Integrity of xylan backbone affects plant responses to drought

Abstract

Drought is a major factor affecting crops, thus efforts are needed to increase plant resilience to this abiotic stress. The overlapping signaling pathways between drought and cell wall integrity maintenance responses create a possibility of increasing drought resistance by modifying cell walls. Here, using herbaceous and woody plant model species, Arabidopsis and hybrid aspen, respectively, we investigated how the integrity of xylan in secondary walls affects the responses of plants to drought stress. Plants, in which secondary wall xylan integrity was reduced by expressing fungal GH10 and GH11 xylanases or by affecting genes involved in xylan backbone biosynthesis, were subjected to controlled drought while their physiological responses were continuously monitored by RGB, fluorescence, and/or hyperspectral cameras. For Arabidopsis, this was supplemented with survival test after complete water withdrawal and analyses of stomatal function and stem conductivity. All Arabidopsis xylan-impaired lines showed better survival upon complete watering withdrawal, increased stomatal density and delayed growth inhibition by moderate drought, indicating increased resilience to moderate drought associated with modified xylan integrity. Subtle differences were recorded between xylan biosynthesis mutants (irx9, irx10 and irx14) and xylanase-expressing lines. irx14 was the most drought resistant genotype, and the only genotype with increased lignin content and unaltered xylem conductivity despite its irx phenotype. Rosette growth was more affected by drought in GH11- than in GH10-expressing plants. In aspen, mild downregulation of GT43B and C genes did not affect drought responses and the transgenic plants grew better than the wild-type in drought and well-watered conditions. Both GH10 and GH11 xylanases strongly inhibited stem elongation and root growth in well-watered conditions but growth was less inhibited by drought in GH11-expressing plants than in wild-type. Overall, plants with xylan integrity impairment in secondary walls were less affected than wild-type by moderately reduced water availability but their responses also varied among genotypes and species. Thus, modifying the secondary cell wall integrity can be considered as a potential strategy for developing crops better suited to withstand water scarcity, but more research is needed to address the underlying molecular causes of this variability.

Keywords: Arabidopsis; Populus; cell wall integrity; drought stress; glucuronoxylan; high-throughput phenotyping; hyperspectral imaging; secondary cell wall.

Figure 1

Watering regimes and image-based phenotyping instruments used to study Arabidopsis (A) and aspen (B) under controlled drought treatment. Different watering regimes were monitored through the maintenance of specific water content in soil by automated watering and weighing units on both platforms. Arrows indicate important dates to define the watering regimes: black arrow – date of change of watering regime; brown arrows – dates of Target Water Content (TCW) in soil corresponding to dates when early drought responses were recorded; orange arrows – dates when late drought responses were recorded. Red-Green-Blue (RGB) camera, chlorophyll fluorescence, hyperspectral (VNIR and SWIR), and thermal imaging units were used to detect changes in plant morphometric parameters and colors, photosynthetic activity, reflectance indices, and surface temperature, respectively. WW, well-watered; MD, moderate drought; SD, severe drought; VNIR, Visible and Near-Infrared; SWIR, Short-Wave Infrared.

Figure 2

Expressing GH10 and GH11 xylanases in xylem cell walls of Arabidopsis induced irregular xylem phenotype similar to phenotype of irx mutants. (A) Transcripts of Aspergillus nidulans GH10 (green bars) and GH11 (blue bars) transgenes were detected in basal stem tissues of two independent lines expressing xylanases under control of wood-specific promoter (WP) (mean relative expression ± se; n=3; p-value according to t-test). (B) GH10:eGFP and GH11:eGFP recombinant proteins were detected in cell walls (arrows) in Arabidopsis root cells plasmolyzed with 20% mannitol. GFP channel (left) and the transmitted light channel (right). Plasmolyzed protoplasts are shown with arrowheads. Scale bar: 10 µm. (C) Stem vascular bundle cross-sections stained with rhodamine B showing irregular xylem phenotype (arrows). Scale bars = 50 μm.

Figure 3

Effect of drought on rosette morphometric parameters of xylan-modified Arabidopsis lines at final stage of plant monitoring (38 days after sowing). (A) Representative images of rosettes under well-watered (WW), moderate drought (MD), and severe drought (SD) conditions (B) Bar graphs representing rosette area and leaf slenderness ratio in the different conditions of watering (top) and in MD or SD relative to the mean of each genotype in WW conditions (bottom). Means ± se; n: 24–37; • - P ≤ 0.1; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001 for comparisons with Col-0 by Dunnett’s test; different letters indicate significant effects of treatments within the same genotype (Tukey’s test, P ≤ 0.05).

Figure 4

Kinetics of rosette growth in well-watered (WW), moderate drought (MD), and severe drought (SD) conditions revealed altered reactions to drought in xylan-modified Arabidopsis lines compared to Col-0. (A) Kinetics of rosette area from the 21^st to the 38^th day after sowing. Vertical gray lines indicate the days when MD-subjected Col-0 became smaller than in WW conditions (dashed line) and when SD-subjected Col-0 became smaller from MD-subjected Col-0 (dotted line) (P_t-test ≤0.05). Dashed and dotted arrows show how these dates differed for other genotypes. (B) Growth rate of the rosette in MD (top) or SD (bottom) relative to rate in WW conditions at early (on the first day of reached target water content) and late (ten days later) stages of drought treatment (see Figure 1 for the dates). The growth rate was calculated as the first derivative at indicated days of the fitted trend lines ( Supplementary Table S3 ). Data are means ± se; n: 24–37; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001 for comparisons with Col-0 by Dunnett’s test. Horizontal brackets and P values indicate significant treatment effects within the same genotype (t-test, P ≤ 0.1).

Figure 5

Parameters determined by fluorescence and reflected light spectra at the final stage of plant monitoring (38 days after sowing) in well-watered (WW), moderate drought (MD), and severe drought (SD) conditions revealed changes in reactions to drought of xylan-modified Arabidopsis lines compared to Col-0. F_v/F_m ratios (A), water loss index (WLI) divided by normalized difference vegetation index (NDVI) (B), and plant senescence reflectance index (PSRI) (C) at different conditions (upper row) and in MD or SD relative to the mean of each genotype in WW conditions (lower row). (D) Percentage of four greenness hues from color segmentation analysis. In clockwise order: hue 3–6 (as shown for Col-0 WW) and hue 1 + 2 + 7 + 8+9 in grey ( Supplementary Figure S4 ). Data in (A–C) are means ± se; n: 24–37; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001 for comparisons with Col-0 by Dunnett’s test; different letters indicate significant treatment effect within the same line (Tukey’s test, P ≤ 0.05).

Figure 6

Clustering of genotypes according to their early and late responses in analyzed parameters to moderate (A) and severe (B) drought. Eighteen traits ( Supplementary Table S2 ) measured on the first day when the target water content level was reached (E; early response) and ten days later (L; late response), expressed as ratios to their average values in well-watered conditions ( Supplementary Table S4 ) were used for clustering with the Ward Linkage method. Values represent averages of 24–37 biological replicates per occasion, genotype, and treatment. Gr, Growth rate; MCARI, Modified Chlorophyll Absorption in Reflectance Index; NDVI, Normalized Difference Vegetation Index; OSAVI, Optimized Soil-Adjusted Vegetation Index; PRI, Photochemical Reflectance Index; PSRI, Plant Senescence Reflectance Index; F_v/F_m, maximum quantum yield of photosystem II; SIPI, Structure Insensitive Pigment Index; WLI, Water Loss index.

Figure 7

Better survival of xylan-modified Arabidopsis lines after complete withholding of watering and subsequent rewatering. (A) Changes in soil water content during water withholding treatment. (B) Cumulative water usage per rosette area during 27 days of growth without watering. (C) Representative images of rosettes grown without watering for 40 days. (D) Survival rate one week after rewatering following 40 days of drought. Data in (A, B, D) are means ± se; n= 10; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001 for comparisons with Col-0 by Dunnett’s test in (B) or Fisher exact test in (D).

Figure 8

Xylan-modified Arabidopsis lines grown in well-watered conditions showed altered physiological parameters related to water evaporation and xylem conductivity. (A) Water loss from detached rosettes of 3-week-old plants. (B) Stomatal density. (C) Stomatal pore area measured in epidermal peels after 2 h light exposure in 50 mM KCl to induce full opening (0 min), followed by 30- and 60-min incubation in 10 μM abscisic acid (ABA) to induce closing. (D, E) Water conductivity in the main stem of 17 cm–tall plants determined by rhodamine uptake. Schematic representation of plants used for the conductivity analysis (D) and rhodamine released to 100 μL of water by 2 mg of tissue of successive stem sections and side-shoots after 1 h of uptake of 0.5 mM rhodamine (E). (F) Representative images of stem cross sections made between sections 3 and 4, as shown with a red arrow in (D), after 1 h of rhodamine uptake. Data in (A–C, E) are means ± se, n=10 for (A), 4 for (B), 4 x 45 (plants x stomates) for (C), and 8 for (E); * - P ≤ 0.05; *** - P ≤ 0.001 for comparisons with Col-0 by Dunnett’s test. Different letters in (C) indicate differences among different durations of ABA treatment within the same line (Tukey’s test, P ≤ 0.05).

Figure 9

Effect of drought on growth of xylan-modified aspen lines. (A) Representative images of the trees and their roots under well-watered (WW) and drought (D) conditions at the end of drought treatment (65 days after planting). (B) Stem height 61 days after planting in WW and D conditions (upper row) and in D relative to mean height in WW conditions (lower row). (C) Rate of stem elongation of D-treated plants relative to average rate in WW condition revealed different responses to drought of GH10- and GH11-expressing lines. The arrow indicates the day when the target drought water content level was reached. (D, E) Morphology of the 20^th leaf from the top 65 days after planting. Leaf blade area (D) and slenderness ratio (E) in WW and D conditions (upper row) and in D relative to WW conditions (lower row). (F) Pseudo-temperature of the canopy from top IR camera 40 days after planting in WW and D conditions (upper row) and in D relative to WW conditions (lower row). Aboveground (G) and belowground (H) biomass 65 days after planting in WW and D conditions (upper row) and in D relative to WW conditions (lower row). Data in (B–H) are means ± se; n= 6–11; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001 for comparisons with wild-type (T89) by Dunnett’s test; significant effects of drought are shown by brackets (t-test, P ≤ 0.05), RU, relative units.

Figure 10

Analysis of stem diameters and wood properties measured at the stem base after 65 days of growth under drought (D) and well-watered (WW) conditions revealed different responses to drought of xylan-modified aspen lines compared to wild-type (T89). (A) Stem diameter measured with a caliper in WW and D conditions (upper graph) and in D relative to WW conditions (lower graph). (B) Diameter of wood core measured by SilviScan in WW and D conditions (upper graph) and in D relative to WW conditions (lower graph). (C, D) Wood properties determined by SilviScan analysis. Wood density in WW and D conditions (upper graph) and in D relative to WW conditions (lower graph) (C), and microfibril angle in WW and D conditions (upper graph) and in D relative to WW conditions (lower graph) (D). Data in (A–D) are means ± se; n= 6–11; * - P ≤ 0.05; ** - P ≤ 0.01; *** - P ≤ 0.001 for comparisons with T89 by Dunnett’s test; brackets show significant effects of drought for each genotype (t-test, P ≤ 0.05); P values above horizontal lines correspond to contrast analysis. ND, not determined.