Cell- and development-specific degradation controls the levels of mixed-linkage glucan in sorghum leaves.

Mixed-linkage glucan (MLG) is a component of the cell wall (CW) of grasses and is composed of glucose monomers linked by ß-1,3 and ß-1,4 bonds. MLG is believed to have several biological functions, such as the mobilizable storage of carbohydrates and structural support of the CW. The extracellular levels of MLG are largely controlled by rates of synthesis mediated by cellulose synthase-like (CSL) enzymes, and turnover by lichenases. Economically important crops like sorghum accumulate MLG to variable levels during development. While in sorghum, like other grasses, there is one major MLG synthase (CSLF6), the identity of lichenases is yet unknown. To fill this gap, we identified three sorghum lichenases (SbLCH1-3) and characterized them in leaves in relation to the expression of SbCSLF6, and the abundance of MLG and starch. We established that SbLCH1-3 are secreted to the apoplast, consistent with a role of degrading MLG extracellularly. Furthermore, while SbCSLF6 expression was associated with cell development, the SbLCH genes exhibited distinct development-, cell-type-specific and diel-regulated expression. Therefore, our study identifies three functional sorghum MLG lichenases and highlights that MLG accumulation in sorghum leaves is likely controlled by the activity of lichenases that tune MLG levels, possibly to suit distinct cell and developmental needs in planta. These findings have important implications for improving the growth, yield, and composition of sorghum as a feedstock.

Disruption of Brachypodium lichenase alters metabolism of mixed-linkage glucan and starch.

Mixed-linkage glucan, which is widely distributed in grasses, is a polysaccharide highly abundant in cell walls of grass endosperm and young vegetative tissues. Lichenases are enzymes that hydrolyze mixed-linkage glucan first identified in mixed-linkage glucan-rich lichens. In this study, we identify a gene encoding a lichenase we name Brachypodium distachyon LICHENASE 1 (BdLCH1), which is highly expressed in the endosperm of germinating seeds and coleoptiles and at lower amounts in mature shoots. RNA in situ hybridization showed that BdLCH1 is primarily expressed in chlorenchyma cells of mature leaves and internodes. Disruption of BdLCH1 resulted in an eight-fold increase in mixed-linkage glucan content in senesced leaves. Consistent with the in situ hybridization data, immunolocalization results showed that mixed-linkage glucan was not removed in chlorenchyma cells of lch1 mutants as it was in wild type and implicate the BdLCH1 enzyme in removing mixed-linkage glucan in chlorenchyma cells in mature vegetative tissues. We also show that mixed-linkage glucan accumulation in lch1 mutants was resistant to dark-induced degradation, and 8-week-old lch1 plants showed a faster rate of starch breakdown than wild type in darkness. Our results suggest a role for BdLCH1 in modifying the cell wall to support highly metabolically active cells.

The synthesis of xyloglucan, an abundant plant cell wall polysaccharide, requires CSLC function.

Xyloglucan (XyG) is an abundant component of the primary cell walls of most plants. While the structure of XyG has been well studied, much remains to be learned about its biosynthesis. Here we employed reverse genetics to investigate the role of Arabidopsis cellulose synthase like-C (CSLC) proteins in XyG biosynthesis. We found that single mutants containing a T-DNA in each of the five Arabidopsis CSLC genes had normal levels of XyG. However, higher-order cslc mutants had significantly reduced XyG levels, and a mutant with disruptions in all five CSLC genes had no detectable XyG. The higher-order mutants grew with mild tissue-specific phenotypes. Despite the apparent lack of XyG, the cslc quintuple mutant did not display significant alteration of gene expression at the whole-genome level, excluding transcriptional compensation. The quintuple mutant could be complemented by each of the five CSLC genes, supporting the conclusion that each of them encodes a XyG glucan synthase. Phylogenetic analyses indicated that the CSLC genes are widespread in the plant kingdom and evolved from an ancient family. These results establish the role of the CSLC genes in XyG biosynthesis, and the mutants described here provide valuable tools with which to study both the molecular details of XyG biosynthesis and the role of XyG in plant cell wall structure and function.

The AGCVIII kinase Dw2 modulates cell proliferation, endomembrane trafficking, and MLG/xylan cell wall localization in elongating stem internodes of Sorghum bicolor.

Stems of bioenergy sorghum (Sorghum bicolor L. Moench.), a drought-tolerant C4 grass, contain up to 50 nodes and internodes of varying length that span 4-5 m and account for approximately 84% of harvested biomass. Stem internode growth impacts plant height and biomass accumulation and is regulated by brassinosteroid signaling, auxin transport, and gibberellin biosynthesis. In addition, an AGCVIII kinase (Dw2) regulates sorghum stem internode growth, but the underlying mechanism and signaling network are unknown. Here we provide evidence that mutation of Dw2 reduces cell proliferation in internode intercalary meristems, inhibits endocytosis, and alters the distribution of heteroxylan and mixed linkage glucan in cell walls. Phosphoproteomic analysis showed that Dw2 signaling influences the phosphorylation of proteins involved in lipid signaling (PLDδ), endomembrane trafficking, hormone, light, and receptor signaling, and photosynthesis. Together, our results show that Dw2 modulates endomembrane function and cell division during sorghum internode growth, providing insight into the regulation of monocot stem development.

In the grass species Brachypodium distachyon, the production of mixed-linkage (1,3;1,4)-ß-glucan (MLG) occurs in the Golgi apparatus.

Mixed-linkage (1,3;1,4)-ß-glucan (MLG) is a glucose polymer with beneficial effects on human health and high potential for the agricultural industry. MLG is present predominantly in the cell wall of grasses and is synthesized by cellulose synthase-like F or H families of proteins, with CSLF6 being the best-characterized MLG synthase. Although the function of this enzyme in MLG production has been established, the site of MLG synthesis in the cell is debated. It has been proposed that MLG is synthesized at the plasma membrane, as occurs for cellulose and callose; in contrast, it has also been proposed that MLG is synthesized in the Golgi apparatus, as occurs for other matrix polysaccharides of the cell wall. Testing these conflicting possibilities is fundamentally important in the general understanding of the biosynthesis of the plant cell wall. Using immuno-localization analyses with MLG-specific antibody in Brachypodium and in barley, we found MLG present in the Golgi, in post-Golgi structures and in the cell wall. Accordingly, analyses of a functional fluorescent protein fusion of CSLF6 stably expressed in Brachypodium demonstrated that the enzyme is localized in the Golgi. We also established that overproduction of MLG causes developmental and growth defects in Brachypodium as also occur in barley. Our results indicated that MLG production occurs in the Golgi similarly to other cell wall matrix polysaccharides, and supports the broadly applicable model in grasses that tight mechanisms control optimal MLG accumulation in the cell wall during development and growth. This work addresses the fundamental question of where mixed linkage (1,3;1,4)-ß-glucan (MLG) is synthesized in plant cells. By analyzing the subcellular localization of MLG and MLG synthase in an endogenous system, we demonstrated that MLG synthesis occurs at the Golgi in Brachypodium and barley. A growth inhibition due to overproduced MLG in Brachypodium supports the general applicability of the model that a tight control of the cell wall polysaccharides accumulation is needed to maintain growth homeostasis during development.

A Trihelix Family Transcription Factor Is Associated with Key Genes in Mixed-Linkage Glucan Accumulation.

Mixed-linkage glucan (MLG) is a polysaccharide that is highly abundant in grass endosperm cell walls and present at lower amounts in other tissues. Cellulose synthase-like F (CSLF) and cellulose synthase-like H genes synthesize MLG, but it is unknown if other genes participate in the production and restructuring of MLG. Using Brachypodium distachyon transcriptional profiling data, we identified a B distachyon trihelix family transcription factor (BdTHX1) that is highly coexpressed with the B distachyon CSLF6 gene (BdCSLF6), which suggests that BdTHX1 is involved in the regulation of MLG biosynthesis. To determine the genes regulated by this transcription factor, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) experiments using immature B distachyon seeds and an anti-BdTHX1 polyclonal antibody. The ChIP-seq experiment identified the second intron of BdCSLF6 as one of the most enriched sequences. The binding of BdTHX1 to the BdCSLF6 intron sequence was confirmed using electrophoretic mobility shift assays (EMSA). ChIP-seq also showed that a gene encoding a grass-specific glycoside hydrolase family 16 endotransglucosylase/hydrolase (BdXTH8) is bound by BdTHX1, and the binding was confirmed by EMSA. Radiochemical transglucanase assays showed that BdXTH8 exhibits predominantly MLG:xyloglucan endotransglucosylase activity, a hetero-transglycosylation reaction, and can thus produce MLG-xyloglucan covalent bonds; it also has a lower xyloglucan:xyloglucan endotransglucosylase activity. B distachyon shoots regenerated from transformed calli overexpressing BdTHX1 showed an abnormal arrangement of vascular tissue and seedling-lethal phenotypes. These results indicate that the transcription factor BdTHX1 likely plays an important role in MLG biosynthesis and restructuring by regulating the expression of BdCSLF6 and BdXTH8.

In Brachypodium a complex signaling is actuated to protect cells from proteotoxic stress and facilitate seed filling.

MAIN CONCLUSION: A conserved UPR machinery is required for Brachypodium ER stress resistance and grain filling. Human and livestock diets depend on the accumulation of cereal storage proteins and carbohydrates, including mixed-linkage glucan (MLG), in the endosperm during seed development. Storage proteins and proteins responsible for the production of carbohydrates are synthesized in the endoplasmic reticulum (ER). Unfavorable conditions during growth that hamper the ER biosynthetic capacity, such as heat, can cause a potentially lethal condition known as ER stress, which activates the unfolded protein response (UPR), a signaling response designed to mitigate ER stress. The UPR relies primarily on a conserved ER-associated kinase and ribonuclease, IRE1, which splices the mRNA of a transcription factor (TF), such as bZIP60 in plants, to produce an active TF that controls the expression of ER resident chaperones. Here, we investigated activation of the UPR in Brachypodium, as a model to study the UPR in seeds of a monocotyledon species, as well as the consequences of heat stress on MLG deposition in seeds. We identified a Brachypodium bZIP60 orthologue and determined a positive correlation between bZIP60 splicing and ER stress induced by chemicals and heat. Each stress condition led to transcriptional modulation of several BiP genes, supporting the existence of condition-specific BiP regulation. Finally, we found that the UPR is elevated at the early stage of seed development and that MLG production is negatively affected by heat stress via modulation of MLG synthase accumulation. We propose that successful accomplishment of seed filling is strongly correlated with the ability of the plant to sustain ER stress via the UPR.

Molecular cloning of the tomato Hairless gene implicates actin dynamics in trichome-mediated defense and mechanical properties of stem tissue.

Trichomes are epidermal structures that provide a first line of defense against arthropod herbivores. The recessive hairless (hl) mutation in tomato (Solanum lycopersicum L.) causes severe distortion of trichomes on all aerial tissues, impairs the accumulation of sesquiterpene and polyphenolic compounds in glandular trichomes, and compromises resistance to the specialist herbivore Manduca sexta Here, we demonstrate that the tomato Hl gene encodes a subunit (SRA1) of the highly conserved WAVE regulatory complex that controls nucleation of actin filaments in a wide range of eukaryotic cells. The tomato SRA1 gene spans a 42-kb region containing both Solyc11g013280 and Solyc11g013290 The hl mutation corresponds to a complex 3-kb deletion that removes the last exon of the gene. Expression of a wild-type SRA1 cDNA in the hl mutant background restored normal trichome development, accumulation of glandular trichome-derived metabolites, and resistance to insect herbivory. These findings establish a role for SRA1 in the development of tomato trichomes and also implicate the actin-cytoskeleton network in cytosolic control of specialized metabolism for plant defense. We also show that the brittleness of hl mutant stems is associated with altered mechanical and cell morphological properties of stem tissue, and demonstrate that this defect is directly linked to the mutation in SRA1.

Transcriptional regulation of the raffinose family oligosaccharides pathway in Sorghum bicolor reveals potential roles in leaf sucrose transport and stem sucrose accumulation.

Bioenergy sorghum hybrids are being developed with enhanced drought tolerance and high levels of stem sugars. Raffinose family oligosaccharides (RFOs) contribute to plant environmental stress tolerance, sugar storage, transport, and signaling. To better understand the role of RFOs in sorghum, genes involved in myo-inositol and RFO metabolism were identified and relative transcript abundance analyzed during development. Genes involved in RFO biosynthesis (SbMIPS1, SbInsPase, SbGolS1, SbRS) were more highly expressed in leaves compared to stems and roots, with peak expression early in the morning in leaves. SbGolS, SbRS, SbAGA1 and SbAGA2 were also expressed at high levels in the leaf collar and leaf sheath. In leaf blades, genes involved in myo-inositol biosynthesis (SbMIPS1, SbInsPase) were expressed in bundle sheath cells, whereas genes involved in galactinol and raffinose synthesis (SbGolS1, SbRS) were expressed in mesophyll cells. Furthermore, SbAGA1 and SbAGA2, genes that encode neutral-alkaline alpha-galactosidases that hydrolyze raffinose, were differentially expressed in minor vein bundle sheath cells and major vein and mid-rib vascular and xylem parenchyma. This suggests that raffinose synthesized from sucrose and galactinol in mesophyll cells diffuses into vascular bundles where hydrolysis releases sucrose for long distance phloem transport. Increased expression (>20-fold) of SbAGA1 and SbAGA2 in stem storage pith parenchyma of sweet sorghum between floral initiation and grain maturity, and higher expression in sweet sorghum compared to grain sorghum, indicates these genes may play a key role in non-structural carbohydrate accumulation in stems.

Relevance of the Unfolded Protein Response to Spaceflight-Induced Transcriptional Reprogramming in Arabidopsis.

Plants are primary producers of food and oxygen on Earth and will likewise be indispensable to the establishment of large-scale sustainable ecosystems and human survival in space. To contribute to the understanding of how plants respond to spaceflight stress, we examined the significance of the unfolded protein response (UPR), a conserved signaling cascade that responds to a number of unfavorable environmental stresses, in the model plant Arabidopsis thaliana. To do so, we performed a large-scale comparative transcriptome profiling in wild type and various UPR-defective mutants during the SpaceX-CRS12 mission to the International Space Station. We established that orbital culture substantially alters the expression of hundreds of stress-related genes compared with ground control conditions. Although expression of those genes varied in the UPR mutants on the ground, it was largely similar across the genotypes in the spaceflight condition. Our results have yielded new information on how plants respond to growth in orbit and support the hypothesis that spaceflight induces the activation of signaling pathways that compensate for the loss of UPR regulators in the control of downstream transcriptional regulatory networks.

Systemic signaling contributes to the unfolded protein response of the plant endoplasmic reticulum.

The unfolded protein response (UPR) of the endoplasmic reticulum constitutes a conserved and essential cytoprotective pathway designed to survive biotic and abiotic stresses that alter the proteostasis of the endoplasmic reticulum. The UPR is typically considered cell-autonomous and it is yet unclear whether it can also act systemically through non-cell autonomous signaling. We have addressed this question using a genetic approach coupled with micro-grafting and a suite of molecular reporters in the model plant species Arabidopsis thaliana. We show that the UPR has a non-cell autonomous component, and we demonstrate that this is partially mediated by the intercellular movement of the UPR transcription factor bZIP60 facilitating systemic UPR signaling. Therefore, in multicellular eukaryotes such as plants, non-cell autonomous UPR signaling relies on the systemic movement of at least a UPR transcriptional modulator.