Mutations of Arabidopsis TBL32 and TBL33 Affect Xylan Acetylation and Secondary Wall Deposition

Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be mono- and di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-O-monoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. These results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls.     

The Arabidopsis heterotrimeric G‐protein β subunit,AGB1, is required for guard cell calcium sensing and calcium‐induced calcium release

Cytosolic calcium concentration ([Ca²⁺]_cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Ca_o) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit, AGB1, is required for four guard cell Ca_o responses: induction of stomatal closure; inhibition of stomatal opening; [Ca²⁺]_cyt oscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Ca_o. By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double‐mutants, as well as those of the agg1agg2 Gγ double‐mutant, were insensitive to Ca_o. These behaviors contrast with ABA‐regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6‐detected [Ca²⁺]_cyt oscillations in response to Ca_o, initiating only a single [Ca²⁺]_cyt spike. Experimentally imposed [Ca²⁺]_cyt oscillations restored stomatal closure in agb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Ca_o induced InsP3 production in Col but not in agb1. In sum, G‐protein signaling via AGB1/AGG1/AGG2 is essential for Ca_o‐regulation of stomatal apertures, and stomatal movements in response to Ca_o apparently require Ca²⁺‐induced Ca²⁺ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.     

Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana

The analysis of the interaction between Arabidopsis thaliana and adapted (PcBMM) and nonadapted (Pc2127) isolates of the necrotrophic fungus Plectosphaerella cucumerina has contributed to the identification of molecular mechanisms controlling plant resistance to necrotrophs. To characterize the pathogenicity bases of the virulence of necrotrophic fungi in Arabidopsis, we developed P. cucumerina functional genomics tools using Agrobacterium tumefaciens‐mediated transformation. We generated PcBMM‐GFP and Pc2127‐GFP transformants constitutively expressing the green fluorescence protein (GFP), and a collection of random T‐DNA insertional PcBMM transformants. Confocal microscopy analyses of the initial stages of PcBMM‐GFP infection revealed that this pathogen, like other necrotrophic fungi, does not form an appressorium or penetrate into plant cells, but causes successive degradation of leaf cell layers. By comparing the colonization of Arabidopsis wild‐type plants and hypersusceptible (agb1‐1 and cyp79B2cyp79B3) and resistant (irx1‐6) mutants by PcBMM‐GFP or Pc2127‐GFP, we found that the plant immune response was already mounted at 12–18 h post‐inoculation, and that Arabidopsis resistance to these fungi correlated with the time course of spore germination and hyphal growth on the leaf surface. The virulence of a subset of the PcBMM T‐DNA insertional transformants was determined in Arabidopsis wild‐type plants and agb1‐1 mutant, and several transformants were identified that showed altered virulence in these genotypes in comparison with that of untransformed PcBMM. The T‐DNA flanking regions in these fungal mutants were successfully sequenced, further supporting the utility of these functional genomics tools in the molecular characterization of the pathogenicity of necrotrophic fungi.     

The heterotrimeric G‐protein β subunit,AGB1, plays multiple roles in the Arabidopsis salinity response

Salinity stress includes both osmotic and ionic toxicity. Sodium homeostasis is influenced by Na⁺ uptake and extrusion, vacuolar Na⁺ compartmentation and root to shoot Na⁺ translocation via transpiration. The knockout mutant of the Arabidopsis heterotrimeric G‐protein Gβ subunit, agb1, is hypersensitive to salt, exhibiting a leaf bleaching phenotype. We show that AGB1 is mainly involved in the ionic toxicity component of salinity stress and plays roles in multiple processes of Na⁺ homeostasis. agb1 mutants accumulate more Na⁺ and less K⁺ in both shoots and roots of hydroponically grown plants, as measured by inductively coupled plasma atomic emission spectrometry. agb1 plants have higher root to shoot translocation rates of radiolabelled ²⁴Na⁺ under transpiring conditions, as a result of larger stomatal apertures and increased stomatal conductance. ²⁴Na⁺ tracer experiments also show that ²⁴Na⁺ uptake rates by excised roots of agb1 and wild type are initially equal, but that agb1 has higher net Na⁺ uptake at 90 min, implicating possible involvement of AGB1 in the regulation of Na⁺ efflux. Calcium alleviates the salt hypersensitivity of agb1 by reducing Na⁺ accumulation to below the toxicity threshold. Our results provide new insights into the regulatory pathways underlying plant responses to salinity stress, an important agricultural problem.     

ESKIMO1 disruption in Arabidopsis alters vascular tissue and impairs water transport

Water economy in agricultural practices is an issue that is being addressed through studies aimed at understanding both plant water-use efficiency (WUE), i.e. biomass produced per water consumed, and responses to water shortage. In the model species Arabidopsis thaliana, the ESKIMO1 (ESK1) gene has been described as involved in freezing, cold and salt tolerance as well as in water economy: esk1 mutants have very low evapo-transpiration rates and high water-use efficiency. In order to establish ESK1 function, detailed characterization of esk1 mutants has been carried out. The stress hormone ABA (abscisic acid) was present at high levels in esk1 compared to wild type, nevertheless, the weak water loss of esk1 was independent of stomata closure through ABA biosynthesis, as combining mutant in this pathway with esk1 led to additive phenotypes. Measurement of root hydraulic conductivity suggests that the esk1 vegetative apparatus suffers water deficit due to a defect in water transport. ESK1 promoter-driven reporter gene expression was observed in xylem and fibers, the vascular tissue responsible for the transport of water and mineral nutrients from the soil to the shoots, via the roots. Moreover, in cross sections of hypocotyls, roots and stems, esk1 xylem vessels were collapsed. Finally, using Fourier-Transform Infrared (FTIR) spectroscopy, severe chemical modifications of xylem cell wall composition were highlighted in the esk1 mutants. Taken together our findings show that ESK1 is necessary for the production of functional xylem vessels, through its implication in the laying down of secondary cell wall components.     

UDP‐Api/UDP‐Xyl synthases affect plant development by controlling the content of UDP‐Api to regulate the RG‐II‐borate complex

Rhamnogalacturonan‐II (RG‐II) is structurally the most complex glycan in higher plants, containing 13 different sugars and 21 distinct glycosidic linkages. Two monomeric RG‐II molecules can form an RG‐II‐borate diester dimer through the two apiosyl (Api) residues of side chain A to regulate cross‐linking of pectin in the cell wall. But the relationship of Api biosynthesis and RG‐II dimer is still unclear. In this study we investigated the two homologous UDP‐D‐apiose/UDP‐D‐xylose synthases (AXSs) in Arabidopsis thaliana that synthesize UDP‐D‐apiose (UDP‐Api). Both AXSs are ubiquitously expressed, while AXS2 has higher overall expression than AXS1 in the tissues analyzed. The homozygous axs double mutant is lethal, while heterozygous axs1/+ axs2 and axs1 axs2/+ mutants display intermediate phenotypes. The axs1/+ axs2 mutant plants are unable to set seed and die. By contrast, the axs1 axs2/+ mutant plants exhibit loss of shoot and root apical dominance. UDP‐Api content in axs1 axs2/+ mutants is decreased by 83%. The cell wall of axs1 axs2/+ mutant plants is thicker and contains less RG‐II‐borate complex than wild‐type Col‐0 plants. Taken together, these results provide direct evidence of the importance of AXSs for UDP‐Api and RG‐II‐borate complex formation in plant growth and development.     

The diversion of 2‐C‐methyl‐d‐erythritol‐2,4‐cyclodiphosphate from the 2‐C‐methyl‐d‐erythritol 4‐phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana

2‐C‐Methyl‐d ‐erythritol‐2,4‐cyclodiphosphate (MEcDP) is an intermediate of the plastid‐localized 2‐C‐methyl‐d ‐erythritol‐4‐phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co‐factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds‐3 mutant, defective in the 1‐hydroxy‐2‐methyl‐2‐(E)‐butenyl‐4‐diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2‐C‐methyl‐d ‐erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds‐3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds‐3 mutant also showed enhanced resistance to the phloem‐feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP‐mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds‐3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.     

Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose

Cell wall hemicelluloses and pectins are O‐acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O‐acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody‐tissue‐specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall‐bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a β‐1,4‐endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1‐expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production.     

The tyrosine‐sulfated peptide receptors PSKR1 and PSY1R modify the immunity of Arabidopsis to biotrophic and necrotrophic pathogens in an antagonistic manner

The tyrosine‐sulfated peptides PSKα and PSY1 bind to specific leucine‐rich repeat surface receptor kinases and control cell proliferation in plants. In a reverse genetic screen, we identified the phytosulfokine (PSK) receptor PSKR1 as an important component of plant defense. Multiple independent loss‐of‐function mutants in PSKR1 are more resistant to biotrophic bacteria, show enhanced pathogen‐associated molecular pattern responses and less lesion formation after infection with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. By contrast, pskr1 mutants are more susceptible to necrotrophic fungal infection with Alternaria brassicicola, show more lesion formation and fungal growth which is not observed on wild‐type plants. The antagonistic effect on biotrophic and necrotrophic pathogen resistance is reflected by enhanced salicylate and reduced jasmonate responses in the mutants, suggesting that PSKR1 suppresses salicylate‐dependent defense responses. Detailed analysis of single and multiple mutations in the three paralogous genes PSKR1, ‐2 and PSY1‐receptor (PSY1R) determined that PSKR1 and PSY1R, but not PSKR2, have a partially redundant effect on plant immunity. In animals and plants, peptide sulfation is catalyzed by a tyrosylprotein sulfotransferase (TPST). Mutants lacking TPST show increased resistance to bacterial infection and increased susceptibility to fungal infection, mimicking the triple receptor mutant phenotypes. Feeding experiments with PSKα in tpst‐1 mutants partially restore the defense‐related phenotypes, indicating that perception of the PSKα peptide has a direct effect on plant defense. These results suggest that the PSKR subfamily integrates growth‐promoting and defense signals mediated by sulfated peptides and modulates cellular plasticity to allow flexible adjustment to environmental changes.     

A role for β‐sitosterol to stigmasterol conversion in plant–pathogen interactions

Upon inoculation with pathogenic microbes, plants induce an array of metabolic changes that potentially contribute to induced resistance or even enhance susceptibility. When analysing leaf lipid composition during the Arabidopsis thaliana–Pseudomonas syringae interaction, we found that accumulation of the phytosterol stigmasterol is a significant plant metabolic process that occurs upon bacterial leaf infection. Stigmasterol is synthesized from β‐sitosterol by the cytochrome P450 CYP710A1 via C22 desaturation. Arabidopsis cyp710A1 mutant lines impaired in pathogen‐inducible expression of the C22 desaturase and concomitant stigmasterol accumulation are more resistant to both avirulent and virulent P. syringae strains than wild‐type plants, and exogenous application of stigmasterol attenuates this resistance phenotype. These data indicate that induced sterol desaturation in wild‐type plants favours pathogen multiplication and plant susceptibility. Stigmasterol formation is triggered through perception of pathogen‐associated molecular patterns such as flagellin and lipopolysaccharides, and through production of reactive oxygen species, but does not depend on the salicylic acid, jasmonic acid or ethylene defence pathways. Isolated microsomal and plasma membrane preparations exhibited a similar increase in the stigmasterol/β‐sitosterol ratio as whole‐leaf extracts after leaf inoculation with P. syringae, indicating that the stigmasterol produced is incorporated into plant membranes. The increased contents of stigmasterol in leaves after pathogen attack do not influence salicylic acid‐mediated defence signalling but attenuate pathogen‐induced expression of the defence regulator flavin‐dependent monooxygenase 1. P. syringae thus promotes plant disease susceptibility through stimulation of sterol C22 desaturation in leaves, which increases the stigmasterol to β‐sitosterol ratio in plant membranes.     

Folding thermodynamics of β‐hairpins studied by replica‐exchange molecular dynamics simulations

We study the differences in folding stability of β‐hairpin peptides, including GB1 hairpin and a point mutant GB1 K10G, as well as tryptophan zippers (TrpZips): TrpZip1, TrpZip2, TrpZip3‐1, and TrpZip4. By performing replica‐exchange molecular dynamics simulations with Amber03* force field (a modified version of Amber ff03) in explicit solvent, we observe ab initio folding of all the peptides except TrpZip3‐1, which is experimentally known to be the least stable among the peptides studied here. By calculating the free energies of unfolding of the peptides at room temperature and folding midpoint temperatures for thermal unfolding of peptides, we find that TrpZip4 and GB1 K10G peptides are the most stable β‐hairpins followed by TrpZip1, GB1, and TrpZip2 in the given order. Hence, the proposed K10G mutation of GB1 peptide results in enhanced stability compared to wild‐type GB1. An important goal of our study is to test whether simulations with Amber 03* model can reproduce experimentally predicted folding stability differences between these peptides. While the stabilities of GB1 and TrpZip1 yield close agreement with experiment, TrpZip2 is found to be less stable than predicted by experiment. However, as heterogenous folding of TrpZip2 may yield divergent thermodynamic parameters by different spectroscopic methods, mismatching of results with previous experimental values are not conclusive of model shortcomings. For most of the cases, molecular simulations with Amber03* can successfully reproduce experimentally known differences between the mutated peptides, further highlighting the predictive capabilities of current state‐of‐the‐art all‐atom protein force fields. Proteins 2015; 83:1307–1315. © 2015 Wiley Periodicals, Inc.     

Two Arabidopsis proteins synthesize acetylated xylan in vitro

Xylan is the third most abundant glycopolymer on earth after cellulose and chitin. As a major component of wood, grain and forage, this natural biopolymer has far‐reaching impacts on human life. This highly acetylated cell wall polysaccharide is a vital component of the plant cell wall, which functions as a molecular scaffold, providing plants with mechanical strength and flexibility. Mutations that impair synthesis of the xylan backbone give rise to plants that fail to grow normally because of collapsed xylem cells in the vascular system. Phenotypic analysis of these mutants has implicated many proteins in xylan biosynthesis; however, the enzymes directly responsible for elongation and acetylation of the xylan backbone have not been unambiguously identified. Here we provide direct biochemical evidence that two Arabidopsis thaliana proteins, IRREGULAR XYLEM 10–L (IRX10‐L) and ESKIMO1/TRICOME BIREFRINGENCE 29 (ESK1/TBL29), catalyze these respective processes in vitro. By identifying the elusive xylan synthase and establishing ESK1/TBL29 as the archetypal plant polysaccharide O‐acetyltransferase, we have resolved two long‐standing questions in plant cell wall biochemistry. These findings shed light on integral steps in the molecular pathways used by plants to synthesize a major component of the world's biomass and expand our toolkit for producing glycopolymers with valuable properties.     

The Arabidopsis LecRK‐VI.2 associates with the pattern‐recognition receptor FLS2 and primes Nicotiana benthamiana pattern‐triggered immunity

Pattern‐triggered immunity (PTI) is broad spectrum and manipulation of PTI is believed to represent an attractive way to engineer plants with broad‐spectrum disease resistance. PTI is activated upon perception of microbe‐associated molecular patterns (MAMPs) by pattern‐recognition receptors (PRRs). We have recently demonstrated that the L‐type lectin receptor kinase‐VI.2 (LecRK‐VI.2) positively regulates Arabidopsis thaliana PTI. Here we show through in vitro pull‐down, bimolecular fluorescence complementation and co‐immunoprecipitation analyses that LecRK‐VI.2 associates with the PRR FLS2. We also demonstrated that LecRK‐VI.2 from the cruciferous plant Arabidopsis remains functional after interfamily transfer to the Solanaceous plant Nicotiana benthamiana. Wild tobacco plants ectopically expressing LecRK‐VI.2 were indeed more resistant to virulent hemi‐biotrophic and necrotrophic bacteria, but not to the fungal pathogen Botrytis cinerea suggesting that, as with Arabidopsis, the LecRK‐VI.2 protective effect in N. benthamiana is bacteria specific. Ectopic expression of LecRK‐VI.2 in N. benthamiana primed PTI‐mediated reactive oxygen species production, mitogen‐activated protein kinase (MAPK) activity, callose deposition and gene expression upon treatment with the MAMP flagellin. Our findings identified LecRK‐VI.2 as a member of the FLS2 receptor complex and suggest that heterologous expression of components of PRR complexes can be used as tools to engineer plant disease resistance to bacteria.     

Mutation by DNA shuffling of 5‐enolpyruvylshikimate‐3‐phosphate synthase from Malus domestica for improved glyphosate resistance

A new 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate‐tolerant plants. However, wild‐type MdEPSPS is not suitable for the development of transgenic glyphosate‐tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate‐resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site‐directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single‐site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate‐resistant crops.     

Sugar homeostasis mediated by cell wall invertase GRAIN INCOMPLETE FILLING 1 (GIF1) plays a role in pre‐existing and induced defence in rice

Sugar metabolism and sugar signalling are not only critical for plant growth and development, but are also important for stress responses. However, how sugar homeostasis is involved in plant defence against pathogen attack in the model crop rice remains largely unknown. In this study, we observed that the grains of gif1, a loss‐of‐function mutant of the cell wall invertase gene GRAIN INCOMPLETE FILLING 1 (GIF1), were hypersusceptible to postharvest fungal pathogens, with decreased levels of sugars and a thinner glume cell wall in comparison with the wild‐type. Interestingly, constitutive expression of GIF1 enhanced resistance to both the rice bacterial pathogen Xanthomonas oryzae pv. oryzae and the fungal pathogen Magnaporthe oryzae. The GIF1‐overexpressing (GIF1‐OE) plants accumulated higher levels of glucose, fructose and sucrose compared with the wild‐type plants. More importantly, higher levels of callose were deposited in GIF1‐OE plants during pathogen infection. Moreover, the cell wall was much thicker in the infection sites of the GIF1‐OE plants when compared with the wild‐type plants. We also found that defence‐related genes were constitutively activated in the GIF1‐OE plants. Taken together, our study reveals that sugar homeostasis mediated by GIF1 plays an important role in constitutive and induced physical and chemical defence.