A mixed-linkage (1,3;1,4)-ß-D-glucan specific hydrolase mediates dark-triggered degradation of this plant cell wall polysaccharide.

The presence of mixed-linkage (1,3;1,4)-ß-d-glucan (MLG) in plant cell walls is a key feature of grass species such as cereals, the main source of calorie intake for humans and cattle. Accumulation of this polysaccharide involves the coordinated regulation of biosynthetic and metabolic machineries. While several components of the MLG biosynthesis machinery have been identified in diverse plant species, degradation of MLG is poorly understood. In this study, we performed a large-scale forward genetic screen for maize (Zea mays) mutants with altered cell wall polysaccharide structural properties. As a result, we identified a maize mutant with increased MLG content in several tissues, including adult leaves and senesced organs, where only trace amounts of MLG are usually detected. The causative mutation was found in the GRMZM2G137535 gene, encoding a GH17 licheninase as demonstrated by an in vitro activity assay of the heterologously expressed protein. In addition, maize plants overexpressing GRMZM2G137535 exhibit a 90% reduction in MLG content, indicating that the protein is not only required, but its expression is sufficient to degrade MLG. Accordingly, the mutant was named MLG hydrolase 1 (mlgh1). mlgh1 plants show increased saccharification yields upon enzymatic digestion. Stacking mlgh1 with lignin-deficient mutations results in synergistic increases in saccharification. Time profiling experiments indicate that wall MLG content is modulated during day/night cycles, inversely associated with MLGH1 transcript accumulation. This cycling is absent in the mlgh1 mutant, suggesting that the mechanism involved requires MLG degradation, which may in turn regulate MLGH1 gene expression.

7-Rhamnosylated Flavonols Modulate Homeostasis of the Plant Hormone Auxin and Affect Plant Development.

Flavonols are a group of secondary metabolites that affect diverse cellular processes. They are considered putative negative regulators of the transport of the phytohormone auxin, by which they influence auxin distribution and concomitantly take part in the control of plant organ development. Flavonols are accumulating in a large number of glycosidic forms. Whether these have distinct functions and diverse cellular targets is not well understood. The rol1-2 mutant of Arabidopsis thaliana is characterized by a modified flavonol glycosylation profile that is inducing changes in auxin transport and growth defects in shoot tissues. To determine whether specific flavonol glycosides are responsible for these phenotypes, a suppressor screen was performed on the rol1-2 mutant, resulting in the identification of an allelic series of UGT89C1, a gene encoding a flavonol 7-O-rhamnosyltransferase. A detailed analysis revealed that interfering with flavonol rhamnosylation increases the concentration of auxin precursors and auxin metabolites, whereas auxin transport is not affected. This finding provides an additional level of complexity to the possible ways by which flavonols influence auxin distribution and suggests that flavonol glycosides play an important role in regulating plant development.

Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth.

BACKGROUND: Leucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive. RESULTS: The LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants. CONCLUSIONS: LRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development.

A large-scale forward genetic screen for maize mutants with altered lignocellulosic properties.

The development of efficient pipelines for the bioconversion of grass lignocellulosic feedstocks is challenging due to the limited understanding of the molecular mechanisms controlling the synthesis, deposition, and degradation of the varying polymers unique to grass cell walls. Here, we describe a large-scale forward genetic approach resulting in the identification of a collection of chemically mutagenized maize mutants with diverse alterations in their cell wall attributes such as crystalline cellulose content or hemicellulose composition. Saccharification yield, i.e. the amount of lignocellulosic glucose (Glc) released by means of enzymatic hydrolysis, is increased in two of the mutants and decreased in the remaining six. These mutants, termed candy-leaf (cal), show no obvious plant growth or developmental defects despite associated differences in their lignocellulosic composition. The identified cal mutants are a valuable tool not only to understand recalcitrance of grass lignocellulosics to enzymatic deconstruction but also to decipher grass-specific aspects of cell wall biology once the genetic basis, i.e. the location of the mutation, has been identified.

Flavonols accumulate asymmetrically and affect auxin transport in Arabidopsis.

Flavonoids represent a class of secondary metabolites with diverse functions in plants including ultraviolet protection, pathogen defense, and interspecies communication. They are also known as modulators of signaling processes in plant and animal systems and therefore are considered to have beneficial effects as nutraceuticals. The rol1-2 (for repressor of lrx1) mutation of Arabidopsis (Arabidopsis thaliana) induces aberrant accumulation of flavonols and a cell-growth phenotype in the shoot. The hyponastic cotyledons, aberrant shape of pavement cells, and deformed trichomes in rol1-2 mutants are suppressed by blocking flavonoid biosynthesis, suggesting that the altered flavonol accumulation in these plants induces the shoot phenotype. Indeed, the identification of several transparent testa, myb, and fls1 (for flavonol synthase1) alleles in a rol1-2 suppressor screen provides genetic evidence that flavonols interfere with shoot development in rol1-2 seedlings. The increased accumulation of auxin in rol1-2 seedlings appears to be caused by a flavonol-induced modification of auxin transport. Quantification of auxin export from mesophyll protoplasts revealed that naphthalene-1-acetic acid but not indole-3-acetic acid transport is affected by the rol1-2 mutation. Inhibition of flavonol biosynthesis in rol1-2 fls1-3 restores naphthalene-1-acetic acid transport to wild-type levels, indicating a very specific mode of action of flavonols on the auxin transport machinery.

Defects in Cell Wall Differentiation of the Arabidopsis Mutant rol1-2 Is Dependent on Cyclin-Dependent Kinase CDK8.

Plant cells are encapsulated by cell walls whose properties largely determine cell growth. We have previously identified the rol1-2 mutant, which shows defects in seedling root and shoot development. rol1-2 is affected in the Rhamnose synthase 1 (RHM1) and shows alterations in the structures of Rhamnogalacturonan I (RG I) and RG II, two rhamnose-containing pectins. The data presented here shows that root tissue of the rol1-2 mutant fails to properly differentiate the cell wall in cell corners and accumulates excessive amounts of callose, both of which likely alter the physical properties of cells. A surr (suppressor of the rol1-2 root developmental defect) mutant was identified that alleviates the cell growth defects in rol1-2. The cell wall differentiation defect is re-established in the rol1-2 surr mutant and callose accumulation is reduced compared to rol1-2. The surr mutation is an allele of the cyclin-dependent kinase 8 (CDK8), which encodes a component of the mediator complex that influences processes central to plant growth and development. Together, the identification of the surr mutant suggests that changes in cell wall composition and turnover in the rol1-2 mutant have a significant impact on cell growth and reveals a function of CDK8 in cell wall architecture and composition.

Flavonol-induced changes in PIN2 polarity and auxin transport in the Arabidopsis thaliana rol1-2 mutant require phosphatase activity.

The phytohormone auxin is a major determinant and regulatory component important for plant development. Auxin transport between cells is mediated by a complex system of transporters such as AUX1/LAX, PIN, and ABCB proteins, and their localization and activity is thought to be influenced by phosphatases and kinases. Flavonols have been shown to alter auxin transport activity and changes in flavonol accumulation in the Arabidopsis thaliana rol1-2 mutant cause defects in auxin transport and seedling development. A new mutation in ROOTS CURL IN NPA 1 (RCN1), encoding a regulatory subunit of the phosphatase PP2A, was found to suppress the growth defects of rol1-2 without changing the flavonol content. rol1-2 rcn1-3 double mutants show wild type-like auxin transport activity while levels of free auxin are not affected by rcn1-3. In the rol1-2 mutant, PIN2 shows a flavonol-induced basal-to-apical shift in polar localization which is reversed in the rol1-2 rcn1-3 to basal localization. In vivo analysis of PINOID action, a kinase known to influence PIN protein localization in a PP2A-antagonistic manner, revealed a negative impact of flavonols on PINOID activity. Together, these data suggest that flavonols affect auxin transport by modifying the antagonistic kinase/phosphatase equilibrium.

The protease-associated domain and C-terminal extension are required for zymogen processing, sorting within the secretory pathway, and activity of tomato subtilase 3 (SlSBT3).

A transgenic plant cell suspension culture was established as a versatile and efficient expression system for the subtilase SlSBT3 from tomato. The recombinant protease was purified to homogeneity from culture supernatants by fractionated ammonium sulfate precipitation, batch adsorption to cation exchange material, and anion exchange chromatography. Purified SlSBT3 was identified as a 79-kDa glycoprotein with both complex and paucimannosidic type glycan chains at Asn(177), Asn(203), Asn(376), Asn(697), and Asn(745). SlSBT3 was found to be a very stable enzyme, being fully active at 60 degrees C and showing highest activity at alkaline conditions with a maximum between pH 7.5 and 8.0. Substrate specificity of SlSBT3 was analyzed in detail, revealing a preference for Gln and Lys in the P(1) and P(2) positions of oligopeptide substrates, respectively. Similar to bacterial, yeast, and mammalian subtilases, SlSBT3 is synthesized as a preproenzyme, and processing of the prodomain in the endoplasmic reticulum is a prerequisite for passage through the secretory pathway. SlSBT3 S538A and S538C active site mutants accumulated intracellularly as unprocessed zymogens, indicating that prodomain cleavage occurs autocatalytically. The wild-type SlSBT3 protein failed to cleave the prodomain of the S538A mutant in trans, demonstrating that zymogen maturation is an intramolecular process. Distinguishing features of plant as compared with mammalian subtilases include the insertion of a large protease-associated domain between the His and Ser residues of the catalytic triad and the C-terminal extension to the catalytic domain. Both features were found to be required for SlSBT3 activity and, consequently, for prodomain processing and secretion.

The modified flavonol glycosylation profile in the Arabidopsis rol1 mutants results in alterations in plant growth and cell shape formation.

Flavonoids are secondary metabolites known to modulate plant growth and development. A primary function of flavonols, a subgroup of flavonoids, is thought to be the modification of auxin fluxes in the plant. Flavonols in the cell are glycosylated, and the repressor of lrx1 (rol1) mutants of Arabidopsis thaliana, affected in rhamnose biosynthesis, have a modified flavonol glycosylation profile. A detailed analysis of the rol1-2 allele revealed hyponastic growth, aberrant pavement cell and stomatal morphology in cotyledons, and defective trichome formation. Blocking flavonoid biosynthesis suppresses the rol1-2 shoot phenotype, suggesting that it is induced by the modified flavonol profile. The hyponastic cotyledons of rol1-2 are likely to be the result of a flavonol-induced increase in auxin concentration. By contrast, the pavement cell, stomata, and trichome formation phenotypes appear not to be induced by the modified auxin distribution. Together, these results suggest that changes in the composition of flavonols can have a tremendous impact on plant development through both auxin-induced and auxin-independent processes.