Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees

In nature, angiosperm trees develop tension wood on the upper side of their leaning trunks and drooping branches. Development of tension wood is one of the straightening mechanisms by which trees counteract leaning or bending of stem and resume upward growth. Tension wood is characterized by the development of a highly crystalline cellulose-enriched gelatinous layer next to the lumen of the tension wood fibers. Thus experimental induction of tension wood provides a system to understand the process of cellulose biosynthesis in trees. Since KORRIGAN endoglucanases (KOR) appear to play an important role in cellulose biosynthesis in Arabidopsis, we cloned PtrKOR, a full-length KOR cDNA from aspen xylem. Using RT-PCR, in situ hybridization, and tissue-print assays, we show that PtrKOR gene expression is significantly elevated on the upper side of the bent aspen stem in response to tension stress while KOR expression is significantly suppressed on the opposite side experiencing compression stress. Moreover, three previously reported aspen cellulose synthase genes, namely, PtrCesA1, PtrCesA2, and PtrCesA3 that are closely associated with secondary cell wall development in the xylem cells exhibited similar tension stress-responsive behavior. Our results suggest that coexpression of these four proteins is important for the biosynthesis of highly crystalline cellulose typically present in tension wood fibers. Their simultaneous genetic manipulation may lead to industrially relevant improvement of cellulose in transgenic crops and trees.Suchita Bhandari and Takeshi Fujino contributed equally to this research.     

Three loblolly pine CesA genes expressed in developing xylem are orthologous to secondary cell wall CesA genes of angiosperms

Specific plant cellulose synthases (CesA), encoded by a multigene family, are necessary for secondary wall synthesis in vascular tissues and are critical to wood production. We obtained full-length clones for the three CesAs that are highly expressed in developing xylem and examined their phylogenetic relationships and expression patterns in loblolly pine tissues. Full-length CesA clones were isolated from cDNA of developing loblolly pine (Pinus taeda) xylem and phylogenetic inferences made from plant CesA protein sequences. Expression of the three genes was examined by Northern blot analysis and semiquantitative RT-PCR. Each of three PtCesA genes is orthologous to one of the three angiosperm secondary cell wall CesAs. The PtCesAs are coexpressed in tissues of loblolly pine with tissues undergoing secondary cell wall biosynthesis showing the highest levels of expression. Phylogenetic and expression analyses suggest that functional roles for these loblolly pine CesAs are analogous to those of orthologs in angiosperm taxa. Based upon evidence from this and other studies, we suggest division of seed plant CesA genes into six major paralogous groups, each containing orthologs from various taxa. Available evidence suggests that paralogous CesA genes and their distinct functional roles evolved before the divergence of gymnosperm and angiosperm lineages.     

The roles of the cytoskeleton during cellulose deposition at the secondary cell wall

During secondary cell wall formation, developing xylem vessels deposit cellulose at specific sites on the plasma membrane. Bands of cortical microtubules mark these sites and are believed to somehow orientate the cellulose synthase complexes. We have used live cell imaging on intact roots of Arabidopsis to explore the relationship between the microtubules, actin and the cellulose synthase complex during secondary cell wall formation. The cellulose synthase complexes are seen to form bands beneath sites of secondary wall synthesis. We find that their maintenance at these sites is dependent upon underlying bundles of microtubules which localize the cellulose synthase complex (CSC) to the edges of developing cell wall thickenings. Thick actin cables run along the long axis of the cells. These cables are essential for the rapid trafficking of complex-containing organelles around the cell. The CSCs appear to be delivered directly to sites of secondary cell wall synthesis and it is likely that transverse actin may mark these sites.     

Functional analysis of complexes with mixed primary and secondary cellulose synthases

In higher plants, cellulose is synthesized by cellulose synthase complexes, which contain multiple isoforms of cellulose synthases (CESAs). Among the total 10 CESA genes in Arabidopsis, recessive mutations at three of them cause the collapse of mature xylem cells in inflorescence stems of Arabidopsis (irx1^cesa8, irx3^cesa7 and irx5^cesa4). These CESA genes are considered secondary cell wall CESAs. The others (the function CESA10 is still unknown) are thought to be specialized for cellulose synthesis in the primary cell wall. A split-ubiquitin membrane yeast two-hybrid system was used to assess interactions among four primary CESAs (CESA1, CESA2, CESA3, CESA6) and three secondary CESAs (CESA4, CESA7, CESA8). Our results showed that primary CESAs could physically interact with secondary CESAs in a limited fashion. Analysis of transgenic lines showed that CESA1 could partially rescue irx1^cesa8 null mutants, resulting in complementation of the plant growth defect, collapsed xylem and cellulose content deficiency. These results suggest that mixed primary and secondary CESA complexes are functional using experimental set-ups.     

Gene expression in Eucalyptus branch wood with marked variation in cellulose microfibril orientation and lacking G-layers

In response to gravitational stresses, angiosperm trees form tension wood in the upper sides of branches and leaning stems in which cellulose content is higher, microfibrils are typically aligned closely with the fibre axis and the fibres often have a thick inner gelatinous cell wall layer (G-layer). Gene expression was studied in Eucalyptus nitens branches oriented at 45 degrees using microarrays containing 4900 xylem cDNAs, and wood fibre characteristics revealed by X-ray diffraction, chemical and histochemical methods. Xylem fibres in tension wood (upper branch) had a low microfibril angle, contained few fibres with G-layers and had higher cellulose and decreased Klason lignin compared with lower branch wood. Expression of two closely related fasciclin-like arabinogalactan proteins and a beta-tubulin was inversely correlated with microfibril angle in upper and lower xylem from branches. Structural and chemical modifications throughout the secondary cell walls of fibres sufficient to resist tension forces in branches can occur in the absence of G-layer enriched fibres and some important genes involved in responses to gravitational stress in eucalypt xylem are identified.     

Histone acetyltransferase GCN5 contributes to cell wall integrity and salt stress tolerance by altering the expression of cellulose synthesis genes

Excess soluble salts in soil are harmful to the growth and development of most plants. Evidence is emerging that the plant cell wall is involved in sensing and responding to salt stress, but the underlying mechanisms are not well understood. We reveal that the histone acetyltransferase General control non‐repressed protein 5 (GCN5) is required for the maintenance of cell wall integrity and salt stress tolerance. The levels of GCN5 mRNA are increased in response to salt stress. The gcn5 mutants exhibited severe growth inhibition and defects in cell wall integrity under salt stress conditions. Combining RNA sequencing and chromatin immunoprecipitation assays, we identified the chitinase‐like gene CTL1, polygalacturonase involved in expansion‐3 (PGX3) and MYB domain protein‐54 (MYB54) as direct targets of GCN5. Acetylation of H3K9 and H3K14 mediated by GCN5 is associated with activation of CTL1, PGX3 and MYB54 under salt stress. Moreover, constitutive expression of CTL1 in the gcn5 mutant restores salt tolerance and cell wall integrity. In addition, the expression of the wheat TaGCN5 gene in Arabidopsis gcn5 mutant plants complemented the salt tolerance and cell wall integrity phenotypes, suggesting that GCN5‐mediated salt tolerance is conserved between Arabidopsis and wheat. Taken together, our data indicate that GCN5 plays a key role in the preservation of salt tolerance via versatile regulation in plants.     

A cellulose synthase‐like protein is required for osmotic stress tolerance in Arabidopsis

Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root‐bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6‐1, which defines a locus essential for osmotic stress tolerance. sos6‐1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase‐like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6‐1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress.     

Spatio‐temporal analysis of cellulose synthesis during cell plate formation in Arabidopsis

During cytokinesis a new crosswall is rapidly laid down. This process involves the formation at the cell equator of a tubulo‐vesicular membrane network (TVN). This TVN evolves into a tubular network (TN) and a planar fenestrated sheet, which extends at its periphery before fusing to the mother cell wall. The role of cell wall polymers in cell plate assembly is poorly understood. We used specific stains and GFP‐labelled cellulose synthases (CESAs) to show that cellulose, as well as three distinct CESAs, accumulated in the cell plate already at the TVN stage. This early presence suggests that cellulose is extruded into the tubular membrane structures of the TVN. Co‐localisation studies using GFP–CESAs suggest the delivery of cellulose synthase complexes (CSCs) to the cell plate via phragmoplast‐associated vesicles. In the more mature TN part of the cell plate, we observed delivery of GFP–CESA from doughnut‐shaped organelles, presumably Golgi bodies. During the conversion of the TN into a planar fenestrated sheet, the GFP–CESA density diminished, whereas GFP–CESA levels remained high in the TVN zone at the periphery of the expanding cell plate. We observed retrieval of GFP–CESA in clathrin‐containing structures from the central zone of the cell plate and from the plasma membrane of the mother cell, which may contribute to the recycling of CESAs to the peripheral growth zone of the cell plate. These observations, together with mutant phenotypes of cellulose‐deficient mutants and pharmacological experiments, suggest a key role for cellulose synthesis already at early stages of cell plate assembly.     

The trafficking of the cellulose synthase complex in higher plants

Background

Cellulose is an important constituent of plant cell walls in a biological context, and is also a material commonly utilized by mankind in the pulp and paper, timber, textile and biofuel industries. The biosynthesis of cellulose in higher plants is a function of the cellulose synthase complex (CSC). The CSC, a large transmembrane complex containing multiple cellulose synthase proteins, is believed to be assembled in the Golgi apparatus, but is thought only to synthesize cellulose when it is localized at the plasma membrane, where CSCs synthesize and extrude cellulose directly into the plant cell wall. Therefore, the delivery and endocytosis of CSCs to and from the plasma membrane are important aspects for the regulation of cellulose biosynthesis.

Scope

Recent progress in the visualization of CSC dynamics in living plant cells has begun to reveal some of the routes and factors involved in CSC trafficking. This review highlights the most recent major findings related to CSC trafficking, provides novel perspectives on how CSC trafficking can influence the cell wall, and proposes potential avenues for future exploration.     

Differential genetic interactions of yeast stress response MAPK pathways

Genetic interaction screens have been applied with great success in several organisms to study gene function and the genetic architecture of the cell. However, most studies have been performed under optimal growth conditions even though many functional interactions are known to occur under specific cellular conditions. In this study, we have performed a large‐scale genetic interaction analysis in Saccharomyces cerevisiae involving approximately 49 × 1,200 double mutants in the presence of five different stress conditions, including osmotic, oxidative and cell wall‐altering stresses. This resulted in the generation of a differential E‐MAP (or dE‐MAP) comprising over 250,000 measurements of conditional interactions. We found an extensive number of conditional genetic interactions that recapitulate known stress‐specific functional associations. Furthermore, we have also uncovered previously unrecognized roles involving the phosphatase regulator Bud14, the histone methylation complex COMPASS and membrane trafficking complexes in modulating the cell wall integrity pathway. Finally, the osmotic stress differential genetic interactions showed enrichment for genes coding for proteins with conditional changes in phosphorylation but not for genes with conditional changes in gene expression. This suggests that conditional genetic interactions are a powerful tool to dissect the functional importance of the different response mechanisms of the cell.     

Development of 15 novel microsatellite markers from cellulose synthase genes in Populus tomentosa (Salicaceae)

? Premise of the study: Microsatellite markers from cellulose synthase genes were developed for the Chinese white poplar, Populus tomentosa, to investigate the genetic diversity of wild germplasm resources and to further identify favorable alleles significantly associated with wood cellulose content. ? Methods and Results: Fifteen microsatellite markers were developed in P. tomentosa by deep sequencing of cellulose synthase genes. Polymorphisms were evaluated in 460 individuals from three climatic regions of P. tomentosa, and all 15 markers revealed polymorphic variation. The number of alleles per locus ranged from two to nine with an average of 4.3; the observed and expected heterozygosity per locus varied from 0.029 to 0.962 and from 0.051 to 0.713, respectively. ? Conclusions: These polymorphic markers will potentially be useful for genetic mapping and in molecular breeding for improvement of wood fiber traits in Populus.