Spatially resolved metabolic analysis reveals a central role for transcriptional control in carbon allocation to wood.

The contribution of transcriptional and post-transcriptional regulation to modifying carbon allocation to developing wood of trees is not well defined. To clarify the role of transcriptional regulation, the enzyme activity patterns of eight central primary metabolism enzymes across phloem, cambium, and developing wood of aspen (Populus tremula L.) were compared with transcript levels obtained by RNA sequencing of sequential stem sections from the same trees. Enzymes were selected on the basis of their importance in sugar metabolism and in linking primary metabolism to lignin biosynthesis. Existing enzyme assays were adapted to allow measurements from ~1 mm3 sections of dissected stem tissue. These experiments provided high spatial resolution of enzyme activity changes across different stages of wood development, and identified the gene transcripts probably responsible for these changes. In most cases, there was a clear positive relationship between transcripts and enzyme activity. During secondary cell wall formation, the increases in transcript levels and enzyme activities also matched with increased levels of glucose, fructose, hexose phosphates, and UDP-glucose, emphasizing an important role for transcriptional regulation in carbon allocation to developing aspen wood. These observations corroborate the efforts to increase carbon allocation to wood by engineering gene regulatory networks.

Neighboring parenchyma cells contribute to Arabidopsis xylem lignification, while lignification of interfascicular fibers is cell autonomous.

Lignin is a critical structural component of plants, providing vascular integrity and mechanical strength. Lignin precursors (monolignols) must be exported to the extracellular matrix where random oxidative coupling produces a complex lignin polymer. The objectives of this study were twofold: to determine the timing of lignification with respect to programmed cell death and to test if nonlignifying xylary parenchyma cells can contribute to the lignification of tracheary elements and fibers. This study demonstrates that lignin deposition is not exclusively a postmortem event, but also occurs prior to programmed cell death. Radiolabeled monolignols were not detected in the cytoplasm or vacuoles of tracheary elements or neighbors. To experimentally define which cells in lignifying tissues contribute to lignification in intact plants, a microRNA against cinnamoyl CoA-reductase1 driven by the promoter from cellulose synthase7 (ProCESA7:miRNA CCR1) was used to silence monolignol biosynthesis specifically in cells developing lignified secondary cell walls. When monolignol biosynthesis in ProCESA7:miRNA CCR1 lines was silenced in the lignifying cells themselves, but not in the neighboring cells, lignin was still deposited in the xylem secondary cell walls. Surprisingly, a dramatic reduction in cell wall lignification of extraxylary fiber cells demonstrates that extraxylary fibers undergo cell autonomous lignification.

O-acetylation of glucuronoxylan in Arabidopsis thaliana wild type and its change in xylan biosynthesis mutants.

O-Acetylglucuronoxylans (AcGX) in Arabidopsis thaliana carry acetyl residues on the 2-O and/or 3-O positions of the xylopyranosyl (Xylp) units, but the distribution of different O-acetylated Xylp units is partly unclear. We studied a possible correlation of xylan acetylation and the activities of different glycosyltransferases involved in xylan biosynthesis by analyzing the distribution of O-acetyl substituents on AcGX from Arabidopsis wild-type and mutants irx7, irx9-1, irx10, irx14 and gux1gux2. The relative contents of the Xylp structural units were determined with quantitative two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. In the wild type, the degree of acetylation (DA) was 60%. Mono- and diacetylated Xylp units constituted 44 and 6% of the AcGX backbone, respectively; while (4-O-methyl)-glucopyranosyluronic acid (1 → 2)-linked Xylp units, most of which also carry 3-O-acetylation, represented 13%. The DA was decreased in irx7, irx9-1 and irx14 due to the decrease in monoacetylation (2-O and 3-O), indicating a relationship between acetylation and other AcGX biosynthetic processes. The possible interactions that could lead to such changes have been discussed. No change in DA was observed in irx10 and gux1gux2, but monoacetylation was nonetheless elevated in gux1gux2. This indicates that acetylation occurs after addition of GlcpA to the xylan backbone. Mass fragmentation analysis suggests that the prevalent acetylation pattern is the acetyl group added on every other Xylp unit.

Deficient sucrose synthase activity in developing wood does not specifically affect cellulose biosynthesis, but causes an overall decrease in cell wall polymers.

The biosynthesis of wood in aspen (Populus) depends on the metabolism of sucrose, which is the main transported form of carbon from source tissues. The largest fraction of the wood biomass is cellulose, which is synthesized from UDP-glucose. Sucrose synthase (SUS) has been proposed previously to interact directly with cellulose synthase complexes and specifically supply UDP-glucose for cellulose biosynthesis. To investigate the role of SUS in wood biosynthesis, we characterized transgenic lines of hybrid aspen with strongly reduced SUS activity in developing wood. No dramatic growth phenotypes in glasshouse-grown trees were observed, but chemical fingerprinting with pyrolysis-GC/MS, together with micromechanical analysis, showed notable changes in chemistry and ultrastructure of the wood in the transgenic lines. Wet chemical analysis showed that the dry weight percentage composition of wood polymers was not changed significantly. However, a decrease in wood density was observed and, consequently, the content of lignin, hemicellulose and cellulose was decreased per wood volume. The decrease in density was explained by a looser structure of fibre cell walls as shown by increased wall shrinkage on drying. The results show that SUS is not essential for cellulose biosynthesis, but plays a role in defining the total carbon incorporation to wood cell walls.

Fructokinase is required for carbon partitioning to cellulose in aspen wood.

Sucrose is the main transported form of carbon in several plant species, including Populus species. Sucrose metabolism in developing wood has therefore a central role in carbon partitioning to stem biomass. Half of the sucrose-derived carbon is in the form of fructose, but metabolism of fructose has received little attention as a factor in carbon partitioning to walls of wood cells. We show that RNAi-mediated reduction of FRK2 activity in developing wood of hybrid aspen (Populus tremula × tremuloides) led to the accumulation of soluble neutral sugars and a decrease in hexose phosphates and UDP-glucose, indicating that carbon flux to cell-wall polysaccharide precursors is decreased. Reduced FRK2 activity also led to thinner fiber cell walls with a reduction in the proportion of cellulose. No pleiotropic effects on stem height or diameter were observed. The results establish a central role for FRK2 activity in carbon flux to wood cellulose.

Development of cellulosic secondary walls in flax fibers requires beta-galactosidase.

Bast (phloem) fibers, tension wood fibers, and other cells with gelatinous-type secondary walls are rich in crystalline cellulose. In developing bast fibers of flax (Linum usitatissimum), a galactan-enriched matrix (Gn-layer) is gradually modified into a mature cellulosic gelatinous-layer (G-layer), which ultimately comprises most of the secondary cell wall. Previous studies have correlated this maturation process with expression of a putative ß-galactosidase. Here, we demonstrate that ß-galactosidase activity is in fact necessary for the dynamic remodeling of polysaccharides that occurs during normal secondary wall development in flax fibers. We found that developing stems of transgenic (LuBGAL-RNAi) flax with reduced ß-galactosidase activity had lower concentrations of free Gal and had significant reductions in the thickness of mature cellulosic G-layers compared with controls. Conversely, Gn-layers, labeled intensively by the galactan-specific LM5 antibody, were greatly expanded in LuBGAL-RNAi transgenic plants. Gross morphology and stem anatomy, including the thickness of bast fiber walls, were otherwise unaffected by silencing of ß-galactosidase transcripts. These results demonstrate a specific requirement for ß-galactosidase in hydrolysis of galactans during formation of cellulosic G-layers. Transgenic lines with reduced ß-galactosidase activity also had biochemical and spectroscopic properties consistent with a reduction in cellulose crystallinity. We further demonstrated that the tensile strength of normal flax stems is dependent on ß-galactosidase-mediated development of the phloem fiber G-layer. Thus, the mechanical strength that typifies flax stems is dependent on a thick, cellulosic G-layer, which itself depends on ß-galactosidase activity within the precursor Gn-layer. These observations demonstrate a novel role for matrix polysaccharides in cellulose deposition; the relevance of these observations to the development of cell walls in other species is also discussed.

Microarray analysis of developing flax hypocotyls identifies novel transcripts correlated with specific stages of phloem fibre differentiation.

BACKGROUND AND AIMS: Hypocotyls are a commonly used model to study primary growth in plants, since post-germinative hypocotyls increase in size by cell elongation rather than cell division. Flax hypocotyls produce phloem fibres in bundles one to two cell layers thick, parallel to the protoxylem poles of the stele. Cell wall deposition within these cells occurs rapidly at a well-defined stage of development. The aim was to identify transcripts associated with distinct stages of hypocotyl and phloem fibre development. METHODS: Stages of flax hypocotyl development were defined by analysing hypocotyl length in relation to fibre secondary wall deposition. Selected stages of development were used in microarray analyses to identify transcripts involved in the transition from elongation to secondary cell wall deposition in fibres. Expression of specific genes was confirmed by qRT-PCR and by enzymatic assays. KEY RESULTS: Genes enriched in the elongation phase included transcripts related to cell-wall modification or primary-wall deposition. Transcripts specifically enriched at the transition between elongation and secondary wall deposition included beta-galactosidase and arabinogalactan proteins. Later stages of wall development showed an increase in secondary metabolism-related transcripts, chitinases and glycosyl hydrolases including KORRIGAN. Microarray analysis also identified groups of transcription factors enriched at one or more stages of fibre development. Subsequent analysis of a differentially expressed beta-galactosidase confirmed that the post-elongation increase in beta-galactosidase enzyme activity was localized to phloem fibres. CONCLUSIONS: Transcripts were identified associated with specific stages of hypocotyl development, in which phloem fibre cells were undergoing thickening of secondary walls. Temporal and spatial regulation of beta-galactosidase activity suggests a role for this enzyme in remodelling of flax bast fibre cell walls during secondary cell wall deposition.

Toward universal coverage in Afghanistan: A multi-stakeholder assessment of capacity investments in the community health worker system.

Global efforts to scale-up the community health workforce have accelerated as a result of the growing evidence of their effectiveness to enhance coverage and health outcomes. Reconstruction efforts in Afghanistan integrated capacity investments for community based service delivery, including the deployment of over 28,000 community health workers (CHWs) to ensure access to basic preventive and curative services. The study aimed to conduct capacity assessments of the CHW system and determine stakeholder perspectives of CHW performance. Structured interviews were conducted on a national sample from 33 provinces and included supervisors, facility providers, patients, and CHWs. Formative assessments were also conducted with national policymakers, community members and health councils in two provinces. Results indicate that more than 70% of the NGO's provide comprehensive training for CHWs, 95% CHWs reported regular supervision, and more than 60% of the health posts had adequate infrastructure and essential commodities. Innovative strategies of paired male and female CHWs, institution of a special cadre of community health supervisors, and community health councils were introduced as systems strengthening mechanisms. Reported barriers included unrealistic and expanding task expectations (14%), unsatisfactory compensation mechanisms (75%), inadequate transport (69%), and lack of commodities (40%). Formative assessments evidenced that CHWs were highly valued as they provided equitable, accessible and affordable 24-h care. Their loyalty, dedication and the ability for women to access care without male family escorts was appreciated by communities. With rising concerns of workforce deficits, insecurity and budget constraints, the health system must enhance the capacity of these frontline workers to improve the continuum of care. The study provides critical insight into the strengths and constraints of Afghanistan's CHW system, warranting further efforts to contextualize service delivery and mechanisms for their support and motivation.

Microarray analysis of flax (Linum usitatissimum L.) stems identifies transcripts enriched in fibre-bearing phloem tissues.

To better understand the molecular processes associated with the development of the unusually long (> 30 mm) and strong bast fibre cells within the phloem of flax stems, we conducted a gene discovery experiment to identify transcripts enriched in fibre-bearing tissues, with the intention that these transcripts would serve as future targets for crop improvement and research in phloem development and cell wall deposition. We produced a library of 9,600 cDNA clones from the peels of flax stems, and selected tissue-specific cDNAs for sequencing based on two series of microarray experiments. In the first microarray series, we compared transcript abundance in stem-peels and leaves, and identified stem-enriched transcripts putatively involved in the processes of polysaccharide and cell wall metabolism. In the second microarray series, we compared gene expression in three segments of the vertical stem axis, which constituted a developmental series for phloem fibres and other cell types. The expression of specific LTP and AGP transcripts was particularly well-correlated with stem segments during either the elongation phase or cell-wall thickening phase of phloem fibre development, and the phloem-specific enrichment of these transcripts was confirmed by qRT-PCR. Transcripts representing multiple, distinct chitinases, beta-galactosidases, arabinogalactan proteins (AGP), and lipid transfer proteins (LTPs) were among the interesting transcripts enriched in specific stages of the developing stem. Considered together, the results of our analyses suggest similarity between the molecular mechanisms underlying phloem fibre development and the gelatinous fibres of tension wood in trees.