A cellulose synthesis inhibitor affects cellulose synthase complex secretion and cortical microtubule dynamics.

P4B (2-phenyl-1-[4-(6-(piperidin-1-yl) pyridazin-3-yl) piperazin-1-yl] butan-1-one) is a novel cellulose biosynthesis inhibitor (CBI) discovered in a screen for molecules to identify inhibitors of Arabidopsis (Arabidopsis thaliana) seedling growth. Growth and cellulose synthesis inhibition by P4B were greatly reduced in a novel mutant for the cellulose synthase catalytic subunit gene CESA3 (cesa3pbr1). Cross-tolerance to P4B was also observed for isoxaben-resistant (ixr) cesa3 mutants ixr1-1 and ixr1-2. P4B has an original mode of action as compared with most other CBIs. Indeed, short-term treatments with P4B did not affect the velocity of cellulose synthase complexes (CSCs) but led to a decrease in CSC density in the plasma membrane without affecting their accumulation in microtubule-associated compartments. This was observed in the wild type but not in a cesa3pbr1 background. This reduced density correlated with a reduced delivery rate of CSCs to the plasma membrane but also with changes in cortical microtubule dynamics and orientation. At longer timescales, however, the responses to P4B treatments resembled those to other CBIs, including the inhibition of CSC motility, reduced growth anisotropy, interference with the assembly of an extensible wall, pectin demethylesterification, and ectopic lignin and callose accumulation. Together, the data suggest that P4B either directly targets CESA3 or affects another cellular function related to CSC plasma membrane delivery and/or microtubule dynamics that is bypassed specifically by mutations in CESA3.

Cell adhesion in plants is under the control of putative O-fucosyltransferases.

Cell-to-cell adhesion in plants is mediated by the cell wall and the presence of a pectin-rich middle lamella. However, we know very little about how the plant actually controls and maintains cell adhesion during growth and development and how it deals with the dynamic cell wall remodeling that takes place. Here we investigate the molecular mechanisms that control cell adhesion in plants. We carried out a genetic suppressor screen and a genetic analysis of cell adhesion-defective Arabidopsis thaliana mutants. We identified a genetic suppressor of a cell adhesion defect affecting a putative O-fucosyltransferase. Furthermore, we show that the state of cell adhesion is not directly linked with pectin content in the cell wall but instead is associated with altered pectin-related signaling. Our results suggest that cell adhesion is under the control of a feedback signal from the state of the pectin in the cell wall. Such a mechanism could be necessary for the control and maintenance of cell adhesion during growth and development.

Rice Sucrose Partitioning Mediated by a Putative Pectin Methyltransferase and Homogalacturonan Methylesterification.

Homogalacturonan (HG) is the main component of pectins. HG methylesterification has recently emerged as a key determinant controlling cell attachment, organ formation, and phyllotaxy. However, whether and how HG methylesterification affects intercellular metabolite transport has rarely been reported. Here, we identified and characterized knockout mutants of the rice (Oryza sativa) OsQUA2 gene encoding a putative pectin methyltransferase. Osqua2 mutants exhibit a remarkable decrease in the degree of methylesterification of HG in the culm-sieve element cell wall and a markedly reduced grain yield. The culm of Osqua2 mutant plants contains excessive sucrose (Suc), and a 13CO2 feeding experiment showed that the Suc overaccumulation in the culm was caused by blocked Suc translocation. These and other findings demonstrate that OsQUA2 is essential for maintaining a high degree of methylesterification of HG in the rice culm-sieve element cell wall, which may be critical for efficient Suc partitioning and grain filling. In addition, our results suggest that the apoplastic pathway is involved in long-distance Suc transport in rice. The identification and characterization of the OsQUA2 gene and its functionality revealed a previously unknown contribution of HG methylesterification and provided insight into how modification of the cell wall regulates intercellular transport in plants.

Arabidopsis thaliana plants lacking the ARP2/3 complex show defects in cell wall assembly and auxin distribution.

Background and Aim: The cytoskeleton plays an important role in the synthesis of plant cell walls. Both microtubules and actin cytoskeleton are known to be involved in the morphogenesis of plant cells through their role in cell wall building. The role of ARP2/3-nucleated actin cytoskeleton in the morphogenesis of cotyledon pavement cells has been described before. Seedlings of Arabidopsis mutants lacking a functional ARP2/3 complex display specific cell wall-associated defects. Methods: In three independent Arabidopsis mutant lines lacking subunits of the ARP2/3 complex, phenotypes associated with the loss of the complex were analysed throughout plant development. Organ size and anatomy, cell wall composition, and auxin distribution were investigated. Key Results: ARP2/3-related phenotype is associated with changes in cell wall composition, and the phenotype is manifested especially in mature tissues. Cell walls of mature plants contain less cellulose and a higher amount of homogalacturonan, and display changes in cell wall lignification. Vascular bundles of mutant inflorescence stems show a changed pattern of AUX1-YFP expression. Plants lacking a functional ARP2/3 complex have decreased basipetal auxin transport. Conclusions: The results suggest that the ARP2/3 complex has a morphogenetic function related to cell wall synthesis and auxin transport.

GDP-L-fucose is required for boundary definition in plants.

The CUP-SHAPED COTYLEDON (CUC) transcription factors control plant boundary formation, thus allowing the emergence of novel growth axes. While the developmental roles of the CUC genes in different organs and across species are well characterized, upstream and downstream events that contribute to their function are still poorly understood. To identify new players in this network, we performed a suppressor screen of CUC2g-m4, a line overexpressing CUC2 that has highly serrated leaves. We identified a mutation that simplifies leaf shape and affects MURUS1 (MUR1), which is responsible for GDP-L-fucose production. Using detailed morphometric analysis, we show that GDP-L-fucose has an essential role in leaf shape acquisition by sustaining differential growth at the leaf margins. Accordingly, reduced CUC2 expression levels are observed in mur1 leaves. Furthermore, genetic analyses reveal a conserved role for GDP-L-fucose in different developmental contexts where it contributes to organ separation in the same pathway as CUC2. Taken together, our results reveal that GDP-L-fucose is necessary for proper establishment of boundary domains in various developmental contexts.

Suppression of Dwarf and irregular xylem Phenotypes Generates Low-Acetylated Biomass Lines in Arabidopsis.

eskimo1-5 (esk1-5) is a dwarf Arabidopsis (Arabidopsis thaliana) mutant that has a constitutive drought syndrome and collapsed xylem vessels, along with low acetylation levels in xylan and mannan. ESK1 has xylan O-acetyltransferase activity in vitro. We used a suppressor strategy on esk1-5 to screen for variants with wild-type growth and low acetylation levels, a favorable combination for ethanol production. We found a recessive mutation in the KAKTUS (KAK) gene that suppressed dwarfism and the collapsed xylem character, the cause of decreased hydraulic conductivity in the esk1-5 mutant. Backcrosses between esk1-5 and two independent knockout kak mutants confirmed suppression of the esk1-5 effect. kak single mutants showed larger stem diameters than the wild type. The KAK promoter fused with a reporter gene showed activity in the vascular cambium, phloem, and primary xylem in the stem and hypocotyl. However, suppression of the collapsed xylem phenotype in esk1 kak double mutants was not associated with the recovery of cell wall O-acetylation or any major cell wall modifications. Therefore, our results indicate that, in addition to its described activity as a repressor of endoreduplication, KAK may play a role in vascular development. Furthermore, orthologous esk1 kak double mutants may hold promise for ethanol production in crop plants.

LACCASE5 is required for lignification of the Brachypodium distachyon Culm.

The oxidation of monolignols is a required step for lignin polymerization and deposition in cell walls. In dicots, both peroxidases and laccases are known to participate in this process. Here, we provide evidence that laccases are also involved in the lignification of Brachypodium distachyon, a model plant for temperate grasses. Transcript quantification data as well as in situ and immunolocalization experiments demonstrated that at least two laccases (LACCASE5 and LACCASE6) are present in lignifying tissues. A mutant with a misspliced LACCASE5 messenger RNA was identified in a targeting-induced local lesion in genome mutant collection. This mutant shows 10% decreased Klason lignin content and modification of the syringyl-to-guaiacyl units ratio. The amount of ferulic acid units ester linked to the mutant cell walls is increased by 40% when compared with control plants, while the amount of ferulic acid units ether linked to lignins is decreased. In addition, the mutant shows a higher saccharification efficiency. These results provide clear evidence that laccases are required for B. distachyon lignification and are promising targets to alleviate the recalcitrance of grass lignocelluloses.

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.

Polysaccharides and phenolics of miscanthus belowground cell walls and their influence on polyethylene composites.

Belowground materials from two miscanthus species were ground into fragments for preparing polyethylene composites. Both species show a lot of similarities in terms of polysaccharides, lignin and cell wall-linked p-coumaric and ferulic acids contents. The structures of polysaccharides and of lignins are markedly different in the miscanthus belowground and aboveground biomass. The non-cellulosic fraction of the samples comprises a high level of xylose, with the arabinose to xylose ratio about twice as high as that observed for analogous stem samples, suggesting that belowground arabinoxylans are more substituted than stem ones. The mechanical properties of the belowground miscanthus-polyethylene composites correlate with several of their compositional traits, with similar trends as for plant stem-polyethylene composites with positive correlations for lignin and p-coumaric acid contents and negative correlations for most non-cellulosic sugars.

Water Deficit-Responsive QTLs for Cell Wall Degradability and Composition in Maize at Silage Stage.

The use of lignocellulosic biomass for animal feed or biorefinery requires the optimization of its degradability. Moreover, biomass crops need to be better adapted to the changing climate and in particular to periods of drought. Although the negative impact of water deficit on biomass yield has often been mentioned, its impact on biomass quality has only been recently reported in a few species. In the present study, we combined the mapping power of a maize recombinant inbred line population with robust near infrared spectroscopy predictive equations to track the response to water deficit of traits associated with biomass quality. The population was cultivated under two contrasted water regimes over 3 consecutive years in the south of France and harvested at silage stage. We showed that cell wall degradability and ß-O-4-linked H lignin subunits were increased in response to water deficit, while lignin and p-coumaric acid contents were reduced. A mixed linear model was fitted to map quantitative trait loci (QTLs) for agronomical and cell wall-related traits. These QTLs were categorized as "constitutive" (QTL with an effect whatever the irrigation condition) or "responsive" (QTL involved in the response to water deficit) QTLs. Fifteen clusters of QTLs encompassed more than two third of the 213 constitutive QTLs and 13 clusters encompassed more than 60% of the 149 responsive QTLs. Interestingly, we showed that only half of the responsive QTLs co-localized with constitutive and yield QTLs, suggesting that specific genetic factors support biomass quality response to water deficit. Overall, our results demonstrate that water deficit favors cell wall degradability and that breeding of varieties that reconcile improved drought-tolerance and biomass degradability is possible.

Influence of the radial stem composition on the thermal behaviour of miscanthus and sorghum genotypes.

The hypothesis made is that thermal resistance of sorghum and miscanthus stem pieces taken at well-defined positions of the stem is simply related to their biochemical composition. For miscanthus, two different genotypes and two internode levels were selected. For each region, the stem was divided into three radial layers. For sorghum, two different genotypes were selected and the stem was divided into the same three radial layers. The results show that the thermal analysis is only sensitive to very large variations of compositions. But aside of such large composition differences, it is impossible to correlate thermal effects to biochemical composition even on very small size, well-identified pieces of plant materials. The interplay between sugar-based components, lignin and minerals is totally blurring the thermal response. Extreme care must be exercised when willing to explain why a given plant material has a thermal behaviour different of another plant material.