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Salinity severely reduces plant growth and limits agricultural productivity. Dynamic changes and rearrangement of the plant cell wall is an important response to salt stress, but relatively little is known about the biological importance of specific cell wall components in the response. Here, we demonstrate a specific function of β-1,4-galactan in salt hypersensitivity. We found that salt stress induces the accumulation of β-1,4-galactan in root cell walls by up regulating the expression of GALACTAN SYNTHASE 1 (GALS1), which encodes a β-1,4-galactan synthase. The accumulation of β-1,4-galactan negatively affects salt tolerance. Exogenous application of D-galactose (D-Gal) causes an increase in β-1,4-galactan levels in the wild type and GALS1 mutants, especially in GALS1 overexpressors, which correlated with the aggravated salt hypersensitivity. Furthermore, we discovered that the BARLEY B RECOMBINANT/BASIC PENTACYSTEINE transcription factors BPC1/BPC2 positively regulate plant salt tolerance by repressing GALS1 expression and β-1,4-galactan accumulation. Genetic analysis suggested that GALS1 is genetically epistatic to BPC1/BPC2 with respect to the control of salt sensitivity as well as accumulation of β-1,4-galactan. Taken together, our results reveal a new regulatory mechanism by which β-1,4-galactan regulated by the BPC1/BPC2-GALS1 module aggravates salt sensitivity in Arabidopsis thaliana., (Copyright © 2020 The Author. Published by Elsevier Inc. All rights reserved.)

Glycosylinositol phosphorylceramides (GIPCs) constitute an important class of plasma-membrane lipids in plants. The complex glycan headgroups of GIPCs vary between plant species and tissues. Recent studies have shown that the structure of the glycan headgroup is important for plant development, abiotic stress tolerance, and interactions with pathogenic and symbiotic microorganisms., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

l-arabinofuranose is a ubiquitous component of the cell wall and various natural products in plants, where it is synthesized from cytosolic UDP-arabinopyranose (UDP-Arap). The biosynthetic machinery long remained enigmatic in terms of responsible enzymes and subcellular localization. With the discovery of UDP-Arap mutase in plant cytosol, the demonstration of its role in cell-wall arabinose incorporation and the identification of UDP-arabinofuranose transporters in the Golgi membrane, it is clear that the cytosolic UDP-Arap mutases are the key enzymes converting UDP-Arap to UDP-arabinofuranose for cell wall and natural product biosynthesis. This has recently been confirmed by several genotype/phenotype studies. In contrast to the solid evidence pertaining to UDP-Arap mutase function in vivo, the molecular features, including enzymatic mechanism and oligomeric state, remain unknown. However, these enzymes belong to the small family of proteins originally identified as reversibly glycosylated polypeptides (RGPs), which has been studied for >20 years. Here, we review the UDP-Arap mutase and RGP literature together, to summarize and systemize reported molecular characteristics and relations to other proteins., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Plants have evolved various strategies to sense and respond to saline environments, which severely reduce plant growth and limit agricultural productivity. Alteration to the cell wall is one strategy that helps plants adapt to salt stress. However, the physiological mechanism of how the cell wall components respond to salt stress is not fully understood. Here, we show that expression of XTH30, encoding xyloglucan endotransglucosylase-hydrolase30, is strongly up-regulated in response to salt stress in Arabidopsis. Loss-of-function of XTH30 leads to increased salt tolerance and overexpression of XTH30 results in salt hypersensitivity. XTH30 is located in the plasma membrane and is highly expressed in the root, flower, stem, and etiolated hypocotyl. The NaCl-induced increase in xyloglucan (XyG)-derived oligosaccharide (XLFG) of the wild type is partly blocked in xth30 mutants. Loss-of-function of XTH30 slows down the decrease of crystalline cellulose content and the depolymerization of microtubules caused by salt stress. Moreover, lower Na+ accumulation in shoot and lower H2O2 content are found in xth30 mutants in response to salt stress. Taken together, these results indicate that XTH30 modulates XyG side chains, altered abundance of XLFG, cellulose synthesis, and cortical microtubule stability, and negatively affecting salt tolerance., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background: Natural rubber is currently produced nearly exclusively from latex of the Para rubber tree, Hevea brasiliensis. The desire to reduce the environmental cost of rubber production, fears of pathogen susceptibility in clonal Hevea plantations, volatility in the price of natural rubber, and increasing labor costs have motivated efforts to diversify the supply of natural rubber by developing alternative rubber crops such as guayule (Parthenium argentatum Gray). In Hevea, latex is produced as an exudate following wounding while in guayule, rubber is deposited within the cortical parenchyma and its production is strongly influenced by environmental conditions., Results: To better understand the enzymology and regulation of guayule rubber biosynthesis and to identify genes with potential uses in the improvement of rubber yields, we conducted de novo transcriptome assembly and differential gene expression analyses of this process in guayule. This analysis supports a role for rubber in the defense against pathogens, identified new enzymes potentially involved in the biosynthesis of rubber as well as transcription factors specifically expressed in rubber-producing tissues., Conclusions: Data presented here will be useful in the improvement of guayule as an alternative source of natural rubber and in better understanding the biosynthesis of this critical polymer. In particular, some of the candidate transcription factors are likely to control the rubber biosynthesis pathway and are good targets for molecular breeding or engineering of guayule plants with higher and more consistent production of rubber.

Water deficit is a key limiting factor that affects the growth, development and productivity of crops. It is vital to understand the mechanisms by which plants respond to drought stress. Here an N -acetylglutamate kinase gene, ZmNAGK , was cloned from maize ( Zea mays ). ZmNAGK was expressed at high levels in maize leaves and at lower levels in root, stem, female flower and male flower. The expression of ZmNAGK was significantly induced by PEG, NaCl, ABA, brassinosteroid and H 2 O 2 . The ectopic expression of ZmNAGK in tobacco resulted in higher tolerance to drought compared to plants transformed with empty vector. Further physiological analysis revealed that overexpression of ZmNAGK could enhance the activities of antioxidant defense enzymes, and decrease malondialdehyde content and leakage of electrolyte in tobacco under drought stress. Moreover, the ZmNAGK transgenic tobacco accumulated more arginine and nitric oxide (NO) than control plants under drought stress. In addition, the ZmNAGK transgenic tobaccos activated drought responses faster than vector-transformed plants. These results indicate that ZmNAGK can play a vital role in enhancing drought tolerance by likely affecting the arginine and NO accumulation, and ZmNAGK could be involved in different strategies in response to drought stress.

Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure-function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4-β-xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4-β-xylan formation, XYS1/IRX10 in plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE-1 (KfXYS1), possesses 1,4-β-xylan synthase activity, and 1,4-β-xylan occurs in the K. flaccidum cell wall. These data suggest that plant 1,4-β-xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide-based plant cell walls., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Pectins are the most complex polysaccharides of the plant cell wall. Based on the number of methylations, acetylations and glycosidic linkages present in their structures, it is estimated that up to 67 transferase activities are involved in pectin biosynthesis. Pectic galactans constitute a major part of pectin in the form of side-chains of rhamnogalacturonan-I. In Arabidopsis, galactan synthase 1 (GALS1) catalyzes the addition of galactose units from UDP-Gal to growing β-1,4-galactan chains. However, the mechanisms for obtaining varying degrees of polymerization remain poorly understood. In this study, we show that AtGALS1 is bifunctional, catalyzing both the transfer of galactose from UDP-α-d-Gal and the transfer of an arabinopyranose from UDP-β-l-Ara p to galactan chains. The two substrates share a similar structure, but UDP-α-d-Gal is the preferred substrate, with a 10-fold higher affinity. Transfer of Ara p to galactan prevents further addition of galactose residues, resulting in a lower degree of polymerization. We show that this dual activity occurs both in vitro and in vivo. The herein described bifunctionality of AtGALS1 may suggest that plants can produce the incredible structural diversity of polysaccharides without a dedicated glycosyltransferase for each glycosidic linkage., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Background: Increasing the oil yield is a major objective for oilseed crop improvement. Oil biosynthesis and accumulation are influenced by multiple genes involved in embryo and seed development. The leafy cotyledon1 ( LEC1 ) is a master regulator of embryo development that also enhances the expression of genes involved in fatty acid biosynthesis. We speculated that seed oil could be increased by targeted overexpression of a master regulating transcription factor for oil biosynthesis, using a downstream promoter for a gene in the oil biosynthesis pathway. To verify the effect of such a combination on seed oil content, we made constructs with maize ( Zea mays ) ZmLEC1 driven by serine carboxypeptidase-like ( SCPL17 ) and acyl carrier protein ( ACP5) promoters, respectively, for expression in transgenic Arabidopsis thaliana and Camelina sativa ., Results: Agrobacterium-mediated transformation successfully generated Arabidopsis and Camelina lines that overexpressed ZmLEC1 under the control of a seed-specific promoter. This overexpression does not appear to be detrimental to seed vigor under laboratory conditions and did not cause observable abnormal growth phenotypes throughout the life cycle of the plants. Overexpression of ZmLEC1 increased the oil content in mature seeds by more than 20% in Arabidopsis and 26% in Camelina., Conclusion: The findings suggested that the maize master regulator, ZmLEC1, driven by a downstream seed-specific promoter, can be used to increase oil production in Arabidopsis and Camelina and might be a promising target for increasing oil yield in oilseed crops.0.

As the major resource of reactive oxygen species (ROS), the NADPH oxidases (Rbohs) have been shown to play important roles in plant cells under normal growth and stress conditions. Although many family members of Rbohs were studied, little is known about the function of RbohI in Arabidopsis thaliana. Here, we report that exogenous ABA application decreases RbohI expression and mannitol significantly increases RbohI expression at transcript level. The RbohI transcripts were strongly detected in dry seeds and roots. The loss-of-function mutant rbohI exhibited sensitivity to ABA and mannitol stress during germination. Furthermore, the lateral root growth of rbohI was severely inhibited after treatment with mannitol stress. Overexpression of RbohI in Arabidopsis significantly improves the drought tolerance. Moreover, more H 2 O 2 accumulated in RbohI overexpressors than in wild type plants in response to mannitol stress. Our conclusion is that AtRbohI functions in drought-stress response in Arabidopsis thaliana., (Copyright © 2017. Published by Elsevier Inc.)

The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein from Oryza sativa (OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low-dose vector data acquisition. Using size-exclusion chromatography, it is shown that the protein is monomeric in solution. Finally, limited proteolysis was employed to demonstrate DTT-enhanced proteolytic digestion, indicating the existence of at least one intramolecular disulfide bridge or, alternatively, a requirement for a structural metal ion.

Aliphatic and aromatic lipids are both essential structural components of the plant cuticle, an important interface between the plant and environment. Although cross links between aromatic and aliphatic or other moieties are known to be associated with the formation of leaf cutin and root and seed suberin, the contribution of aromatic lipids to the biosynthesis of anther cuticles and pollen walls remains elusive. In this study, we characterized the rice (Oryza sativa) male sterile mutant, defective pollen wall 2 (dpw2), which showed an abnormal anther cuticle, a defective pollen wall, and complete male sterility. Compared with the wild type, dpw2 anthers have increased amounts of cutin and waxes and decreased levels of lipidic and phenolic compounds. DPW2 encodes a cytoplasmically localized BAHD acyltransferase. In vitro assays demonstrated that recombinant DPW2 specifically transfers hydroxycinnamic acid moieties, using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs as acyl donors. Thus, The cytoplasmic hydroxycinnamoyl-CoA:ω-hydroxy fatty acid transferase DPW2 plays a fundamental role in male reproduction via the biosynthesis of key components of the anther cuticle and pollen wall., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi and is required for proper plant growth and development.

Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense and enhance the tolerance of plants to drought stress. However, its downstream molecular events are poorly understood. Here, we identify a NAC transcription factor, ZmNAC84, in maize (Zea mays), which physically interacts with ZmCCaMK in vitro and in vivo. ZmNAC84 displays a partially overlapping expression pattern with ZmCCaMK after ABA treatment, and H2O2 is required for ABA-induced ZmNAC84 expression. Functional analysis reveals that ZmNAC84 is essential for ABA-induced antioxidant defense in a ZmCCaMK-dependent manner. Furthermore, ZmCCaMK directly phosphorylates Ser-113 of ZmNAC84 in vitro, and Ser-113 is essential for the ABA-induced stimulation of antioxidant defense by ZmCCaMK. Moreover, overexpression of ZmNAC84 in tobacco (Nicotiana tabacum) can improve drought tolerance and alleviate drought-induced oxidative damage of transgenic plants. These results define a mechanism for ZmCCaMK function in ABA-induced antioxidant defense, where ABA-produced H2O2 first induces expression of ZmCCaMK and ZmNAC84 and activates ZmCCaMK. Subsequently, the activated ZmCCaMK phosphorylates ZmNAC84 at Ser-113, thereby inducing antioxidant defense by activating downstream genes., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Background: Pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth., Results: T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wild type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content., Conclusions: Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.

Most glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as posttranslational modifications and polysaccharide biosynthesis. Most plant cell wall polysaccharides are biosynthesized in the Golgi apparatus from cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters (NSTs). An exception is UDP-xylose, which is biosynthesized in both the cytosol and the Golgi lumen by a family of UDP-xylose synthases. The NST-based transport of UDP-xylose into the Golgi lumen would appear to be redundant. However, employing a recently developed approach, we identified three UDP-xylose transporters in the Arabidopsis thaliana NST family and designated them UDP-XYLOSE TRANSPORTER1 (UXT1) to UXT3. All three transporters localize to the Golgi apparatus, and UXT1 also localizes to the endoplasmic reticulum. Mutants in UXT1 exhibit ∼30% reduction in xylose in stem cell walls. These findings support the importance of the cytosolic UDP-xylose pool and UDP-xylose transporters in cell wall biosynthesis., (© 2015 American Society of Plant Biologists. All rights reserved.)

Xylans constitute the main non-cellulosic polysaccharide in the secondary cell walls of plants. Several genes predicted to encode glycosyltransferases are required for the synthesis of the xylan backbone even though it is a homopolymer consisting entirely of β-1,4-linked xylose residues. The putative glycosyltransferases IRX9, IRX14, and IRX10 (or the paralogs IRX9L, IRX14L, and IRX10L) are required for xylan backbone synthesis in Arabidopsis. To investigate the function of IRX9, IRX9L, and IRX14, we identified amino acid residues known to be essential for catalytic function in homologous mammalian proteins and generated modified cDNA clones encoding proteins where these residues would be mutated. The mutated gene constructs were used to transform wild-type Arabidopsis plants and the irx9 and irx14 mutants, which are deficient in xylan synthesis. The ability of the mutated proteins to complement the mutants was investigated by measuring growth, determining cell wall composition, and microscopic analysis of stem cross-sections of the transgenic plants. The six different mutated versions of IRX9 and IRX9-L were all able to complement the irx9 mutant phenotype, indicating that residues known to be essential for glycosyltransferases function in homologous proteins are not essential for the biological function of IRX9/IRX9L. Two out of three mutated IRX14 complemented the irx14 mutant, including a mutant in the predicted catalytic amino acid. A IRX14 protein mutated in the substrate-binding DxD motif did not complement the irx14 mutant. Thus, substrate binding is important for IRX14 function but catalytic activity may not be essential for the function of the protein. The data indicate that IRX9/IRX9L have an essential structural function, most likely by interacting with the IRX10/IRX10L proteins, but do not have an essential catalytic function. Most likely IRX14 also has primarily a structural role, but it cannot be excluded that the protein has an important enzymatic activity.

Glycosyl inositol phosphorylceramide (GIPC) sphingolipids are a major class of lipids in fungi, protozoans, and plants. GIPCs are abundant in the plasma membrane in plants, comprising around a quarter of the total lipids in these membranes. Plant GIPCs contain unique glycan decorations that include a conserved glucuronic acid (GlcA) residue and various additional sugars; however, no proteins responsible for glycosylating GIPCs have been identified to date. Here, we show that the Arabidopsis thaliana protein INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE1 (IPUT1) transfers GlcA from UDP-GlcA to GIPCs. To demonstrate IPUT1 activity, we introduced the IPUT1 gene together with genes for a UDP-glucose dehydrogenase from Arabidopsis and a human UDP-GlcA transporter into a yeast mutant deficient in the endogenous inositol phosphorylceramide (IPC) mannosyltransferase. In this engineered yeast strain, IPUT1 transferred GlcA to IPC. Overexpression or silencing of IPUT1 in Nicotiana benthamiana resulted in an increase or a decrease, respectively, in IPC glucuronosyltransferase activity in vitro. Plants in which IPUT1 was silenced accumulated IPC, the immediate precursor, as well as ceramides and glucosylceramides. Plants overexpressing IPUT1 showed an increased content of GIPCs. Mutations in IPUT1 are not transmitted through pollen, indicating that these sphingolipids are essential in plants., (© 2014 American Society of Plant Biologists. All rights reserved.)

Plant cells are surrounded by a rigid wall made up of cellulose microfibrils, pectins, hemicelluloses, and lignin. This cell wall provides structure and protection for plant cells. In grasses and in dicot secondary cell walls, the major hemicellulose is a polymer of β-(1,4)-linked xylose units called xylan. Unlike cellulose--which is synthesized by large complexes at the plasma membrane--xylan is synthesized by enzymes in the Golgi apparatus. Xylan synthesis thus requires the coordinated action and regulation of these synthetic enzymes as well as others that synthesize and transport substrates into the Golgi. Recent research has identified several genes involved in xylan synthesis, some of which have already been used in engineering efforts to create plants that are better suited for biofuel production., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double, triple, and quadruple loss-of-function mutants of all four members of the RWA family in Arabidopsis (Arabidopsis thaliana). In contrast to rwa2, the triple and quadruple rwa mutants display severe growth phenotypes revealing the importance of wall acetylation for plant growth and development. The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco)mannan, and xyloglucan as well as overall cell wall acetylation is affected differently in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell differentiation of cell types with secondary cell walls.

Brassinosteroid (BR)-induced antioxidant defence has been shown to enhance stress tolerance. In this study, the role of the maize 65 kDa microtubule-associated protein (MAP65), ZmMAP65-1a, in BR-induced antioxidant defence was investigated. Treatment with BR increased the expression of ZmMAP65-1a in maize (Zea mays) leaves and mesophyll protoplasts. Transient expression and RNA interference silencing of ZmMAP65-1a in mesophyll protoplasts further revealed that ZmMAP65-1a is required for the BR-induced increase in expression and activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX). Both exogenous and BR-induced endogenous H2O2 increased the expression of ZmMAP65-1a. Conversely, transient expression of ZmMAP65-1a in maize mesophyll protoplasts enhanced BR-induced H2O2 accumulation, while transient silencing of ZmMAP65-1a blocked the BR-induced expression of NADPH oxidase genes and inhibited BR-induced H2O2 accumulation. Inhibiting the activity and gene expression of ZmMPK5 significantly prevented the BR-induced expression of ZmMAP65-1a. Likewise, transient expression of ZmMPK5 enhanced BR-induced activities of the antioxidant defence enzymes SOD and APX in a ZmMAP65- 1a-dependent manner. ZmMPK5 directly interacted with ZmMAP65-1a in vivo and phosphorylated ZmMAP65-1a in vitro. These results suggest that BR-induced antioxidant defence in maize operates through the interaction of ZmMPK5 with ZmMAP65-1a. Furthermore, ZmMAP65-1a functions in H2O2 self-propagation via regulation of the expression of NADPH oxidase genes in BR signalling.

β-1,4-Galactans are abundant polysaccharides in plant cell walls, which are generally found as side chains of rhamnogalacturonan I. Rhamnogalacturonan I is a major component of pectin with a backbone of alternating rhamnose and galacturonic acid residues and side chains that include α-1,5-arabinans, β-1,4-galactans, and arabinogalactans. Many enzymes are required to synthesize pectin, but few have been identified. Pectin is most abundant in primary walls of expanding cells, but β-1,4-galactan is relatively abundant in secondary walls, especially in tension wood that forms in response to mechanical stress. We investigated enzymes in glycosyltransferase family GT92, which has three members in Arabidopsis thaliana, which we designated GALACTAN SYNTHASE1, (GALS1), GALS2 and GALS3. Loss-of-function mutants in the corresponding genes had a decreased β-1,4-galactan content, and overexpression of GALS1 resulted in plants with 50% higher β-1,4-galactan content. The plants did not have an obvious growth phenotype. Heterologously expressed and affinity-purified GALS1 could transfer Gal residues from UDP-Gal onto β-1,4-galactopentaose. GALS1 specifically formed β-1,4-galactosyl linkages and could add successive β-1,4-galactosyl residues to the acceptor. These observations confirm the identity of the GT92 enzyme as β-1,4-galactan synthase. The identification of this enzyme could provide an important tool for engineering plants with improved bioenergy properties.

Background: Cost-efficient generation of second-generation biofuels requires plant biomass that can easily be degraded into sugars and further fermented into fuels. However, lignocellulosic biomass is inherently recalcitrant toward deconstruction technologies due to the abundant lignin and cross-linked hemicelluloses. Furthermore, lignocellulosic biomass has a high content of pentoses, which are more difficult to ferment into fuels than hexoses. Engineered plants with decreased amounts of xylan in their secondary walls have the potential to render plant biomass a more desirable feedstock for biofuel production., Results: Xylan is the major non-cellulosic polysaccharide in secondary cell walls, and the xylan deficient irregular xylem (irx) mutants irx7, irx8 and irx9 exhibit severe dwarf growth phenotypes. The main reason for the growth phenotype appears to be xylem vessel collapse and the resulting impaired transport of water and nutrients. We developed a xylan-engineering approach to reintroduce xylan biosynthesis specifically into the xylem vessels in the Arabidopsis irx7, irx8 and irx9 mutant backgrounds by driving the expression of the respective glycosyltransferases with the vessel-specific promoters of the VND6 and VND7 transcription factor genes. The growth phenotype, stem breaking strength, and irx morphology was recovered to varying degrees. Some of the plants even exhibited increased stem strength compared to the wild type. We obtained Arabidopsis plants with up to 23% reduction in xylose levels and 18% reduction in lignin content compared to wild-type plants, while exhibiting wild-type growth patterns and morphology, as well as normal xylem vessels. These plants showed a 42% increase in saccharification yield after hot water pretreatment. The VND7 promoter yielded a more complete complementation of the irx phenotype than the VND6 promoter., Conclusions: Spatial and temporal deposition of xylan in the secondary cell wall of Arabidopsis can be manipulated by using the promoter regions of vessel-specific genes to express xylan biosynthetic genes. The expression of xylan specifically in the xylem vessels is sufficient to complement the irx phenotype of xylan deficient mutants, while maintaining low overall amounts of xylan and lignin in the cell wall. This engineering approach has the potential to yield bioenergy crop plants that are more easily deconstructed and fermented into biofuels.

Xylan is a major component of the plant cell wall and the most abundant noncellulosic component in the secondary cell walls that constitute the largest part of plant biomass. Dicot glucuronoxylan consists of a linear backbone of β(1,4)-linked xylose residues substituted with α(1,2)-linked glucuronic acid (GlcA). Although several genes have been implicated in xylan synthesis through mutant analyses, the biochemical mechanisms responsible for synthesizing xylan are largely unknown. Here, we show evidence for biochemical activity of GUX1 (for GlcA substitution of xylan 1), a member of Glycosyltransferase Family 8 in Arabidopsis (Arabidopsis thaliana) that is responsible for adding the glucuronosyl substitutions onto the xylan backbone. GUX1 has characteristics typical of Golgi-localized glycosyltransferases and a K(m) for UDP-GlcA of 165 μm. GUX1 strongly favors xylohexaose as an acceptor over shorter xylooligosaccharides, and with xylohexaose as an acceptor, GlcA is almost exclusively added to the fifth xylose residue from the nonreducing end. We also show that several related proteins, GUX2 to GUX5 and Plant Glycogenin-like Starch Initiation Protein6, are Golgi localized and that only two of these proteins, GUX2 and GUX4, have activity as xylan α-glucuronosyltransferases.

Glycosyltransferase complexes are known to be involved in plant cell wall biosynthesis, as for example in cellulose. It is not known to what extent such complexes are involved in biosynthesis of pectin as well. To address this question, work was initiated on ARAD1 (ARABINAN DEFICIENT 1) and its close homolog ARAD2 of glycosyltransferase family GT47. Using bimolecular fluorescence complementation, Förster resonance energy transfer and non-reducing gel electrophoresis, we show that ARAD1 and ARAD2 are localized in the same Golgi compartment and form homo-and heterodimeric intermolecular dimers when expressed transiently in Nicotiana benthamiana. Biochemical analysis of arad2 cell wall or fractions hereof showed no difference in the monosaccharide composition, when compared with wild type. The double mutant arad1 arad2 had an arad1 cell wall phenotype and overexpression of ARAD2 did not complement the arad1 phenotype, indicating that ARAD1 and ARAD2 are not redundant enzymes. To investigate the cell wall structure of the mutants in detail, immunohistochemical analyses were carried out on arad1, arad2 and arad1 arad2 using the arabinan-specific monoclonal antibody LM13. In roots, the labeling pattern of arad2 was distinct from both that of wild type, arad1 and arad1 arad2. Likewise, in epidermal cell walls of inflorescence stems, LM13 binding differed between arad2 and WILD TYPE, arad1 or arad1 arad2. Altogether, these data show that ARAD2 is associated with arabinan biosynthesis, not redundant with ARAD1, and that the two glycosyltransferases may function in complexes held together by disulfide bridges.

A mung bean (Vigna radiata) pectin acetyl esterase (CAA67728) was heterologously expressed in tubers of potato (Solanum tuberosum) under the control of the granule-bound starch synthase promoter or the patatin promoter in order to probe the significance of O-acetylation on cell wall and tissue properties. The recombinant tubers showed no apparent macroscopic phenotype. The enzyme was recovered from transgenic tubers using a high ionic strength buffer and the extract was active against a range of pectic substrates. Partial in vivo de-acetylation of cell wall polysaccharides occurred in the transformants, as shown by a 39% decrease in the degree of acetylation (DA) of tuber cell wall material (CWM). Treatment of CWM using a combination of endo-polygalacturonase and pectin methyl esterase extracted more pectin polymers from the transformed tissue compared to wild type. The largest effect of the pectin acetyl esterase (68% decrease in DA) was seen in the residue from this extraction, suggesting that the enzyme is preferentially active on acetylated pectin that is tightly bound to the cell wall. The effects of acetylation on tuber mechanical properties were investigated by tests of failure under compression and by determination of viscoelastic relaxation spectra. These tests suggested that de-acetylation resulted in a stiffer tuber tissue and a stronger cell wall matrix, as a result of changes to a rapidly relaxing viscoelastic component. These results are discussed in relation to the role of pectin acetylation in primary cell walls and its implications for industrial uses of potato fibres.

The cuticle is a complex aliphatic polymeric layer connected to the cell wall and covers surfaces of all aerial plant organs. The cuticle prevents nonstomatal water loss, regulates gas exchange, and acts as a barrier against pathogen infection. The cuticle is synthesized by epidermal cells and predominantly consists of an aliphatic polymer matrix (cutin) and intracuticular and epicuticular waxes. Cutin monomers are primarily C(16) and C(18) unsubstituted, ω-hydroxy, and α,ω-dicarboxylic fatty acids. Phenolics such as ferulate and p-coumarate esters also contribute to a minor extent to the cutin polymer. Here, we present the characterization of a novel acyl-coenzyme A (CoA)-dependent acyl-transferase that is encoded by a gene designated Deficient in Cutin Ferulate (DCF). The DCF protein is responsible for the feruloylation of ω-hydroxy fatty acids incorporated into the cutin polymer of aerial Arabidopsis (Arabidopsis thaliana) organs. The enzyme specifically transfers hydroxycinnamic acids using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs, preferentially feruloyl-CoA and sinapoyl-CoA, as acyl donors in vitro. Arabidopsis mutant lines carrying DCF loss-of-function alleles are devoid of rosette leaf cutin ferulate and exhibit a 50% reduction in ferulic acid content in stem insoluble residues. DCF is specifically expressed in the epidermis throughout all green Arabidopsis organs. The DCF protein localizes to the cytosol, suggesting that the feruloylation of cutin monomers takes place in the cytoplasm.

Spike mosses are among the most basal vascular plants, and one species, Selaginella moellendorffii, was recently selected for full genome sequencing by the Joint Genome Institute (JGI). Glycosyltransferases (GTs) are involved in many aspects of a plant life, including cell wall biosynthesis, protein glycosylation, primary and secondary metabolism. Here, we present a comparative study of the S. moellendorffii genome across 92 GT families and an additional family (DUF266) likely to include GTs. The study encompasses the moss Physcomitrella patens, a non-vascular land plant, while rice and Arabidopsis represent commelinid and non-commelinid seed plants. Analysis of the subset of GT-families particularly relevant to cell wall polysaccharide biosynthesis was complemented by a detailed analysis of S. moellendorffii cell walls. The S. moellendorffii cell wall contains many of the same components as seed plant cell walls, but appears to differ somewhat in its detailed architecture. The S. moellendorffii genome encodes fewer GTs (287 GTs including DUF266s) than the reference genomes. In a few families, notably GT51 and GT78, S. moellendorffii GTs have no higher plant orthologs, but in most families S. moellendorffii GTs have clear orthologies with Arabidopsis and rice. A gene naming convention of GTs is proposed which takes orthologies and GT-family membership into account. The evolutionary significance of apparently modern and ancient traits in S. moellendorffii is discussed, as is its use as a reference organism for functional annotation of GTs.

The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP) and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER). The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology). We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes.

Glycosyltransferases of the Cellulose Synthase Like D (CSLD) subfamily have been reported to be involved in tip growth and stem development in Arabidopsis. The csld2 and csld3 mutants are root hair defective and the csld5 mutant has reduced stem growth. In this study, we produced double and triple knockout mutants of CSLD2, CSLD3, and CSLD5. Unlike the single mutants and the csld2/csld3 double mutant, the csld2/csld5, csld3/csld5, and csld2/ csld3/csld5 mutants were dwarfed and showed severely reduced viability. This demonstrates that the cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development, and that they are involved in important processes besides the specialized role in tip growth. The mutant phenotypes indicate that CSLD2 and CSLD3 have overlapping functions with CSLD5 in early plant development, whereas the CSLD2 and CSLD3 proteins are non-redundant. To determine the biochemical function of CSLD proteins, we used transient expression in tobacco leaves. Microsomes containing heterologously expressed CSLD5 transferred mannose from GDP-mannose onto endogenous acceptors. The same activity was detected when CSLD2 and CSLD3 were co-expressed but not when they were expressed separately. With monosaccharides as exogenous acceptors, microsomal preparations from CSLD5-expressing plants mediated the transfer of mannose from GDP-mannose onto mannose. These results were supported by immunodetection studies that showed reduced levels of a mannan epitope in the cell walls of stem interfascicular fibers and xylem vessels of the csld2/csld3/csld5 mutant., (© The Author 2011. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.)

Cellulose Synthase Like (CSL) proteins are a group of plant glycosyltransferases that are predicted to synthesize β-1,4-linked polysaccharide backbones. CSLC, CSLF and CSLH families have been confirmed to synthesize xyloglucan and mixed linkage β-glucan, while CSLA family proteins have been shown to synthesize mannans. The polysaccharide products of the five remaining CSL families have not been determined. Five CSLD genes have been identified in Arabidopsis thaliana and a role in cell wall biosynthesis has been demonstrated by reverse genetics. We have extended past research by producing a series of double and triple Arabidopsis mutants and gathered evidence that CSLD2, CSLD3 and CSLD5 are involved in mannan synthesis and that their products are necessary for the transition between early developmental stages in Arabidopsis. Moreover, our data revealed a complex interaction between the three glycosyltransferases and brought new evidence regarding the formation of non-cellulosic polysaccharides through multimeric complexes.

Vascular plants appeared ~410 million years ago, then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes. We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first nonseed vascular plant genome reported. By comparing gene content in evolutionarily diverse taxa, we found that the transition from a gametophyte- to a sporophyte-dominated life cycle required far fewer new genes than the transition from a nonseed vascular to a flowering plant, whereas secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in posttranscriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the trans-acting small interfering RNA pathway, and extensive RNA editing of organellar genes.

L-Ara, an important constituent of plant cell walls, is found predominantly in the furanose rather than in the thermodynamically more stable pyranose form. Nucleotide sugar mutases have been demonstrated to interconvert UDP-Larabinopyranose (UDP-Arap) and UDP-L-arabinofuranose (UDP-Araf) in rice (Oryza sativa). These enzymes belong to a small gene family encoding the previously named Reversibly Glycosylated Proteins (RGPs). RGPs are plant-specific cytosolic proteins that tend to associate with the endomembrane system. In Arabidopsis thaliana, the RGP protein family consists of five closely related members. We characterized all five RGPs regarding their expression pattern and subcellular localizations in transgenic Arabidopsis plants. Enzymatic activity assays of recombinant proteins expressed in Escherichia coli identified three of the Arabidopsis RGP protein family members as UDP-L-Ara mutases that catalyze the formation of UDP-Araf from UDP-Arap. Coimmunoprecipitation and subsequent liquid chromatography-electrospray ionization-tandem mass spectrometry analysis revealed a distinct interaction network between RGPs in different Arabidopsis organs. Examination of cell wall polysaccharide preparations from RGP1 and RGP2 knockout mutants showed a significant reduction in total L-Ara content (12–31%) compared with wild-type plants. Concomitant downregulation of RGP1 and RGP2 expression results in plants almost completely deficient in cell wall–derived L-Ara and exhibiting severe developmental defects.

Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea.

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.

Plant cell walls are composites of various carbohydrates, proteins and other compounds. Cell walls provide plants with strength and protection, and also represent the most abundant source of renewable biomass. Despite the importance of plant cell walls, comparatively little is known about the identities of genes and functions of proteins involved in their biosynthesis. The model plant Arabidopsis and the availability of its genome sequence have been invaluable for the identification and functional characterization of genes encoding enzymes involved in plant cell-wall biosynthesis. This review covers recent progress in the identification and characterization of genes encoding proteins involved in the biosynthesis of Arabidopsis cell-wall polysaccharides and arabinogalactan proteins. These studies have improved our understanding of both the mechanisms of cell-wall biosynthesis and the functions of various cell-wall polymers, and have highlighted areas where further research is needed.

Polysaccharides derived from plant cell wall materials play an important role in our diet. Dietary fibers work as prebiotics in the human gut, and some plant polysaccharides are known to have more direct beneficial impacts on human health. In the food industry, plant fiber materials, such as gums are widely used as structural ingredients. Complex polysaccharides account for most of the plant cell wall, and their biosynthetic pathways and their regulation are largely unknown. Systematic collaborative efforts for defining the structure and impact on human health of beneficial fibers and elucidating the biosynthetic pathways and their regulation will open a great potential for biotechnological applications.

Hemicelluloses are polysaccharides in plant cell walls that have beta-(1-->4)-linked backbones with an equatorial configuration. Hemicelluloses include xyloglucans, xylans, mannans and glucomannans, and beta-(1-->3,1-->4)-glucans. These types of hemicelluloses are present in the cell walls of all terrestrial plants, except for beta-(1-->3,1-->4)-glucans, which are restricted to Poales and a few other groups. The detailed structure of the hemicelluloses and their abundance vary widely between different species and cell types. The most important biological role of hemicelluloses is their contribution to strengthening the cell wall by interaction with cellulose and, in some walls, with lignin. These features are discussed in relation to widely accepted models of the primary wall. Hemicelluloses are synthesized by glycosyltransferases located in the Golgi membranes. Many glycosyltransferases needed for biosynthesis of xyloglucans and mannans are known. In contrast, the biosynthesis of xylans and beta-(1-->3,1-->4)-glucans remains very elusive, and recent studies have led to more questions than answers.

Two Arabidopsis xylosyltransferases, designated RGXT1 and RGXT2, were recently expressed in Baculovirus transfected insect cells and by use of the free sugar assay shown to catalyse transfer of D-xylose from UDP-alpha-D-xylose to L-fucose and derivatives hereof. We have now examined expression of RGXT1 and RGXT2 in Pichia pastoris and compared the two expression systems. Pichia transformants, expressing soluble, secreted forms of RGXT1 and RGXT2 with an N- or C-terminal Flag-tag, accumulated recombinant, hyper-glycosylated proteins at levels between 6 and 16 mg protein * L(-1) in the media fractions. When incubated with 0.5 M L-fucose and UDP-D-xylose all four RGXT1 and RGXT2 variants catalyzed transfer of D-xylose onto L-fucose with estimated turnover numbers between 0.15 and 0.3 sec(-1), thus demonstrating that a free C-terminus is not required for activity. N- and O-glycanase treatment resulted in deglycosylation of all four proteins, and this caused a loss of xylosyltransferase activity for the C-terminally but not the N-terminally Flag-tagged proteins. The RGXT1 and RGXT2 proteins displayed an absolute requirement for Mn(2+) and were active over a broad pH range. Simple dialysis of media fractions or purification on phenyl Sepharose columns increased enzyme activities 2-8 fold enabling direct verification of the product formed in crude assay mixtures using electrospray ionization mass spectrometry. Pichia expressed and dialysed RGXT variants yielded activities within the range 0.011 to 0.013 U (1 U = 1 nmol conversion of substrate * min(-1) * microl medium(-1)) similar to those of RGXT1 and RGXT2 expressed in Baculovirus transfected insect Sf9 cells. In summary, the data presented suggest that Pichia is an attractive host candidate for expression of plant glycosyltransferases.

A cytoplasmically inherited chlorophyll-deficient mutant of barley (Hordeum vulgare) termed cytoplasmic line 3 (CL3), displaying a viridis (homogeneously light-green colored) phenotype, has been previously shown to be affected by elevated temperatures. In this article, biochemical, biophysical, and molecular approaches were used to study the CL3 mutant under different temperature and light conditions. The results lead to the conclusion that an impaired assembly of photosystem I (PSI) under higher temperatures and certain light conditions is the primary cause of the CL3 phenotype. Compromised splicing of ycf3 transcripts, particularly at elevated temperature, resulting from a mutation in a noncoding region (intron 1) in the mutant ycf3 gene results in a defective synthesis of Ycf3, which is a chaperone involved in PSI assembly. The defective PSI assembly causes severe photoinhibition and degradation of PSII.

KORRIGAN1 (KOR1) is a membrane-bound cellulase implicated in cellulose biosynthesis. PttCel9A1 from hybrid aspen (Populus tremula L. x tremuloides Michx.) has high sequence similarity to KOR1 and we demonstrate here that it complements kor1-1 mutants, indicating that it is a KOR1 ortholog. We investigated the function of PttCel9A1/KOR1 in Arabidopsis secondary growth using transgenic lines expressing 35S::PttCel9A1 and the KOR1 mutant line irx2-2. The presence of elevated levels of PttCel9A1/KOR1 in secondary walls of 35S::PttCel9A1 lines was confirmed by in muro visualization of cellulase activity. Compared with the wild type, 35S::PttCel9A1 lines had higher trifluoroacetic acid (TFA)-hydrolyzable glucan contents, similar Updegraff cellulose contents and lower cellulose crystallinity indices, as determined by (13)C solid-state nuclear magnetic resonance (NMR) spectroscopy. irx2-2 mutants had wild-type TFA-hydrolyzable glucan contents, but reduced Updegraff cellulose contents and higher than wild-type cellulose crystallinity indices. The data support the hypothesis that PttCel9A1/KOR1 activity is present in cell walls, where it facilitates cellulose biosynthesis in a way that increases the amount of non-crystalline cellulose.

Thylakoid-soluble phosphoprotein of 9 kDa, TSP9, is an intrinsically unstructured plant-specific protein [Song, J., et al. (2006) Biochemistry 45, 15633-15643] with unknown function but established associations with light-harvesting proteins and peripheries of both photosystems [Hansson, M., et al. (2007) J. Biol. Chem. 282, 16214-16222]. To investigate the function of this protein, we used a combination of reverse genetics and biochemical and fluorescence measurement methods in Arabidopsis thaliana. Differential gene expression analysis of plants with a T-DNA insertion in the TSP9 gene using an array of 24000 Arabidopsis genes revealed disappearance of high light-dependent induction of a specific set of mostly signaling and unknown proteins. TSP9-deficient plants had reduced levels of in vivo phosphorylation of light-harvesting complex II polypeptides. Recombinant TSP9 was phosphorylated in light by thylakoid membranes isolated from the wild-type and mutant plants lacking STN8 protein kinase but not by the thylakoids deficient in STN7 kinase, essential for photosynthetic state transitions. TSP9-lacking mutant and RNAi plants with downregulation of TSP9 showed reduced ability to perform state transitions. The nonphotochemical quenching of chlorophyll fluorescence at high light intensities was also less efficient in the mutant compared to wild-type plants. Blue native electrophoresis of thylakoid membrane protein complexes revealed that TSP9 deficiency increased relative stability of photosystem II dimers and supercomplexes. It is concluded that TSP9 regulates plant light harvesting acting as a membrane-binding protein facilitating dissociation of light-harvesting proteins from photosystem II.

A reverse genetic approach was used to investigate the functions of three members of the cellulose synthase superfamily in Arabidopsis (Arabidopsis thaliana), CELLULOSE SYNTHASE-LIKE D1 (CSLD1), CSLD2, and CSLD4. CSLD2 is required for normal root hair growth but has a different role from that previously described for CSLD3 (KOJAK). CSLD2 is required during a later stage of hair development than CSLD3, and CSLD2 mutants produce root hairs with a range of abnormalities, with many root hairs rupturing late in development. Remarkably, though, it was often the case that in CSLD2 mutants, tip growth would resume after rupturing of root hairs. In silico, semiquantitative reverse transcription-polymerase chain reaction, and promoter-reporter construct analyses indicated that the expression of both CSLD2 and CSLD3 is elevated at reduced temperatures, and the phenotypes of mutants homozygous for insertions in these genes were partially rescued by reduced temperature growth. However, this was not the case for a double mutant homozygous for insertions in both CSLD2 and CSLD3, suggesting that there may be partial redundancy in the functions of these genes. Mutants in CSLD1 and CSLD4 had a defect in male transmission, and plants heterozygous for insertions in CSLD1 or CSLD4 were defective in their ability to produce pollen tubes, although the number and morphology of pollen grains was normal. We propose that the CSLD family of putative glycosyltransferases synthesize a polysaccharide that has a specialized structural role in the cell walls of tip-growing cells.

Summary: AtCYP38 is a thylakoid lumen protein comprising the immunophilin domain and the phosphatase inhibitor module. Here we show the association of AtCYP38 with the photosystem II (PSII) monomer complex and address its functional role using AtCYP38-deficient mutants. The dynamic greening process of etiolated leaves failed in the absence of AtCYP38, due to specific problems in the biogenesis of PSII complexes. Also the development of leaves under short-day conditions was severely disturbed. Detailed biophysical and biochemical analysis of mature AtCYP38-deficient plants from favorable growth conditions (long photoperiod) revealed: (i) intrinsic malfunction of PSII, which (ii) occurred on the donor side of PSII and (iii) was dependent on growing light intensity. AtCYP38 mutant plants also showed decreased accumulation of PSII, which was shown not to originate from impaired D1 synthesis or assembly of PSII monomers, dimers and supercomplexes as such but rather from the incorrect fine-tuning of the oxygen-evolving side of PSII. This, in turn, rendered PSII centers extremely susceptible to photoinhibition. AtCYP38 deficiency also drastically decreased the in vivo phosphorylation of PSII core proteins, probably related to the absence of the AtCYP38 phosphatase inhibitor domain. It is proposed that during PSII assembly AtCYP38 protein guides the proper folding of D1 (and CP43) into PSII, thereby enabling the correct assembly of the water-splitting Mn(4)-Ca cluster even with high turnover of PSII.

Xylogalacturonan (XGA) is a class of pectic polysaccharide found in plant cell walls. The Arabidopsis thaliana locus At5g33290 encodes a predicted Type II membrane protein, and insertion mutants of the At5g33290 locus had decreased cell wall xylose. Immunological studies, enzymatic extraction of polysaccharides, monosaccharide linkage analysis, and oligosaccharide mass profiling were employed to identify the affected cell wall polymer. Pectic XGA was reduced to much lower levels in mutant than in wild-type leaves, indicating a role of At5g33290 in XGA biosynthesis. The mutated gene was designated xylogalacturonan deficient1 (xgd1). Transformation of the xgd1-1 mutant with the wild-type gene restored XGA to wild-type levels. XGD1 protein heterologously expressed in Nicotiana benthamiana catalyzed the transfer of xylose from UDP-xylose onto oligogalacturonides and endogenous acceptors. The products formed could be hydrolyzed with an XGA-specific hydrolase. These results confirm that the XGD1 protein is a XGA xylosyltransferase. The protein was shown by expression of a fluorescent fusion protein in N. benthamiana to be localized in the Golgi vesicles as expected for a glycosyltransferase involved in pectin biosynthesis.

Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed.

Photosystem I (PSI) is a multisubunit protein complex located in the thylakoid membranes of green plants and algae, where it initiates one of the first steps of solar energy conversion by light-driven electron transport. In this review, we discuss recent progress on several topics related to the functioning of the PSI complex, like the protein composition of the complex in the plant Arabidopsis thaliana, the function of these subunits and the mechanism by which nuclear-encoded subunits can be inserted into or transported through the thylakoid membrane. Furthermore, the structure of the native PSI complex in several oxygenic photosynthetic organisms and the role of the chlorophylls and carotenoids in the antenna complexes in light harvesting and photoprotection are reviewed. The special role of the 'red' chlorophylls (chlorophyll molecules that absorb at longer wavelength than the primary electron donor P700) is assessed. The physiology and mechanism of the association of the major light-harvesting complex of photosystem II (LHCII) with PSI during short term adaptation to changes in light quality and quantity is discussed in functional and structural terms. The mechanism of excitation energy transfer between the chlorophylls and the mechanism of primary charge separation is outlined and discussed. Finally, a number of regulatory processes like acclimatory responses and retrograde signalling is reviewed with respect to function of the thylakoid membrane. We finish this review by shortly discussing the perspectives for future research on PSI.