the high accumulation of Fe in roots and shoots, mir plants triggered the expression of Fe-deficiency-inducible genes, indicating that mir may not be able to utilize Fe for physiological functions. These results clearly suggest that MIR is a rice-specific mitochondrial protein, recently evolved, and plays a significant role in Fe homeostasis.

Analysis of Transcriptome Changes Induced by Ptr ToxA in Wheat Provides Insights into the Mechanisms of Plant Susceptibility

Tue, 22 Sep 2009 14:14:16 PDT

To obtain greater insight into the molecular events underlying plant disease susceptibility, we studied transcriptome changes induced by a host-selective toxin of Pyrenophora tritici-repentis, Ptr ToxA (ToxA), on its host plant, wheat. Transcriptional profiling of ToxA-treated leaves of a ToxA-sensitive wheat cultivar was performed using the GeneChip^® Wheat Genome Array. An improved and up-to-date annotation of the wheat microarray was generated and a new tool for array data analysis (BRAT) was developed, and both are available for public use via a web-based interface. Our data indicate that massive transcriptional reprogramming occurs due to ToxA treatment, including cellular responses typically associated with defense. In addition, this study supports previous results indicating that ToxA-induced cell death is triggered by impairment of the photosynthetic machinery and accumulation of reactive oxygen species. Based on results of this study, we propose that ToxA acts as both an elicitor and a virulence factor.

Virus-Induced Gene Silencing in the Culinary Ginger (Zingiber officinale): An Effective Mechanism for Down-Regulating Gene Expression in Tropical Monocots

Renner, T., Bragg, J., Driscoll, H. E., Cho, J., Jackson, A. O., Specht, C. D. — Tue, 22 Sep 2009 14:14:16 PDT

Virus-induced gene silencing (VIGS) has been shown to be effective for transient knockdown of gene expression in plants to analyze the effects of specific genes in development and stress-related responses. VIGS is well established for studies of model systems and crops within the Solanaceae, Brassicaceae, Leguminaceae, and Poaceae, but only recently has been applied to plants residing outside these families. Here, we have demonstrated that barley stripe mosaic virus (BSMV) can infect two species within the Zingiberaceae, and that BSMV–VIGS can be applied to specifically down-regulate phytoene desaturase in the culinary ginger Zingiber officinale. These results suggest that extension of BSMV–VIGS to monocots other than cereals has the potential for directed genetic analyses of many important temperate and tropical crop species.

Post-Translational Regulation of AtFER2 Ferritin in Response to Intracellular Iron Trafficking during Fruit Development in Arabidopsis

Tue, 22 Sep 2009 14:14:16 PDT

Ferritins are major players in plant iron homeostasis. Surprisingly, their overexpression in transgenic plants led only to a moderate increase in seed iron content, suggesting the existence of control checkpoints for iron loading and storage in seeds. This work reports the identification of two of these checkpoints. First, measurement of seed metal content during fruit development in Arabidopsis thaliana reveals a similar dynamic of loading for Fe, Mn, Cu, and Zn. The step controlling metal loading into the seed occurs by the regulation of transport from the hull to the seed. Second, metal loading and ferritin abundance were monitored in different genetic backgrounds affected in vacuolar iron transport (AtVIT1, AtNRAMP3, AtNRAMP4) or plastid iron storage (AtFER1 to 4). This approach revealed (1) a post-translational regulation of ferritin accumulation in seeds, and (2) that ferritin stability depends on the balance of iron allocation between vacuoles and plastids. Thus, the success of ferritin overexpression strategies for iron biofortification, a promising approach to reduce iron-deficiency anemia in developing countries, would strongly benefit from the identification and engineering of mechanisms enabling the translocation of high amounts of iron into seed plastids.

Global Analysis of Gene Expression Profiles in Brassica napus Developing Seeds Reveals a Conserved Lipid Metabolism Regulation with Arabidopsis thaliana

Tue, 22 Sep 2009 14:14:16 PDT

In order to study Brassica napus fatty acid (FA) metabolism and relevant regulatory networks, a systematic identification of fatty acid (FA) biosynthesis-related genes was conducted. Following gene identification, gene expression profiles during B. napus seed development and FA metabolism were performed by cDNA chip hybridization (>8000 EST clones from seed). The results showed that FA biosynthesis and regulation, and carbon flux, were conserved between B. napus and Arabidopsis. However, a more critical role of starch metabolism was detected for B. napus seed FA metabolism and storage-component accumulation when compared with Arabidopsis. In addition, a crucial stage for the transition of seed-to-sink tissue was 17–21 d after flowering (DAF), whereas FA biosynthesis-related genes were highly expressed primarily at 21 DAF. Hormone (auxin and jasmonate) signaling is found to be important for FA metabolism. This study helps to reveal the global regulatory network of FA metabolism in developing B. napus seeds.

Corrigendum: Editorial

Tue, 22 Sep 2009 14:14:16 PDT

Corrigendum: Molecular Evolution of VEF-Domain-Containing PcG Genes in Plants

Chen, L.-J., Diao, Z.-Y., Specht, C., Sung, Z. R. — Tue, 22 Sep 2009 14:14:16 PDT