Compensatory guaiacyl lignin biosynthesis at the expense of syringyl lignin in 4CL1-knockout poplar

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in poplar (Populus tremula x alba) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5-hydroxylases (F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1. Upregulation of caffeoyl-CoA O-methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis

Metabolite ratios of source leaves (LPI 15) of wild-type and transgenic <i>Populus.</i>

1<p>Pairwise comparisons were conducted using Student’s <i>t-</i>test.</p

The Tonoplast-Localized Sucrose Transporter in <em>Populus</em> (PtaSUT4) Regulates Whole-Plant Water Relations, Responses to Water Stress, and Photosynthesis

<div><p>The <em>Populus</em> sucrose (Suc) transporter 4 (PtaSUT4), like its orthologs in other plant taxa, is tonoplast localized and thought to mediate Suc export from the vacuole into the cytosol. In source leaves of <em>Populus</em>, <em>SUT4</em> is the predominantly expressed gene family member, with transcript levels several times higher than those of plasma membrane SUTs. A hypothesis is advanced that SUT4-mediated tonoplast sucrose fluxes contribute to the regulation of osmotic gradients between cellular compartments, with the potential to mediate both sink provisioning and drought tolerance in <em>Populus</em>. Here, we describe the effects of <em>PtaSUT4-</em>RNA interference (RNAi) on sucrose levels and raffinose family oligosaccharides (RFO) induction, photosynthesis, and water uptake, retention and loss during acute and chronic drought stresses. Under normal water-replete growing conditions, SUT4-RNAi plants had generally higher shoot water contents than wild-type plants. In response to soil drying during a short-term, acute drought, RNAi plants exhibited reduced rates of water uptake and delayed wilting relative to wild-type plants. SUT4-RNAi plants had larger leaf areas and lower photosynthesis rates than wild-type plants under well-watered, but not under chronic water-limiting conditions. Moreover, the magnitude of shoot water content, height growth, and photosynthesis responses to contrasting soil moisture regimes was greater in RNAi than wild-type plants. The concentrations of stress-responsive RFOs increased in wild-type plants but were unaffected in SUT4-RNAi plants under chronically dry conditions. We discuss a model in which the subcellular compartmentalization of sucrose mediated by PtaSUT4 is regulated in response to both sink demand and plant water status in <em>Populus</em>.</p> </div

Gas exchange in source leaves (LPI 15) of wild-type and transgenic <i>Populus</i> from the chronic drought experiment.

1<p>Determined at saturating light intensity of 1500 µmol/m²/sec.</p>2<p>WUE = A_max/Transpiration.</p>3<p>Chlorophyll concentrations determined using equations presented in Porra et al. 1989.</p>4<p>Specific leaf area calculations were used to present the Chl data per unit leaf area.</p>5<p>Determined by Student’s <i>t-</i>test.</p

Leaf gas exchange properties of <i>Populus</i> wild-type and RNAi plants.

<p>Light response curves for (A) Photosynthetic CO₂ fixation, and (B) Leaf transpiration (solid lines) and stomatal conductance (dashed lines). A fully expanded source leaf (LPI-10) was used for the measurements. Data points are means ± SD of 19 WT, 10 line G, and 12 line F plants. * <i>p</i>≤0.05 for each transgenic line compared to wild type as determined by Student’s <i>t</i>-test.</p

Area, water content, and specific leaf area of source leaves (LPI 10) from wild-type and transgenic <i>Populus</i> under contrasting long-term soil moisture regimes.

<p>Values are means ± SD of n = 6–8 replicates per treatment group. Bold values are <i>p<</i>0.05, italics are 0.05<<i>p</i><0.1 as determined by Student’s <i>t-</i>test.</p

Effects of water availability on <i>Populus</i> stem growth and water concentrations.

<p>(A) height growth rate, (B) stem diameter growth rate, (C) wood water concentrations (g/g×100), and (D) bark water concentration (g/g×100). Light and dark bars represent high and low soil moisture, respectively. For (A) and (B), asterisk denotes statistical significance between high and low soil moisture within a line. For (C) and (D), asterisks and <i>p</i>-values represent pairwise comparisons between high soil moisture groups delineated by the arrows (i.e., wild type vs. one of the transgenic lines). Bars represent means ± SD of 6–8 replicate plants. *<i>p</i>≤0.05, **0.001<<i>p</i>≤0.01, ***<i>p</i>≤0.001 as determined by Student’s <i>t</i>-test.</p