Carbon for cellulose biosynthesis is derived from sucrose. Cellulose is synthesized from uridine 5′-diphosphoglucose (UDP-glucose), but the enzyme(s) responsible for the initial sucrose cleavage and ...the source of UDP-glucose for cellulose biosynthesis in developing wood have not been defined.
We investigated the role of CYTOSOLIC INVERTASEs (CINs) during wood formation in hybrid aspen (Populus tremula × tremuloides) and characterized transgenic lines with reduced CIN activity during secondary cell wall biosynthesis.
Suppression of CIN activity by 38–55% led to a 9–13% reduction in crystalline cellulose. The changes in cellulose were reflected in reduced diameter of acid-insoluble cellulose microfibrils and increased glucose release from wood upon enzymatic digestion of cellulose. Reduced CIN activity decreased the amount of the cellulose biosynthesis precursor UDP-glucose in developing wood, pointing to the likely cause of the cellulose phenotype.
The findings suggest that CIN activity has an important role in the cellulose biosynthesis of trees, and indicate that cellulose biosynthesis in wood relies on a quantifiable UDP-glucose pool. The results also introduce a concept of altering cellulose microfibril properties by modifying substrate supply to cellulose biosynthesis.
All photosynthetic organisms balance CO2 assimilation with growth and carbon storage. Stored carbon is used for growth at night and when demand exceeds assimilation. Gaining a mechanistic ...understanding of carbon partitioning between storage and growth in trees is important for biological studies and for estimating the potential of terrestrial photosynthesis to sequester anthropogenic CO2 emissions.1,2 Starch represents the main carbon storage in plants.3,4 To examine the carbon storage mechanism and role of starch during tree growth, we generated and characterized low-starch hybrid aspen (Populus tremula × tremuloides) trees using CRISPR-Cas9-mediated gene editing of two PHOSPHOGLUCOMUTASE (PGM) genes coding for plastidial PGM isoforms essential for starch biosynthesis. We demonstrate that starch deficiency does not reduce tree growth even in short days, showing that starch is not a critical carbon reserve during diel growth of aspen. The low-starch trees assimilated up to ∼30% less CO2 compared to the wild type under a range of irradiance levels, but this did not reduce growth or wood density. This implies that aspen growth is not limited by carbon assimilation under benign growth conditions. Moreover, the timing of bud set and bud flush in the low-starch trees was not altered, implying that starch reserves are not critical for the seasonal growth-dormancy cycle. The findings are consistent with a passive starch storage mechanism that contrasts with the annual Arabidopsis and indicate that the capacity of the aspen to absorb CO2 is limited by the rate of sink tissue growth.
•Aspen trees employ a passive starch-storage mechanism during growth•Carbon assimilation is not limiting growth of aspen trees under benign conditions•Starch is not required for bud set and bud flush or its timing in aspen trees
Wang et al. create low-starch aspen mutants and discover that aspen trees employ a passive investing strategy to save carbon for future needs and that tree growth is not carbon limited under benign conditions.
The evolution of the plant vasculature was essential for the emergence of terrestrial life. Xylem vessels are solute-transporting elements in the vasculature that possess secondary wall thickenings ...deposited in intricate patterns. Evenly dispersed microtubule (MT) bands support the formation of these wall thickenings, but how the MTs direct cell wall synthesis during this process remains largely unknown. Cellulose is the major secondary wall constituent and is synthesized by plasma membrane-localized cellulose synthases (CesAs) whose catalytic activity propels them through the membrane. We show that the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1)/POM2 is necessary to align the secondary wall CesAs and MTs during the initial phase of xylem vessel development in Arabidopsis thaliana and rice (Oryza sativa). Surprisingly, these MT-driven patterns successively become imprinted and sufficient to sustain the continued progression of wall thickening in the absence of MTs and CSI1/POM2 function. Hence, two complementary principles underpin wall patterning during xylem vessel development.
Summary
Despite the ecological and industrial importance of biomass accumulation in wood, the control of carbon (C) allocation to this tissue and to other tree tissues remain poorly understood.
We ...studied sucrose synthase (SUS) to clarify its role in biomass formation and C metabolism at the whole tree level in hybrid aspen (Populus tremula × tremuloides). To this end, we analysed source leaves, phloem, developing wood, and roots of SUSRNAi trees using a combination of metabolite profiling, 13CO2 pulse labelling experiments, and long‐term field experiments.
The glasshouse grown SUSRNAi trees exhibited a mild stem phenotype together with a reduction in wood total C. The 13CO2 pulse labelling experiments showed an alteration in the C flow in all the analysed tissues, indicating that SUS affects C metabolism at the whole tree level. This was confirmed when the SUSRNAi trees were grown in the field over a 5‐yr period; their stem height, diameter and biomass were substantially reduced.
These results establish that SUS influences C allocation to developing wood, and that it affects C metabolism at the whole tree level.
See also the Commentary on this article by Gessler, 229: 8–10.
Sugar phosphates are important intermediates of central carbon metabolism in biological systems, with roles in glycolysis, the pentose-phosphate pathway, tricarboxylic acid (TCA) cycle, and many ...other biosynthesis pathways. Understanding central carbon metabolism requires a simple, robust and comprehensive analytical method. However, sugar phosphates are notoriously difficult to analyze by traditional reversed phase liquid chromatography.
Here, we show a two-step derivatization of sugar phosphates by methoxylamine and propionic acid anhydride after chloroform/methanol (3:7) extraction from
leaf and developing wood that improves separation, identification and quantification of sugar phosphates by ultra high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS). Standard curves of authentic sugar phosphates were generated for concentrations from pg to ng/μl with a correlation coefficient
> 0.99. The method showed high sensitivity and repeatability with relative standard deviation (RSD) < 20% based on repeated extraction, derivatization and detection. The analytical accuracy for
leaf extracts, determined by a two-level spiking approach of selected metabolites, was 79-107%.
The results show the reliability of combined reversed phase liquid chromatography-tandem mass spectrometry for sugar phosphate analysis and demonstrate the presence of two unknown sugar phosphates in
extracts.
SUMMARY
Cellulose microfibrils synthesized by CELLULOSE SYNTHASE COMPLEXES (CSCs) are the main load‐bearing polymers in wood. CELLULOSE SYNTHASE INTERACTING1 (CSI1) connects CSCs with cortical ...microtubules, which align with cellulose microfibrils. Mechanical properties of wood are dependent on cellulose microfibril alignment and structure in the cell walls, but the molecular mechanism(s) defining these features is unknown. Herein, we investigated the role of CSI1 in hybrid aspen (Populus tremula × Populus tremuloides) by characterizing transgenic lines with significantly reduced CSI1 transcript abundance. Reduction in leaves (50–80%) caused leaf twisting and misshaped pavement cells, while reduction (70–90%) in developing xylem led to impaired mechanical wood properties evident as a decrease in the elastic modulus and rupture. X‐ray diffraction measurements indicate that microfibril angle was not impacted by the altered CSI1 abundance in developing wood fibres. Instead, the augmented wood phenotype of the transgenic trees was associated with a reduced cellulose degree of polymerization. These findings establish a function for CSI1 in wood mechanics and in defining leaf cell shape. Furthermore, the results imply that the microfibril angle in wood is defined by CSI1 independent mechanism(s).
Significance statement
Mechanical properties of wood allow trees to grow tall and withstand a variety of environmental stresses. Understanding of the molecular machinery defining the mechanical properties of wood is important for tree breeding and wood industry. By combining functional genetics and mechanics we report here a new molecular link between cellulose biosynthesis and wood mechanics.
Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface- and wall-associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the ...relationships among their glycosylation levels, processing, and functionalities are poorly understood. Here, we report the identification and functional characterization of two Golgi-localized exo-beta-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family inArabidopsis thaliana. GH43 loss-of-function mutants exhibited root cell expansion defects in sugar-containing growth media. This root phenotype was associated with an increase in the extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. Characterization of recombinant GH43 variants revealed that the exo-beta-1,3-galactosidase activity of GH43 enzymes is hindered by beta-1,6 branches on beta-1,3-galactans. In line with this steric hindrance, the recombinant GH43 variants did not release galactose from cell wall-extracted glycoproteins or AGP-rich gum arabic. These results indicate that the lack of exo-beta-1,3-galactosidase activity alters cell wall extensibility in roots, a phenotype that could be explained by the involvement of galactosidases in AGP glycan biosynthesis.
Plant cell wall associated hydroxyproline-rich glycoproteins (HRGPs) are involved in several aspects of plant growth and development, including wood formation in trees. HRGPs such as ...arabinogalactan-proteins (AGPs), extensins (EXTs), and proline rich proteins (PRPs) are important for the development and architecture of plant cell walls. Analysis of publicly available gene expression data revealed that many
encoding genes show tight spatio-temporal expression patterns in the developing wood of
that are indicative of specific functions during wood formation. Similar results were obtained for the expression of glycosyl transferases putatively involved in HRGP glycosylation.
immunolabelling of transverse wood sections using AGP and EXT antibodies revealed the cell type specificity of different epitopes. In mature wood AGP epitopes were located in xylem ray cell walls, whereas EXT epitopes were specifically observed between neighboring xylem vessels, and on the ray cell side of the vessel walls, likely in association with pits. Molecular mass and glycan analysis of AGPs and EXTs in phloem/cambium, developing xylem, and mature xylem revealed clear differences in glycan structures and size between the tissues. Separation of AGPs by agarose gel electrophoresis and staining with β-D-glucosyl Yariv confirmed the presence of different AGP populations in phloem/cambium and xylem. These results reveal the diverse changes in HRGP-related processes that occur during wood formation at the gene expression and HRGP glycan biosynthesis levels, and relate HRGPs and glycosylation processes to the developmental processes of wood formation.
Bioelectronic devices that convert biochemical signals to electronic readout enable biosensing with high spatiotemporal resolution. These technologies have been primarily applied in biomedicine while ...in plants sensing is mainly based on invasive methods that require tissue sampling, hindering in-vivo detection and having poor spatiotemporal resolution. Here, we developed enzymatic biosensors based on organic electrochemical transistors (OECTs) for in-vivo and real-time monitoring of sugar fluctuations in the vascular tissue of trees. The glucose and sucrose OECT-biosensors were implanted into the vascular tissue of trees and were operated through a low-cost portable unit for 48hr. Our work consists a proof-of-concept study where implantable OECT-biosensors not only allow real-time monitoring of metabolites in plants but also reveal new insights into diurnal sugar homeostasis. We anticipate that this work will contribute to establishing bioelectronic technologies as powerful minimally invasive tools in plant science, agriculture and forestry.
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•In vivo, real-time monitoring of sugars fluctuations in trees with OECTs for 48hr•OECTs reveal previously uncharacterized diurnal sucrose fluctuations in aspen•Multienzyme functionalization of OECT for detection of sucrose•Operation of sensors with low-cost portable unit
Biotechnology; Bioelectronics; Plant Physiology
Cellulose is synthesized at the plasma membrane by cellulose synthase complexes (CSCs) containing cellulose synthases (CESAs). Genetic analysis and CESA isoform quantification indicate that cellulose ...in the secondary cell walls of Arabidopsis (Arabidopsis thaliana) is synthesized by isoforms CESA4, CESA7, and CESA8 in equimolar amounts. Here, we used quantitative proteomics to investigate whether the CSC model based on Arabidopsis secondary cell wall CESA stoichiometry can be applied to the angiosperm tree aspen (Populus tremula) and the gymnosperm tree Norway spruce (Picea abies). In the developing xylem of aspen, the secondary cell wall CESA stoichiometry was 3:2:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b, while in Norway spruce, the stoichiometry was 1:1:1, as observed previously in Arabidopsis. Furthermore, in aspen tension wood, the secondary cell wall CESA stoichiometry changed to 8:3:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b. PtCESA8b represented 73% of the total secondary cell wall CESA pool, and quantitative polymerase chain reaction analysis of CESA transcripts in cryosectioned tension wood revealed increased PtCESA8b expression during the formation of the cellulose-enriched gelatinous layer, while the transcripts of PtCESA4, PtCESA7a/b, and PtCESA8a decreased. A wide-angle x-ray scattering analysis showed that the shift in CESA stoichiometry in tension wood coincided with an increase in crystalline cellulose microfibril diameter, suggesting that the CSC CESA composition influences microfibril properties. The aspen CESA stoichiometry results raise the possibility of alternative CSC models and suggest that homomeric PtCESA8b complexes are responsible for cellulose biosynthesis in the gelatinous layer in tension wood.
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