A metabolomic assessment of NAC154 transcription factor overexpression in field grown poplar stem wood
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Bud-break is an economically and environmentally important process in trees and shrubs from boreal and temperate latitudes, but its molecular mechanisms are poorly understood. Here, we show that two previously reported transcription factors, EARLY BUD BREAK 1 (EBB1) and SHORT VEGETATIVE PHASE-Like (SVL) directly interact to control bud-break. EBB1 is a positive regulator of bud-break, whereas SVL is a negative regulator of bud-break. EBB1 directly and negatively regulates SVL expression. We further report the identification and characterization of the EBB3 gene. EBB3 is a temperature-responsive, epigenetically-regulated, positive regulator of bud-break that provides a direct link to activation of the cell cycle during bud-break. EBB3 is an AP2/ERF transcription factor that positively and directly regulates CYCLIND3.1 gene. Our results reveal the architecture of a putative regulatory module that links temperature-mediated control of bud-break with activation of cell cycle.
Although primarily studied in relation to photorespiration, serine metabolism in chloroplasts may play a key role in plant CO 2 fertilization responses by linking CO 2 assimilation with growth. Here, we show that the phosphorylated serine pathway is part of a 'photosynthetic C 1 pathway' and demonstrate its high activity in foliage of a C 3 tree where it rapidly integrates photosynthesis and C 1 metabolism contributing to new biomass via methyl transfer reactions, imparting a large natural 13 C-depleted signature. Using 13 CO 2 -labelling, we show that leaf serine, the S-methyl group of leaf methionine, pectin methyl esters, and the associated methanol released during cell wall expansion during growth, are directly produced from photosynthetically-linked C 1 metabolism, within minutes of light exposure. We speculate that the photosynthetic C 1 pathway is highly conserved across the photosynthetic tree of life, is responsible for synthesis of the greenhouse gas methane, and may have evolved with oxygenic photosynthesis by providing a mechanism of directly linking carbon and ammonia assimilation with growth. Although the rise in atmospheric CO 2 inhibits major metabolic pathways like photorespiration, our results suggest that the photosynthetic C 1 pathway may accelerate and represents a missing link between enhanced photosynthesis and plant growth rates during CO 2 fertilization under a changing climate.
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Lignin as the only natural resource of aromatics has been extracted in methanol and catalytically upgraded to three monomeric phenol products.
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The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. In this study, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ~100 kb in Populus tremula × Populus alba. We demonstrated efficient multiplex editing with one single guide RNA in 42 transgenic lines. The mutation profiles ranged from small insertions and deletions and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid future functional characterization.
Leaf osmotic adjustment by the active accrual of compatible organic solutes (e.g. sucrose) contributes to drought tolerance throughout the plant kingdom. In Populus tremula x alba, PtaSUT4 encodes a tonoplast sucrose–proton symporter, whose downregulation by chronic mild drought or transgenic manipulation is known to increase leaf sucrose and turgor. While this may constitute a single drought tolerance mechanism, we now report that other adjustments which can occur during a worsening water deficit are damped when PtaSUT4 is constitutively downregulated. Specifically, we report that starch use and leaf relative water content (RWC) dynamics were compromised when plants with constitutively downregulated PtaSUT4 were subjected to a water deficit. Leaf RWC decreased more in wild-type and vector control lines than in transgenic PtaSUT4-RNAi (RNA-interference) or CRISPR (clustered regularly interspersed short palindromic repeats) knockout (KO) lines. The control line RWC decrease was accompanied by increased PtaSUT4 transcript levels and a mobilization of sucrose from the mesophyll-enriched leaf lamina into the midvein. The findings suggest that changes in SUT4 expression can increase turgor or decrease RWC as different tolerance mechanisms to reduced water availability. Evidence is presented that PtaSUT4-mediated sucrose partitioning between the vacuole and the cytosol is important not only for overall sucrose abundance and turgor, but also for reactive oxygen species (ROS) and antioxidant dynamics. Interestingly, the reduced capacity for accelerated starch breakdown under worsening water-deficit conditions was correlated with reduced ROS in the RNAi and KO lines. A role for PtaSUT4 in the orchestration of ROS, antioxidant, starch utilization and RWC dynamics during water stress and its importance in trees especially, with their high hydraulic resistances, is considered.
Abstract Nonstructural carbohydrate reserves of stems and roots underpin overall tree fitness and productivity under short-rotation management practices such as coppicing for bioenergy. While sucrose and starch comprise the predominant stem carbohydrate reserves of Populus, utilization for fitness and agricultural productivity is understood primarily in terms of starch turnover. The tonoplast sucrose transport protein SUT4 modulates sucrose export from source leaves to distant sinks during photoautotrophic growth, but the possibility of its involvement in remobilizing carbohydrates from storage organs during heterotrophic growth has not been explored. Here, we used PtaSUT4-knockout mutants of Populus tremula × P. alba (INRA 717-1B4) in winter (cool) and summer (warm) glasshouse coppicing experiments to assess SUT4 involvement in reserve utilization. Conditions preceding and supporting summer sprouting were considered favorable for growth, while those preceding and supporting cool temperature sprouting were suboptimal akin to conditions associated with coppicing as generally practiced. Epicormic bud emergence was delayed in sut4 mutants following lower temperature ‘winter’ but not summer coppicing. Winter xylem hexose increases were observed in control but not in sut4 stumps after coppicing. The magnitude of starch and sucrose reserve depletion was similar in control and sut4 stumps during the winter and did not explain the sprouting and xylem hexose differences. However, winter maintenance costs appeared higher in sut4 based partly on Krebs cycle intermediate levels. In control plants, bark accrual of abundant defense metabolites, including salicinoids and condensed tannins, was higher in summer than in winter, but this increase of summer defense allocations was attenuated in sut4 mutants. Temperature-sensitive trade-offs between growth and other priorities may therefore depend on SUT4 in Populus.
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