Cell-Specific Suppression of 4-Coumarate-CoA Ligase Gene Reveals Differential Effect of Lignin on Cell Physiological Function in Populus

Abstract

Lignin is a main component of the secondary cell wall in vessels and fibers of xylem tissue. However, the significance of lignin in cell physiology during plant growth is unclear. In this study, we generated lignin-modified Populus via cell-specific downregulation of the 4-coumarate-CoA ligase gene (4CL). The transgenic plants with selective lignin modification in vessel elements or fiber cells allowed us to investigate how lignin affects the physiology of vessel or fiber cells in relation to plant growth. Results showed that vessel-specific suppression of lignin biosynthesis resulted in deformed vessels and normal fibers, while fiber-specific suppression of lignin biosynthesis led to less-lignified fibers and normal vessels. Further analyses revealed that the efficiency of long distance water transport was severely affected in transgenics with vessel-specific lignin modification, while minimal effect was detected in transgenics with fiber-specific lignin modification. Vessel-specific lignin reduction led to high susceptibility to drought stress and poor growth in field, likely due to vessel defects in long distance transport of water. The distinct physiological significance of lignin in different cell types provides insights into the selective modification of lignin for improvement of lignocellulosic biomass utilization.

Keywords: 4-coumarate-CoA ligase gene; Populus; fiber cell; lignin; vessel; xylem.

FIGURE 1

Phenotypes of the vessel-specific and fiber-specific down-regulation of 4CL1 expression in Populus. (A) Phenotypes of the transgenics through fiber-specific (F-4CL-A) and vessel-specific (V-4CL-A) downregulation of 4CL1 expression grown in phytotron at 2 months old. Scale bar, 5 cm. (B) Expression of 4CL1 gene in 4CL1 antisense transgenic plants. Gene expression in the control was set as 1. Results are means ± SE of three biological replicates. (C–G) Plant height (C), internode length (D), stem diameter (E), leaf blade length (F), and leaf blade width (G) of the transgenic and control plants were measured at 2 months old. Plant height and stem diameter: means ± SE of 5 clonally propagated plants; the other parameters: means ± SE of 20 internodes from 5 plants. Different lowercase letters in (B) indicate significant differences at p < 0.01 by ANOVA.

FIGURE 2

Vessel-specific and fiber-specific downregulation of 4CL1 expression resulted in reduction of lignin biosynthesis in vessels and fibers, respectively. (A–F) Cross sections (11th internode) of the wild-type control (A), F-4CL-A (B), and V-4CL-A (C) stained for lignin using phloroglucinol-HCl. Close-up images of the control (D), F-4CL-A (E), and V-4CL-A (F) indicated with a rectangle in the upper panel (A–C). Scale bar, 50 μm. (G) The percentage of deformed xylem vessels in the WT, F-4CL-A, and V-4CL-A trees. Five biological replicates from two independent lines were examined for the count. (H,I) Lignin content (H) and crystalline cellulose content (I) in the stems of the 2-month-old plants. Results are means ± SE of three biological replicates of three independent lines. ABSL, acetyl bromide-soluble lignin. Different lowercase letters in (G,H) indicate significant differences at p < 0.01 by ANOVA.

FIGURE 3

Continuous phenotyping of the transgenic Populus under greenhouse conditions. (A,B) The transgenic Populus through vessel-specific or fiber-specific downregulation of 4CL1 expression were clonally propagated (12 copes for each transgenic) and grown in a greenhouse at 2 months old (A) and at 4 months old (B). (C) Plant height was recorded in a period of a continuous 2 months. Results are means ± SE of 12 clonally propagated plants from 3 independent lines.

FIGURE 4

Vessel-specific downregulation of 4CL1 expression affected stem sap flow and water conductance. (A) Stem sap flow was recorded continuously for 3 days, and three biological replicates were carried out. One representative graph is shown. (B) Hydraulic conductance was measured using a High Pressure Flowmeter (HPFM). The hydraulic conductance in control plants was set as 1. Relative hydraulic conductance: means ± SE of 12 clonally propagated plants from 3 independent lines. (C) Water content distribution was estimated based on the near-infrared chemometric imaging. The distribution of low water content and high water content was calculated based on the stem near-infrared (NIR) images. The values represent means ± SE of 12 clonally propagated plants from 3 independent lines. Different lowercase letters in (B,C) indicate significant differences at p < 0.01 by ANOVA.

FIGURE 5

Vessel-specific downregulation of 4CL1 expression resulted in susceptibility to drought stress. (A) The transgenic response to drought condition. (B) Zoomed-in image of the leaf blade in rectangled area in (A). (C) Measurement of the stem sap flow under drought condition and three biological replicates were carried out. One representative graph is shown. (D) Water loss rate of leaves in different transgenics. Water loss was calculated at different times after detaching from the tree at room temperature. The values represent means ± SE of 30 leaves from 6 clonally propagated plants from 3 independent lines.

FIGURE 6

Field performance of the transgenic Populus. (A) The vessel-specific (V-4CL-A) and fiber-specific (F-4CL-A) downregulation of 4CL1 transgenics were grown in field for 1 year. Scale bar, 50 cm. (B) The hydraulic conductance in control plants was set as 1. Relative hydraulic conductance: means ± SE of six clonally propagated plants from three independent lines. (C–E) Plant height (C), stem diameter (D), and aboveground fresh weight (E). Results are means ± SE of 12 clonally propagated plants from 3 independent lines. (F,G) Lignin content (F) and crystalline cellulose content (G) in the stem of the filed grown trees. Results are means ± SE of three biological replicates (lines). (H) Modulus of rupture (MOR) of the xylem tissue in stems. The values represent means ± SE of three biological replicates (lines). Different lowercase letters indicate significant differences at p < 0.01 by ANOVA. (I) The survival rate was calculated based on the initial planted trees (each line with 30 individuals) in field.