Molecular Plant - recent issues

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

Editorial

Tue, 21 Jul 2009 10:28:07 PDT

Control of the Transition to Flowering by Chromatin Modifications

He, Y. — Tue, 21 Jul 2009 10:28:07 PDT

The timing of floral transition is critical to reproductive success in angiosperms and is genetically controlled by a network of flowering genes. In Arabidopsis, expression of certain flowering genes is regulated by various chromatin modifications, among which are two central regulators of flowering, namely FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT). Recent studies have revealed that a number of chromatin-modifying components are involved in activation or repression of FLC expression. Activation of FLC expression is associated with various ‘active’ chromatin modifications including acetylation of core histone tails, histone H3 lysine-4 (H3K4) methylation, H2B monoubiquitination, H3 lysine-36 (H3K36) di- and tri-methylation and deposition of the histone variant H2A.Z, whereas various ‘repressive’ histone modifications are associated with FLC repression, including histone deacetylation, H3K4 demethylation, histone H3 lysine-9 (H3K9) and H3 lysine-27 (H3K27) methylation, and histone arginine methylation. In addition, recent studies have revealed that Polycomb group gene-mediated transcriptional-silencing mechanism not only represses FLC expression, but also directly represses FT expression. Regulation of FLC expression provides a paradigm for control of the expression of other developmental genes in plants through chromatin mechanisms.

The Beauty of Being a Variant: H2A.Z and the SWR1 Complex in Plants

March-Diaz, R., Reyes, J. C. — Tue, 21 Jul 2009 10:28:07 PDT

Numerous studies have shown that the nucleosome is a dynamic structure that strongly influences gene expression. Dynamism concerns different nucleosomal characteristics, including position, posttranslational modifications, and histone composition. Thus, within the nucleosome, canonical histones can be exchanged by histone variant proteins with specific functions—a process known as ‘histone replacement’. The histone variant H2A.Z has an important function in transcription and, during the last few years, its role in plant development and immune response has become evident. Compiling genetic and biochemical studies from several laboratories has revealed that plants contain a multiprotein complex, similar to the SWR1/SRCAP complex from yeast and animals, involved in H2A.Z deposition. Despite intense research in different organisms, the mechanism by which H2A.Z influences transcription is still unknown. However, recent results from Arabidopsis have shown a strong inverse correlation between H2A.Z and DNA methylation, suggesting that H2A.Z might protect genes from silencing.

Paramutation: A Heritable Change in Gene Expression by Allelic Interactions In Trans

Stam, M. — Tue, 21 Jul 2009 10:28:07 PDT

Epigenetic gene regulation involves the stable propagation of gene activity states through mitotic, and sometimes even meiotic, cell divisions without changes in DNA sequence. Paramutation is an epigenetic phenomenon involving changes in gene expression that are stably transmitted through mitosis as well as meiosis. These heritable changes are mediated by in trans interactions between homologous DNA sequences on different chromosomes. During these in trans interactions, epigenetic information is transferred from one allele of a gene to another allele of the same gene, resulting in a change in gene expression. Although paramutation was initially discovered in plants, it has recently been observed in mammals as well, suggesting that the mechanisms underlying paramutation might be evolutionarily conserved. Recent findings point to a crucial role for small RNAs in the paramutation process. In mice, small RNAs appear sufficient to induce paramutation, whereas in maize, it seems not to be the only player in the process. In this review, potential mechanisms are discussed in relation to the various paramutation phenomena.

Epigenetic Programming: The Challenge to Species Hybridization

Ishikawa, R., Kinoshita, T. — Tue, 21 Jul 2009 10:28:07 PDT

In many organisms, the genomes of individual species are isolated by a range of reproductive barriers that act before or after fertilization. Successful mating between species results in the presence of different genomes within a cell (hybridization), which can lead to incompatibility in cellular events due to adverse genetic interactions. In addition to such genetic interactions, recent studies have shown that the epigenetic control of the genome, silencing of transposons, control of non-additive gene expression and genomic imprinting might also contribute to reproductive barriers in plant and animal species. These genetic and epigenetic mechanisms play a significant role in the prevention of gene flow between species. In this review, we focus on aspects of epigenetic control related to hybrid incompatibility during species hybridization, and also consider key mechanism(s) in the interaction between different genomes.

Chromatin Remodeling in Stem Cell Maintenance in Arabidopsis thaliana

Shen, W.-H., Xu, L. — Tue, 21 Jul 2009 10:28:07 PDT

Pluripotent stem cells are able to both self-renew and generate undifferentiated cells for the formation of new tissues and organs. In higher plants, stem cells found in the shoot apical meristem (SAM) and the root apical meristem (RAM) are origins of organogenesis occurring post-embryonically. It is important to understand how the regulation of stem cell fate is coordinated to enable the meristem to constantly generate different types of lateral organs. Much knowledge has accumulated on specific transcription factors controlling SAM and RAM activity. Here, we review recent evidences for a role of chromatin remodeling in the maintenance of stable expression states of transcription factor genes and the control of stem cell activity in Arabidopsis.

An Epigenetic Perspective on Developmental Regulation of Seed Genes

Zhang, H., Ogas, J. — Tue, 21 Jul 2009 10:28:07 PDT

The developmental program of seeds is promoted by master regulators that are expressed in a seed-specific manner. Ectopic expression studies reveal that expression of these master regulators and other transcriptional regulators is sufficient to promote seed-associated traits, including generation of somatic embryos. Recent work highlights the importance of chromatin-associated factors in restricting expression of seed-specific genes, in particular PcG proteins and ATP-dependent remodelers. This review summarizes what is known regarding factors that promote zygotic and/or somatic embryogenesis and the chromatin machinery that represses their expression. Characterization of the regulation of seed-specific genes reveals that plant chromatin-based repression systems exhibit broad conservation with and surprising differences from animal repression systems.

From Decision to Commitment: The Molecular Memory of Flowering

Adrian, J., Torti, S., Turck, F. — Tue, 21 Jul 2009 10:28:07 PDT

During the floral transition the shoot apical meristem changes its identity from a vegetative to an inflorescence state. This change in identity can be promoted by external signals, such as inductive photoperiod conditions or vernalization, and is accompanied by changes in expression of key developmental genes. The change in meristem identity is usually not reversible, even if the inductive signal occurs only transiently. This implies that at least some of the key genes must possess an intrinsic memory of the newly acquired expression state that ensures irreversibility of the process. In this review, we discuss different molecular scenarios that may underlie a molecular memory of gene expression.

Temporal and Spatial Requirement of EMF1 Activity for Arabidopsis Vegetative and Reproductive Development

Sanchez, R., Kim, M. Y., Calonje, M., Moon, Y.-H., Sung, Z. R. — Tue, 21 Jul 2009 10:28:07 PDT

EMBRYONIC FLOWER (EMF) genes are required to maintain vegetative development via repression of flower homeotic genes in Arabidopsis. Removal of EMF gene function caused plants to flower upon germination, producing abnormal and sterile flowers. The pleiotropic effect of emf1 mutation suggests its requirement for gene programs involved in diverse developmental processes. Transgenic plants harboring EMF1 promoter::glucuronidase (GUS) reporter gene were generated to investigate the temporal and spatial expression pattern of EMF1. These plants displayed differential GUS activity in vegetative and flower tissues, consistent with the role of EMF1 in regulating multiple gene programs. EMF1::GUS expression pattern in emf mutants suggests organ-specific auto-regulation. Sense- and antisense (as) EMF1 cDNA were expressed under the control of stage- and tissue-specific promoters in transgenic plants. Characterization of these transgenic plants showed that EMF1 activity is required in meristematic as well as differentiating tissues to rescue emf mutant phenotype. Temporal removal or reduction of EMF1 activity in the embryo or shoot apex of wild-type seedlings was sufficient to cause early flowering and terminal flower formation in adult plants. Such reproductive cell memory is reflected in the flower MADS-box gene activity expressed prior to flowering in these early flowering plants. However, temporal removal of EMF1 activity in flower meristem did not affect flower development. Our results are consistent with EMF1’s primary role in repressing flowering in order to allow for vegetative growth.

Control of PHERES1 Imprinting in Arabidopsis by Direct Tandem Repeats

Villar, C. B. R., Erilova, A., Makarevich, G., Trosch, R., Kohler, C. — Tue, 21 Jul 2009 10:28:08 PDT

Genomic imprinting is an epigenetic phenomenon that causes monoallelic expression of specific genes dependent on the parent-of-origin. Imprinting of the Arabidopsis gene PHERES1 requires the function of the FERTILIZATION INDEPENDENT SEED (FIS) Polycomb group complex as well as a distally located methylated region containing a tandem triple repeat sequence. In this study, we investigated the regulation of the close PHERES1 homolog PHERES2. We found that PHERES2 is also a direct target gene of the FIS Polycomb group complex, but, in contrast to PHERES1, PHERES2 is equally expressed from maternal and paternal alleles. Thus, PHERES2 is not regulated by genomic imprinting, correlating with the lack of tandem repeats at PHERES2. Eliminating tandem repeats from the PHERES1 locus abolishes PHERES1 imprinting, demonstrating that tandem repeats are essential for PHERES1 imprinting. Taking these results together, our study shows that the recently duplicated genes PHERES1 and PHERES2 are both target genes of the FIS Polycomb group complex but only PHERES1 is regulated by genomic imprinting, which is likely caused by the presence of repeat sequences in the proximity of the PHERES1 locus.

Defining the Functional Network of Epigenetic Regulators in Arabidopsis thaliana

Luo, C., Durgin, B. G., Watanabe, N., Lam, E. — Tue, 21 Jul 2009 10:28:08 PDT

Development of ChIP-chip and ChIP-seq technologies has allowed genome-wide high-resolution profiling of chromatin-associated marks and binding sites for epigenetic regulators. However, signals for directing epigenetic modifiers to their target sites are not understood. In this paper, we tested the hypothesis that genome location can affect the involvement of epigenetic regulators using Chromatin Charting (CC) Lines, which have an identical transgene construct inserted at different locations in the Arabidopsis genome. Four CC lines that showed evidence for epigenetic silencing of the luciferase reporter gene were transformed with RNAi vectors individually targeting epigenetic regulators LHP1, MOM1, CMT3, DRD1, DRM2, SUVH2, CLF, and HD1. Involvement of a particular epigenetic regulator in silencing the transgene locus in a CC line was determined by significant alterations in luciferase expression after suppression of the regulator's expression. Our results suggest that the targeting of epigenetic regulators can be influenced by genome location as well as sequence context. In addition, the relative importance of an epigenetic regulator can be influenced by tissue identity. We also report a novel approach to predict interactions between epigenetic regulators through clustering analysis of the regulators using alterations in gene expression of putative downstream targets, including endogenous loci and transgenes, in epigenetic mutants or RNAi lines. Our data support the existence of a complex and dynamic network of epigenetic regulators that serves to coordinate and control global gene expression in higher plants.

Arabidopsis MSI1 Is Required for Negative Regulation of the Response to Drought Stress

Alexandre, C., Moller-Steinbach, Y., Schonrock, N., Gruissem, W., Hennig, L. — Tue, 21 Jul 2009 10:28:08 PDT

Arabidopsis MSI1 has fundamental functions in plant development. MSI1 is a subunit of Polycomb group protein complexes and Chromatin assembly factor 1, and it interacts with the Retinoblastoma-related protein 1. Altered levels of MSI1 result in pleiotropic phenotypes, reflecting the complexity of MSI1 protein functions. In order to uncover additional functions of MSI1, we performed transcriptional profiling of wild-type and plants with highly reduced MSI1 levels (msi1-cs). Surprisingly, the known functions of MSI1 could only account for a minor part of the transcriptional changes in msi1-cs plants. One of the most striking unexpected observations was the up-regulation of a subset of ABA-responsive genes eliciting the response to drought and salt stress. We report that MSI1 can bind to the chromatin of the drought-inducible downstream target RD20 and suggest a new role for MSI1 in the negative regulation of the Arabidopsis drought-stress response.

A Truncated Arabidopsis NUCLEOSOME ASSEMBLY PROTEIN 1, AtNAP1;3T, Alters Plant Growth Responses to Abscisic Acid and Salt in the Atnap1;3-2 Mutant

Liu, Z.-Q., Gao, J., Dong, A.-W., Shen, W.-H. — Tue, 21 Jul 2009 10:28:08 PDT

Chromatin remodeling is thought to have crucial roles in plant adaptive response to environmental stimulus. Here, we report that, in Arabidopsis, the evolutionarily conserved histone chaperone, NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1), is involved in plant response to abscisic acid (ABA), a phytohormone important in stress adaptation. We show that simultaneous loss-of-function of AtNAP1;1, AtNAP1;2, and AtNAP1;3 (the triple mutant m123-1) caused a slight hypersensitive response to ABA in seedling growth. Strikingly, the other triple mutant m123-2 containing a different mutant allele of AtNAP1;3, the Atnap1;3-2 allele, showed a hyposensitive response to ABA and a decreased tolerance to salt stress. This ABA-hyposensitive and salt response phenotype specifically associated with the Atnap1;3-2 mutant allele. We show that this mutant allele produced a truncated protein, AtNAP1;3T, which lacks 34 amino acids at the C-terminus compared to the wild-type protein AtNAP1;3. We further show that the heterozygous plants containing the Atnap1;3-2 mutant allele as well as transgenic plants overexpressing AtNAP1;3T exhibit ABA-hyposensitive phenotype. It thus indicates that AtNAP1;3T functions as a dominant negative factor in ABA response. The expression of some ABA-responsive genes, including genes encoding protein kinases and transcription regulators, was found perturbed in the mutant and in the AtNAP1;3T transgenic plants. Taken together, our study uncovered AtNAP1 proteins as positive regulators and AtNAP1;3T as a negative regulator in ABA signaling pathways, providing a novel link of chromatin remodeling to hormonal and stress responses.

RNA Polymerase V Functions in Arabidopsis Interphase Heterochromatin Organization Independently of the 24-nt siRNA-Directed DNA Methylation Pathway

Pontes, O., Costa-Nunes, P., Vithayathil, P., Pikaard, C. S. — Tue, 21 Jul 2009 10:28:08 PDT

In Arabidopsis, pericentromeric repeats, retroelements, and silenced rRNA genes are assembled into heterochromatin within nuclear structures known as chromocenters. The mechanisms governing higher-order heterochromatin organization are poorly understood but 24-nt small interfering RNAs (siRNAs) are known to play key roles in heterochromatin formation. Nuclear RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), and DICER-LIKE 3 (DCL3) are required for biogenesis of 24-nt siRNAs that associate with ARGONAUTE 4 (AGO4). Nuclear RNA polymerase V (Pol V) collaborates with DRD1 (DEFICIENT IN RNA-DEPENDENT DNA METHYLATION 1) to generate transcripts at heterochromatic loci that are hypothesized to bind to siRNA-AGO4 complexes and subsequently recruit the de-novo DNA methylation and/or histone modifying machinery. Here, we report that decondensation of the major pericentromeric repeats and depletion of the heterochromatic mark histone H3 lysine 9 dimethylation at chromocenters occurs specifically in pol V and drd1 mutants. Disruption of pericentromeric repeats condensation is coincident with transcriptional reactivation of specific classes of pericentromeric 180-bp repeats. We further demonstrate that Pol V functions independently of Pol IV, RDR2, and DCL3-mediated siRNA production to affect interphase heterochromatin organization, possibly by involving RNAs that recruit structural or chromatin-modifying proteins.

Expression, Imprinting, and Evolution of Rice Homologs of the Polycomb Group Genes

Luo, M., Platten, D., Chaudhury, A., Peacock, W.J., Dennis, E. S. — Tue, 21 Jul 2009 10:28:08 PDT

Polycomb group proteins (PcG) play important roles in epigenetic regulation of gene expression. Some core PcG proteins, such as Enhancer of Zeste (E(z)), Suppressor of Zeste (12) (Su(z)12), and Extra Sex Combs (ESC), are conserved in plants. The rice genome contains two E(z)-like genes, OsiEZ1 and OsCLF, two homologs of Su(z)12, OsEMF2a and OsEMF2b, and two ESC-like genes, OsFIE1 and OsFIE2. OsFIE1 is expressed only in endosperm; the maternal copy is expressed while the paternal copy is not active. Other rice PcG genes are expressed in a wide range of tissues and are not imprinted in the endosperm. The two E(z)-like genes appear to have duplicated before the separation of the dicots and monocots; the two homologs of Su(z)12 possibly duplicated during the evolution of the Gramineae and the two ESC-like genes are likely to have duplicated in the ancestor of the grasses. No homologs of the Arabidopsis seed-expressed PcG genes MEA and FIS2 were identified in the rice genome. We have isolated T-DNA insertion lines in the rice homologs of three PcG genes. There is no autonomous endosperm development in these T-DNA insertion lines. One line with a T-DNA insertion in OsEMF2b displays pleiotropic phenotypes including altered flowering time and abnormal flower organs, suggesting important roles in rice development for this gene.

Histone Acetylation, VERNALIZATION INSENSITIVE 3, FLOWERING LOCUS C, and the Vernalization Response

Bond, D. M., Dennis, E. S., Pogson, B. J., Finnegan, E. J. — Tue, 21 Jul 2009 10:28:08 PDT

The quantitative induction of VIN3 by low temperatures is required for PRC2 repression of FLC and promotion of flowering (vernalization) in Arabidopsis. Histone acetylation, a chromatin modification commonly associated with gene transcription, increased on VIN3 chromatin in two spatially and temporally distinct phases in response to low temperatures. During short-term cold exposure, histone H3 acetylation at the transcription start site rapidly increased, implying that it is required for VIN3 induction. Subsequent changes in histone H3 and H4 acetylation occurred following continued VIN3 transcription during prolonged cold exposure. Members of the SAGA-like transcriptional adaptor complex, including the histone acetyltransferase GCN5, which induces expression of the cold acclimation pathway genes, do not regulate VIN3 induction during cold exposure, indicating that the cold acclimation pathway and the cold-induction of VIN3 are regulated by different transcriptional mechanisms. Mutations in the other 11 histone acetyltransferase genes did not affect VIN3 induction. However, nicotinamide, a histone deacetylase inhibitor, induced VIN3 and altered histone acetylation at the VIN3 locus. VIN3 induction was proportional to the length of nicotinamide treatment, which was associated with an early-flowering phenotype and repression of FLC. However, unlike vernalization, the repression of FLC was independent of VIN3 activity. Nicotinamide treatment did not cause a change in the expression of any genes in the autonomous pathway or members of the PRC2 complex, the well characterized repressors of FLC. Our data suggest that FLC is repressed via a novel pathway involving the SIR2 class of histone deacetylases.

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

Chen, L.-J., Diao, Z.-Y., Specht, C., Sung, Z. R. — Tue, 21 Jul 2009 10:28:08 PDT

Arabidopsis VERNALIZATION2 (VRN2), EMBRYONIC FLOWER2 (EMF2), and FERTILIZATION-INDEPENDENT SEED2 (FIS2) are involved in vernalization-mediated flowering, vegetative development, and seed development, respectively. Together with Arabidopsis VEF-L36, they share a VEF domain that is conserved in plants and animals. To investigate the evolution of VEF-domain-containing genes (VEF genes), we analyzed sequences related to VEF genes across land plants. To date, 24 full-length sequences from 11 angiosperm families and 54 partial sequences from another nine families were identified. The majority of the full-length sequences identified share greatest sequence similarity with and possess the same major domain structure as Arabidopsis EMF2. EMF2-like sequences are not only widespread among angiosperms, but are also found in genomic sequences of gymnosperms, lycophyte, and moss. No FIS2- or VEF-L36-like sequences were recovered from plants other than Arabidopsis, including from rice and poplar for which whole genomes have been sequenced. Phylogenetic analysis of the full-length sequences showed a high degree of amino acid sequence conservation in EMF2 homologs of closely related taxa. VRN2 homologs are recovered as a clade nested within the larger EMF2 clade. FIS2 and VEF-L36 are recovered in the VRN2 clade. VRN2 clade may have evolved from an EMF2 duplication event that occurred in the rosids prior to the divergence of the eurosid I and eurosid II lineages. We propose that dynamic changes in genome evolution contribute to the generation of the family of VEF-domain-containing genes. Phylogenetic analysis of the VEF domain alone showed that VEF sequences continue to evolve following EMF2/VRN2 divergence in accordance with species relationship. Existence of EMF2-like sequences in animals and across land plants suggests that a prototype form of EMF2 was present prior to the divergence of the plant and animal lineages. A proposed sequence of events, based on domain organization and occurrence of intermediate sequences throughout angiosperms, could explain VRN2 evolution from an EMF2-like ancestral sequence, possibly following duplication of the ancestral EMF2. Available data further suggest that VEF-L36 and FIS2 were derived from a VRN2-like ancestral sequence. Thus, the presence of VEF-L36 and FIS2 in a genome may ultimately be dependent upon the presence of a VRN2-like sequence.

Genome-Wide Analysis Revealed the Complex Regulatory Network of Brassinosteroid Effects in Photomorphogenesis

Song, L., Zhou, X.-Y., Li, L., Xue, L.-J., Yang, X., Xue, H.-W. — Tue, 21 Jul 2009 10:28:08 PDT

Light and brassinosteroids (BRs) have been proved to be crucial in regulating plant growth and development; however, the mechanism of how they synergistically function is still largely unknown. To explore the underlying mechanisms in photomorphogenesis, genome-wide analyses were carried out through examining the gene expressions of the dark-grown WT or BR biosynthesis-defective mutant det2 seedlings in the presence of light stimuli or exogenous Brassinolide (BL). Results showed that BR deficiency stimulates, while BL treatment suppresses, the expressions of light-responsive genes and photomorphogenesis, confirming the negative effects of BR in photomorphogenesis. This is consistent with the specific effects of BR on the expression of genes involved in cell wall modification, cellular metabolism and energy utilization during dark–light transition. Further analysis revealed that hormone biosynthesis and signaling-related genes, especially those of auxin, were altered under BL treatment or light stimuli, indicating that BR may modulate photomorphogenesis through synergetic regulation with other hormones. Additionally, suppressed ubiquitin-cycle pathway during light–dark transition hinted the presence of a complicated network among light, hormone, and protein degradation. The study provides the direct evidence of BR effects in photomorphogenesis and identified the genes involved in BR and light signaling pathway, which will help to elucidate the molecular mechanism of plant photomorphogenesis.

Camouflage Patterning in Maize Leaves Results from a Defect in Porphobilinogen Deaminase

Tue, 21 Jul 2009 10:28:08 PDT

Maize leaves are produced from polarized cell divisions that result in clonal cell lineages arrayed along the long axis of the leaf. We utilized this stereotypical division pattern to identify a collection of mutants that form chloroplast pigmentation sectors that violate the clonal cell lineages. Here, we describe the camouflage1 (cf1) mutant, which develops nonclonal, yellow–green sectors in its leaves. We cloned the cf1 gene by transposon tagging and determined that it encodes porphobilinogen deaminase (PBGD), an enzyme that functions early in chlorophyll and heme biosynthesis. While PBGD has been characterized biochemically, no viable mutations in this gene have been reported in plants. To investigate the in vivo function of PBGD, we characterized the cf1 mutant. Histological analyses revealed that cf1 yellow sectors display the novel phenotype of bundle sheath cell-specific death. Light-shift experiments determined that constant light suppressed cf1 sector formation, a dark/light transition is required to induce yellow sectors, and that sectors form only during a limited time of leaf development. Biochemical experiments determined that cf1 mutant leaves have decreased PBGD activity and increased levels of the enzyme substrate in both green and yellow regions. Furthermore, the cf1 yellow regions displayed a reduction in catalase activity. A threshold model is hypothesized to explain the cf1 variegation and incorporates photosynthetic cell differentiation, reactive oxygen species scavenging, and PBGD function.

CPC, a Single-Repeat R3 MYB, Is a Negative Regulator of Anthocyanin Biosynthesis in Arabidopsis

Zhu, H.-F., Fitzsimmons, K., Khandelwal, A., Kranz, R. G. — Tue, 21 Jul 2009 10:28:08 PDT

Single-repeat R3 MYB transcription factors like CPC (CAPRICE) are known to play roles in developmental processes such as root hair differentiation and trichome initiation. However, none of the six Arabidopsis single-repeat R3 MYB members has been reported to regulate flavonoid biosynthesis. We show here that CPC is a negative regulator of anthocyanin biosynthesis. In the process of using CPC to test GAL4-dependent driver lines, we observed a repression of anthocyanin synthesis upon GAL4-mediated CPC overexpression. We demonstrated that this is not due to an increase in nutrient uptake because of more root hairs. Rather, CPC expression level tightly controls anthocyanin accumulation. Microarray analysis on the whole genome showed that, of 37 000 features tested, 85 genes are repressed greater than three-fold by CPC overexpression. Of these 85, seven are late anthocyanin biosynthesis genes. Also, anthocyanin synthesis genes were shown to be down-regulated in 35S::CPC overexpression plants. Transient expression results suggest that CPC competes with the R2R3–MYB transcription factor PAP1/2, which is an activator of anthocyanin biosynthesis genes. This report adds anthocyanin biosynthesis to the set of programs that are under CPC control, indicating that this regulator is not only for developmental programs (e.g. root hairs, trichomes), but can influence anthocyanin pigment synthesis.

Transcriptome Analysis of Arabidopsis Wild-Type and gl3-sst sim Trichomes Identifies Four Additional Genes Required for Trichome Development

Marks, M. D., Wenger, J. P., Gilding, E., Jilk, R., Dixon, R. A. — Tue, 21 Jul 2009 10:28:08 PDT

Transcriptome analyses have been performed on mature trichomes isolated from wild-type Arabidopsis leaves and on leaf trichomes isolated from the gl3–sst sim double mutant, which exhibit many attributes of immature trichomes. The mature trichome profile contained many highly expressed genes involved in cell wall synthesis, protein turnover, and abiotic stress response. The most highly expressed genes in the gl3–sst sim profile encoded ribosomal proteins and other proteins involved in translation. Comparative analyses showed that all but one of the genes encoding transcription factors previously found to be important for trichome formation, and many other trichome-important genes, were preferentially expressed in gl3–sst sim trichomes. The analysis of genes preferentially expressed in gl3–sst sim led to the identification of four additional genes required for normal trichome development. One of these was the HDG2 gene, which is a member of the HD–ZIP IV transcription factor gene family. Mutations in this gene did not alter trichome expansion, but did alter mature trichome cell walls. Mutations in BLT resulted in a loss of trichome branch formation. The relationship between blt and the phenotypically identical mutant, sti, was explored. Mutations in PEL3, which was previously shown to be required for development of the leaf cuticle, resulted in the occasional tangling of expanding trichomes. Mutations in another gene encoding a protein with an unknown function altered trichome branch formation.

Expression of PIN Genes in Rice (Oryza sativa L.): Tissue Specificity and Regulation by Hormones

Wang, J.-R., Hu, H., Wang, G.-H., Li, J., Chen, J.-Y., Wu, P. — Tue, 21 Jul 2009 10:28:08 PDT

Twelve genes of the PIN family in rice were analyzed for gene and protein structures and an evolutionary relationship with reported AtPINs in Arabidopsis. Four members of PIN1 (designated as OsPIN1a–d), one gene paired with AtPIN2 (OsPIN2), three members of PIN5 (OsPIN5a–c), one gene paired with AtPIN8 (OsPIN8), and three monocot-specific PINs (OsPIN9, OsPIN10a, and b) were identified from the phylogenetic analysis. Tissue-specific expression patterns of nine PIN genes among them were investigated using RT–PCR and GUS reporter. The wide variations in the expression domain in different tissues of the PIN genes were observed. In general, PIN genes are up-regulated by exogenous auxin, while different responses of different PIN genes to other hormones were found.

Expression of the Arabidopsis thaliana Histone Gene AtHTA1 Enhances Rice Transformation Efficiency

Zheng, Y., He, X.-W., Ying, Y.-H., Lu, J.-F., Gelvin, S. B., Shou, H.-X. — Tue, 21 Jul 2009 10:28:08 PDT

We expressed the Arabidopsis thaliana histone AtHTA1 in rice under the control of the maize ubiquitin promoter. Transformation efficiencies of rice plants that constitutively expressed AtHTA1 were 28–44% higher than calli containing an empty vector control. Furthermore, co-infection of rice calli with a vector containing AtHTA1 and another vector with the target gene increased transformation by 27–50%. Thus, expression of AtHTA1 either transiently or in stably transformed cells improved rice transformation efficiency.

Erratum: Activation of Defense Response Pathways by OGs and Flg22 Elicitors in Arabidopsis Seedlings

Tue, 21 Jul 2009 10:28:08 PDT

Editorial

Buchanan, B. B., Dietz, K.-J., Geigenberger, P., Jacquot, J.-P., Schurmann, P. — Wed, 20 May 2009 00:01:40 PDT

Redox Changes during the Legume-Rhizobium Symbiosis

Chang, C., Damiani, I., Puppo, A., Frendo, P. — Wed, 20 May 2009 00:01:40 PDT

Reactive Oxygen Species (ROS) are continuously produced as a result of aerobic metabolism or in response to biotic and abiotic stresses. ROS are not only toxic by-products of aerobic metabolism, but are also signaling molecules involved in plant growth and environmental adaptation. Antioxidants can protect the cell from oxidative damage by scavenging the ROS. Thus, they play an important role in optimizing cell function by regulating cellular redox state and modifying gene expression. This article aims to review recent studies highlighting the role of redox signals in establishing and maintaining symbiosis between rhizobia and legumes.

From Proteomics to Structural Studies of Cytosolic/Mitochondrial-Type Thioredoxin Systems in Barley Seeds

Shahpiri, A., Svensson, B., Finnie, C. — Wed, 20 May 2009 00:01:40 PDT

Thioredoxins (Trx) are ubiquitous proteins that participate in thiol disulfide reactions via two active site cysteine residues, allowing Trx to reduce disulfide bonds in target proteins. Recent progress in proteome analysis has resulted in identification of a wide range of potential target proteins for Trx, indicating that Trx plays a key role in several aspects of cell metabolism. In contrast to other organisms, plants contain multiple forms of Trx that are classified based on their primary structures and sub-cellular localization. The reduction of cytosolic and mitochondrial types of Trx is dependent on NADPH and catalyzed by NADPH-dependent thioredoxin reductase (NTR). In barley, two isoforms each of Trx and NTR have been identified and investigated using proteomics, gene expression, and structural studies. This review outlines the diverse roles suggested for cytosolic/mitochondrial-type Trx systems in cereal seeds and summarizes the current knowledge of the barley system including recent data on function, regulation, interactions, and structure. Directions for future research are discussed.

The Metabolic Response of Arabidopsis Roots to Oxidative Stress is Distinct from that of Heterotrophic Cells in Culture and Highlights a Complex Relationship between the Levels of Transcripts, Metabolites, and Flux

Wed, 20 May 2009 00:01:40 PDT

Metabolic adjustments are a significant, but poorly understood, part of the response of plants to oxidative stress. In a previous study (Baxter et al., 2007), the metabolic response of Arabidopsis cells in culture to induction of oxidative stress by menadione was characterized. An emergency survival strategy was uncovered in which anabolic primary metabolism was largely down-regulated in favour of catabolic and antioxidant metabolism. The response in whole plant tissues may be different and we have therefore investigated the response of Arabidopsis roots to menadione treatment, analyzing the transcriptome, metabolome and key metabolic fluxes with focus on primary as well as secondary metabolism. Using a redox-sensitive GFP, it was also shown that menadione causes redox perturbation, not just in the mitochondrion, but also in the cytosol and plastids of roots. In the first 30 min of treatment, the response was similar to the cell culture: there was a decrease in metabolites of the TCA cycle and amino acid biosynthesis and the transcriptomic response was dominated by up-regulation of DNA regulatory proteins. After 2 and 6 h of treatment, the response of the roots was different to the cell culture. Metabolite levels did not remain depressed, but instead recovered and, in the case of pyruvate, some amino acids and aliphatic glucosinolates showed a steady increase above control levels. However, no major changes in fluxes of central carbon metabolism were observed and metabolic transcripts changed largely independently of the corresponding metabolites. Together, the results suggest that root tissues can recover metabolic activity after oxidative inhibition and highlight potentially important roles for glycolysis and the oxidative pentose phosphate pathway.

The Interaction of Spinach Nitrite Reductase with Ferredoxin: A Site-Directed Mutation Study

Wed, 20 May 2009 00:01:40 PDT

A series of site-directed mutants of the ferredoxin-dependent spinach nitrite reductase has been characterized and several amino acids have been identified that appear to be involved in the interaction of the enzyme with ferredoxin. In a complementary study, binding constants to nitrite reductase and steady-state kinetic parameters of site-directed mutants of ferredoxin were determined in an attempt to identify ferredoxin amino acids involved in the interaction with nitrite reductase. The results have been interpreted in terms of an in-silico docking model for the 1:1 complex of ferredoxin with nitrite reductase.

The Role of Phosphorylation in Redox Regulation of Photosynthesis Genes psaA and psbA during Photosynthetic Acclimation of Mustard

Steiner, S., Dietzel, L., Schroter, Y., Fey, V., Wagner, R., Pfannschmidt, T. — Wed, 20 May 2009 00:01:40 PDT

The long-term response (LTR) to light-quality gradients improves performance and survival of plants in dense stands. It involves redox-controlled transcriptional regulation of the plastome-encoded genes psaAB (encoding the P700 apoproteins of photosystem I) and psbA (encoding the D1 protein of photosystem II) and requires the action of plastid-localized kinases. To study the potential impact of phosphorylation events on plastid gene expression during the LTR, we analyzed mustard seedlings acclimated to light sources favoring either photosystem I or photosystem II. Primer extension analyses of psaA transcripts indicate that the redox regulation occurs at the principal bacterial promoters, suggesting that the plastid encoded RNA polymerase (PEP) is the target for redox signals. Chloroplast protein fractions containing PEP and other DNA-binding proteins were purified from mustard via heparin-Sepharose chromatography. The biochemical properties of these fractions were analyzed with special emphasis on promoter recognition and specificity, phosphorylation state, and kinase activity. The results demonstrate that the LTR involves the action of small DNA-binding proteins; three of them exhibit specific changes in the phosphorylation state. Auto-phosphorylation assays, in addition, exhibit large differences in the activity of endogenous kinase activities. Chloroplast run-on transcription experiments with the kinase inhibitor H7 and the reductant DTT indicate that phosphorylation events are essential for the mediation of redox signals toward psaA and psbA transcription initiation, but require the synergistic action of a thiol redox signal. The data support the idea that redox signals from the thylakoid membrane are linked to gene expression via phosphorylation events; however, this mediation appears to require a complex network of interacting proteins rather than a simple phosphorelay.

The Level of Expression of Thioredoxin is Linked to Fundamental Properties and Applications of Wheat Seeds

Wed, 20 May 2009 00:01:40 PDT

Work with cereals (barley and wheat) and a legume (Medicago truncatula) has established thioredoxin h (Trx h) as a central regulatory protein of seeds. Trx h acts by reducing disulfide (S–S) groups of diverse seed proteins (storage proteins, enzymes, and enzyme inhibitors), thereby facilitating germination. Early in vitro protein studies were complemented with experiments in which barley seeds with Trx h overexpressed in the endosperm showed accelerated germination and early or enhanced expression of associated enzymes (-amylase and pullulanase). The current study extends the transgenic work to wheat. Two approaches were followed to alter the expression of Trx h genes in the endosperm: (1) a hordein promoter and its protein body targeting sequence led to overexpression of Trx h5, and (2) an antisense construct of Trx h9 resulted in cytosolic underexpression of that gene (Arabidopsis designation). Underexpression of Trx h9 led to effects opposite to those observed for overexpression Trx h5 in barley—retardation of germination and delayed or reduced expression of associated enzymes. Similar enzyme changes were observed in developing seeds. The wheat lines with underexpressed Trx showed delayed preharvest sprouting when grown in the greenhouse or field without a decrease in final yield. Wheat with overexpressed Trx h5 showed changes commensurate with earlier in vitro work: increased solubility of disulfide proteins and lower allergenicity of the gliadin fraction. The results are further evidence that the level of Trx h in cereal endosperm determines fundamental properties as well as potential applications of the seed.

Pyridine Nucleotide Cycling and Control of Intracellular Redox State in Relation to Poly (ADP-Ribose) Polymerase Activity and Nuclear Localization of Glutathione during Exponential Growth of Arabidopsis Cells in Culture

Wed, 20 May 2009 00:01:40 PDT

Pyridine nucleotides, ascorbate and glutathione are major redox metabolites in plant cells, with specific roles in cellular redox homeostasis and the regulation of the cell cycle. However, the regulation of these metabolite pools during exponential growth and their precise functions in the cell cycle remain to be characterized. The present analysis of the abundance of ascorbate, glutathione, and pyridine nucleotides during exponential growth of Arabidopsis cells in culture provides evidence for the differential regulation of each of these redox pools. Ascorbate was most abundant early in the growth cycle, but glutathione was low at this point. The cellular ascorbate to dehydroascorbate and reduced glutathione (GSH) to glutathione disulphide ratios were high and constant but the pyridine nucleotide pools were largely oxidized over the period of exponential growth and only became more reduced once growth had ceased. The glutathione pool increased in parallel with poly (ADP-ribose) polymerase (PARP) activities and with increases in the abundance of PARP1 and PARP2 mRNAs at a time of high cell cycle activity as indicated by transcriptome information. Marked changes in the intracellular partitioning of GSH between the cytoplasm and nucleus were observed. Extension of the exponential growth phase by dilution or changing the media led to increases in the glutathione and nicotinamide adenine dinucleotide, oxidized form (NAD)-plus-nicotinamide adenine dinucleotide, reduced form (NADH) pools and to higher NAD/NADH ratios but the nicotinamide adenine dinucleotide phosphate, oxidized form (NADP)-plus-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) pool sizes, and NAPD/NADPH ratios were much less affected. The ascorbate, glutathione, and pyridine nucleotide pools and PARP activity decreased before the exponential growth phase ended. We conclude that there are marked changes in intracellular redox state during the growth cycle but that redox homeostasis is maintained by interplay of the major redox pyridine nucleotides, glutathione, and ascorbate pools. The correlation between PARP expression and activity and GSH accumulation and the finding that GSH can be recruited to the nucleus suggest a relationship between redox regulation and nuclear enzyme activity.

The Quaternary Structure of NADPH Thioredoxin Reductase C Is Redox-Sensitive

Perez-Ruiz, J. M., Gonzalez, M., Spinola, M. C., Sandalio, L. M., Cejudo, F. J. — Wed, 20 May 2009 00:01:40 PDT

NADPH thioredoxin reductase C (NTRC) is a chloroplast enzyme able to conjugate NADPH thioredoxin reductase (NTR) and thioredoxin (TRX) activities for the efficient reduction of 2-Cys peroxiredoxin (2-Cys PRX). Because NADPH can be produced in chloroplasts during darkness, NTRC plays a key role for plant peroxide detoxification during the night. Here, it is shown that the quaternary structure of NTRC is highly dependent on its redox status. In vitro, most of the enzyme adopted an oligomeric state that disaggregated in dimers upon addition of NADPH, NADH, or DTT. Gel filtration and Western blot analysis of protein extracts from Arabidopsis chloroplast stroma showed that native NTRC forms aggregates, which are sensitive to NADPH and DTT, suggesting that the aggregation state might be a significant aspect of NTRC activity in vivo. Moreover, the enzyme is localized in clusters in Arabidopsis chloroplasts. NTRC triple and double mutants, A164G-V182E-R183F and A164G-R183F, replacing key residues of NADPH binding site, showed reduced activity but were still able to dimerize though with an increase in intermediary forms. Based on these results, we propose that the catalytically active form of NTRC is the dimer, which formation is induced by NADPH.

The Presence and Localization of Thioredoxins in Diatoms, Unicellular Algae of Secondary Endosymbiotic Origin

Weber, T., Gruber, A., Kroth, P. G. — Wed, 20 May 2009 00:01:41 PDT

Diatoms are unicellular algae of great ecological importance. So far, very little is known about the regulation of carbon fixation in these algae; however, there are strong indications that in diatom plastids, the ferredoxin/thioredoxin system might play a minor role in redox regulation of the photosynthetic reactions compared to land plants. Until now, it is unknown whether there are fewer or other target enzymes of thioredoxins in diatoms. Only a single potential target enzyme for thioredoxin, the plastidic fructose-1,6-bisphosphatase, has yet been identified. Nevertheless, during the annotation of the genome of the diatom Phaeodactylum tricornutum, we identified several genes encoding different thioredoxins. Utilizing in vivo expression of GFP:presequence fusion proteins in P. tricornutum, we were able to show that these thioredoxins are targeted either into plastids, mitochondria, or remain in the cytosol. Surprisingly, two of the three usually cytosolic thioredoxin h proteins are apparently plastid associated and, together with a thioredoxin reductase, putatively located in the periplastidic compartment. This is one of the few indications for so far unknown enzymatic reactions in the space between the two pairs of diatom plastid envelope membranes.

Between a Rock and a Dry Place: The Water-Stressed Moss

Charron, A. J., Quatrano, R. S. — Wed, 20 May 2009 00:01:41 PDT

The earliest land plants faced a suite of abiotic stresses largely unknown to their aquatic algal ancestors. The descendants of these plants evolved two general mechanisms for survival in the relatively arid aerial environment. While the vascular plants or ‘tracheophytes’ developed tissue specializations to transport and retain water, the other main lineages of land plants, the bryophytes, retained a simple, nonvascular morphology. The bryophytes—mosses, hornworts, and liverworts—continually undergo a co-equilibration of their water content with the surrounding environment and rely to a great extent on intrinsic cellular mechanisms to mitigate damage due to water stress. This short review will focus on the cellular and molecular responses to dehydration and rehydration in mosses, and offer insights into general plant responses to water stress.

A Novel ABA Insensitive Mutant of Lotus japonicus with a Wilty Phenotype Displays Unaltered Nodulation Regulation

Biswas, B., Chan, P. K., Gresshoff, P. M. — Wed, 20 May 2009 00:01:41 PDT

An ABA insensitive mutant, Beyma, was isolated in Lotus japonicus MG-20 from an EMS mutagenesis population using root growth inhibition to applied ABA as the screening criterion. (The name ‘Beyma’ was taken from the Australian Aboriginal language, Wagiman, beyma, meaning ‘drying up’.) The stable mutant that segregates as a dominant Mendelian mutation is insensitive to ABA induced inhibition of germination, vegetative growth, stomatal opening, as well as nodulation. Tissue ABA levels were normal, suggesting a sensitivity rather than biosynthesis mutation. It is slow-growing (50–70% of wild-type MG-20) and has a near-constitutive wilty phenotype associated with its inability to regulate stomatal opening. Whilst showing a wide range of ABA insensitive phenotypes, Beyma did not show alteration of nodule number control, as, in the absence of added ABA, the number and patterning (but not size) of nodules formed in the mutant were similar to that of MG-20. Split root experiments on MG-20 showed that application of ABA on one side of the root inhibited nodulation locally but not systemically. We propose that ABA is not involved directly in systemic autoregulation of nodulation (AON).

Small RNA Pathways Are Present and Functional in the Angiosperm Male Gametophyte

Grant-Downton, R., Hafidh, S., Twell, D., Dickinson, H. G. — Wed, 20 May 2009 00:01:41 PDT

Small non-coding RNAs are essential for development of the sporophyte, the somatic diploid phase of flowering plants. They are integral to key cellular processes such as defense, generation of chromatin structure, and regulation of native gene expression. Surprisingly, very little is known of their presence and function in the male haploid phase of plant development (male gametophyte/pollen grain), where dramatic cell fate changes leading to gametogenesis occur over just two mitotic divisions. We show that critical components of small RNA pathways are expressed throughout pollen development, but in a pattern that differs from the sporophyte. We also demonstrate that mature pollen accumulates a range of mature microRNAs, the class of small RNA most frequently involved in post-transcriptional regulation of endogenous gene expression. Significantly, these miRNAs cleave their target transcripts in developing pollen—a process that seemingly contributes to the purging of key regulatory transcripts from the mature pollen grain. Small RNAs are thus likely to make a hitherto unappreciated contribution to male gametophyte gene expression patterns, pollen development, and gametogenesis.

Arabidopsis Extra Large G-Protein 2 (XLG2) Interacts with the G{beta} Subunit of Heterotrimeric G Protein and Functions in Disease Resistance

Zhu, H., Li, G.-J., Ding, L., Cui, X., Berg, H., Assmann, S. M., Xia, Y. — Wed, 20 May 2009 00:01:41 PDT

Heterotrimeric GTP-binding proteins, which consist of G, Gβ, and G subunits, play important roles in transducing extracellular signals perceived by cell surface receptors into intracellular physiological responses. In addition to a single prototypical G protein (GPA1), Arabidopsis has three unique G-like proteins, known as XLG1, XLG2, and XLG3, that have been found to be localized in nuclei, although their functions and mode of action remain largely unknown. Through a transcriptomic analysis, we found that XLG2 and XLG3 were rapidly induced by infection with the bacterial pathogen Pseudomonas syringae, whereas the XLG1 transcript level was not affected by pathogen infection. A reverse genetic screen revealed that the xlg2 loss-of-function mutation causes enhanced susceptibility to P. syringae. Transcriptome profiling revealed that the xlg2 mutation affects pathogen-triggered induction of a small set of defense-related genes. However, xlg1 and xlg3 mutants showed no difference from wild-type plants in resistance to P. syringae. In addition, the xlg2 xlg3 double mutant and the xlg1 xlg2 xlg3 triple mutant were not significantly different from the xlg2 single mutant in the disease resistance phenotype, suggesting that the roles of XLG1 and XLG3 in defense, if any, are less significant than for XLG2. Constitutive overexpression of XLG2 leads to the accumulation of abnormal transcripts from multiple defense-related genes. Through co-immunoprecipitation assays, XLG2 was found to interact with AGB1, the sole Gβ subunit in Arabidopsis, which has previously been found to be a positive regulator in resistance to necrotrophic fungal pathogens. However, no significant difference was found between three xlg single mutants, the xlg2 xlg3 double mutant, the xlg triple mutant, and wild-type plants in resistance to the necrotrophic fungal pathogens Botrytis cinerea or Alternaria brassicicola. These results suggest that XLG2 and AGB1 are components of a G-protein complex different from the prototypical heterotrimeric G-protein and may have distinct functions in modulating defense responses.

Prefoldins 3 and 5 Play an Essential Role in Arabidopsis Tolerance to Salt Stress

Rodriguez-Milla, M. A., Salinas, J. — Wed, 20 May 2009 00:01:41 PDT

During the last years, our understanding of the mechanisms that control plant response to salt stress has been steadily progressing. Pharmacological studies have allowed the suggestion that the cytoskeleton may be involved in regulating such a response. Nevertheless, genetic evidence establishing that the cytoskeleton has a role in plant tolerance to salt stress has not been reported yet. Here, we have characterized Arabidopsis T-DNA mutants for genes encoding proteins orthologous to prefoldin (PFD) subunits 3 and 5 from yeast and mammals. In these organisms, PFD subunits, also known as Genes Involved in Microtubule biogenesis (GIM), form a heterohexameric PFD complex implicated in tubulin and actin folding. We show that, indeed, PFD3 and PFD5 can substitute for the loss of their yeast orthologs, as they are able to complement yeast gim2 and gim5 mutants, respectively. Our results indicate that pfd3 and pfd5 mutants have reduced levels of - and β-tubulin compared to the wild-type plants when growing under both control and salt-stress conditions. In addition, pfd3 and pfd5 mutants display alterations in their developmental patterns and microtubule organization, and, more importantly, are hypersensitive to high concentrations of NaCl but not of LiCl or mannitol. These results demonstrate that the cytoskeleton plays an essential role in plant tolerance to salt stress.

The Phosphate Transporter PHT4;6 Is a Determinant of Salt Tolerance that Is Localized to the Golgi Apparatus of Arabidopsis

Wed, 20 May 2009 00:01:41 PDT

Insertion mutations that disrupt the function of PHT4;6 (At5g44370) cause NaCl hypersensitivity of Arabidopsis seedlings that is characterized by reduced growth of the primary root, enhanced lateral branching, and swelling of root tips. Mutant phenotypes were exacerbated by sucrose, but not by equiosmolar concentrations of mannitol, and attenuated by low inorganic phosphate in the medium. Protein PHT4;6 belongs to the Major Facilitator Superfamily of permeases that shares significant sequence similarity to mammalian type-I Pi transporters and vesicular glutamate transporters, and is a member of the PHT4 family of putative intracellular phosphate transporters of plants. PHT4;6 localizes to the Golgi membrane and transport studies indicate that PHT4;6 facilitates the selective transport of Pi but not of chloride or inorganic anions. Phenotypic similarities with other mutants displaying root swelling suggest that PHT4;6 likely functions in protein N-glycosylation and cell wall biosynthesis, which are essential for salt tolerance. Together, our results indicate that PHT4;6 transports Pi out of the Golgi lumenal space for the re-cycling of the Pi released from glycosylation processes.