Patterned Deposition of Xylan and Lignin is Independent from that of the Secondary Wall Cellulose of Arabidopsis Xylem Vessels

Abstract

The secondary cell wall (SCW) of xylem vessel cells provides rigidity and strength that enables efficient water conduction throughout the plant. To gain insight into SCW deposition, we mutagenized Arabidopsis thaliana VASCULAR-RELATED NAC-DOMAIN7-inducible plant lines, in which ectopic protoxylem vessel cell differentiation is synchronously induced. The baculites mutant was isolated based on the absence of helical SCW patterns in ectopically-induced protoxylem vessel cells, and mature baculites plants exhibited an irregular xylem (irx) mutant phenotype in mature plants. A single nucleic acid substitution in the CELLULOSE SYNTHASE SUBUNIT 7 (CESA7) gene in baculites was identified: while the mutation was predicted to produce a C-terminal truncated protein, immunoblot analysis revealed that cesa7^bac mutation results in loss of production of CESA7 proteins, indicating that baculites is a novel cesa7 loss-of-function mutant. In cesa7^bac , despite a lack of patterned cellulose deposition, the helically-patterned deposition of other SCW components, such as the hemicellulose xylan and the phenolic polymer lignin, was not affected. Similar phenotypes were found in another point mutation mutant cesa7^mur10-2 , and an established knock-out mutant, cesa7^irx3-4 Taken together, we propose that the spatio-temporal deposition of different SCW components, such as xylan and lignin, is not dependent on cellulose patterning.

Figure 1.

baculites Is a Mutant with Impaired SCW Deposition Patterning. (A) to (D) Typical differential interference contrast (DIC) images of VND7-VP16-GR-induced SCW in hypocotyls of wild-type ([A] and [B]) and baculites ([C] and [D]) cells in 6-day-old seedlings. Dashed area in (A) and (C) represents close-up in (B) and (D), respectively. (E) Monosaccharide composition of cell walls from seedlings with vector control VP16-GR (vector control), wild-type VND7-VP16-GR (wild type) and baculites VND7-VP16-GR (baculites), with (+) or without (−) DEX treatment, as determined by HPLC. Results are means ±sd (n = 4). Ara, arabinose; Rha, rhamnose; Gal, galactose; Glu, glucose; Xyl, xylose; Man, mannose. Asterisks indicate statistically significant differences (Welch’s t test; *P < 0.05 and **P < 0.01) between the presence and absence of DEX treatment for each genotype. (F) to (M) Visualization of cell wall components in VND7-VP16-GR-induced SCW of wild-type ([F], [H], and [J]) and baculites ([G], [I], and [K]) cotyledon cells. Xylan was detected by immunostaining using LM10 antibody ([F] and [G]), and cellulose was stained with S4B ([H] and [I]). Merged views are shown in ([J] and [K]). (L) and (M) Lignin autofluorescence signals detected with multi-photon microscopy in VND7-VP16-GR-induced SCW of wild type (L) and baculites (M). (N) Measurement of cellulose content of 6-day-old seedlings of wild-type and baculites before VND7-VP16-GR induction, and 3 days after treatment with (+) or without (−) DEX. Results are means ±sd (n = 5). Asterisks indicate statistically significant differences (Welch’s t test, **P < 0.01). Bars = 100 µm ([A] to [D]), 30 µm ([F] to [K]) and 10 µm ([L] and [M]).

Figure 2.

CESA7 Is Responsible for the baculites Phenotype. (A) RT-qPCR analysis of secondary cell wall-related genes. Total RNA was extracted from the 6-day-old seedlings of the vector control VP16-GR (vector control), wild type VND7-VP16-GR (wild type), and baculites VND7-VP16-GR (baculites), which were treated with (+) or without (−) DEX. UBQ10 was used for the internal control, and the results shown are relative values to the expression level of VP16-GR without DEX treatment. Results are mean ±sd (n = 3). Statistically significant differences were found in CESA7 expression between VND7-VP16-GR and baculites (*P<0.05; Student's t test). (B) to (D) Transverse sections of roots of 20-day-old plants of the vector control VP16-GR (B), wild-type VND7-VP16-GR (C), and baculites VND7-VP16-GR (D). Black arrows indicate collapsed xylem vessels. Bars = 20 μm. (E) Two-month-old plants of vector control VP16-GR (left), wild-type VND7-VP16-GR (middle), and baculites VND7-VP16-GR (right). (F) and (G) Single nucleic acid substitution (G2937 to A transition; indicated in red) was found at the acceptor site of the 8th intron of CESA7 in the genome of baculites, resulting in a shift on the acceptor site 13 bp downstream to the next AG (indicated in bold in [F]). This change was presumed to produce the premature stop codon in baculites (G). (H) to (N) Introduction of CESA7pro::YFP-CESA7 rescued the irx phenotype of baculites. (H) to (J) Transverse sections of roots of VND7-VP16-GR in wild-type (H), baculites (I), and baculites/CESA7pro::YFP-CESA7 backgrounds (J). (K) Growth phenotype of 2-month-old plants with either vector control VP16-GR (left) or VND7-VP16-GR in (left to right) wild-type, baculites, baculites/CESA7pro::YFP-CESA7 backgrounds. (L) to (N) DIC images of ectopic VND7-VP16-GR-induced SCW of wild type (L), baculites (M), and baculites/CESA7pro::YFP-CESA7 (N). Bars = 20 µm ([B] to [D]), 10 cm ([E], [K], and [L] to [N]), 20 µm ([H] to [J]).

Figure 3.

baculites Is a Null Mutant of CESA7 and the Effects of other cesa7 Mutations on SCW Deposition during VND7-VP16-GR-Induced Ectopic Xylem Vessel Cell Differentiation. (A) to (C) Equal amounts (30 µg) of protein from the wild type VND7-VP16-GR (WT) (lane 1, 2, 3, 7, and 8) and baculites VND7-VP16-GR (bac) (lane 4, 5, 6, 9, and 10) with DEX (lane 1 to 6) or mock (lane 7 to 10) treatments were separated by SDS-PAGE and immunoblotted to detect the SCW-type CESA proteins. Each lane showed an independent biological replicate sample. (A) and (B) The immunodetection of CESA7 proteins by anti-CESA7 antibody. View of extended exposure of (A) was shown in (B). Black and white arrows ([A] and [B]) indicate the bands corresponding to full-length CESA7 proteins and the expected size (∼66 kD) of truncated CESA7 in baculites by nonsense mutation, respectively. (C) Immunodetection of CESA4, CESA7, and CESA8 using specific antibodies. (D) to (L) Hypocotyl cells of 6-day-old seedlings with VND7-VP16-GR in wild-type ([D], [G] and [L]), mur10-2 ([E], [H], and [K]), and irx3-4 ([F], [I], and [L]) backgrounds treated with DEX for 3 days. Xylan ([D], [E], and [F]) and cellulose ([G], [H], and [I]) were visualized by immunolabeling using LM10 antibody and S4B staining, respectively. Merged view of xylan and cellulose were shown in ([J], [K], and [L]). Bars = 10 µm.

Figure 4.

Time-Course Analysis of Cellulose and Xylan Deposition during Ectopic Xylem Vessel Cell Formation. Six-day-old VND7-VP16-GR seedlings in wild type ([A] to [H]) and baculites ([I] to [P]) were treated with DEX, and then sampled between 0 and 6 h ([A], [B], [I], and [J]), between 6 and 7.5 h ([C], [D], [K], and [L]), between 7.5 and 15 h ([E], [F], [M], and [N]), and between 15 and 24 h ([G], [H], [O], and [P]) after DEX treatment. Cellulose ([A], [C], [E], [G], [I], [K], [M], and [O]) and xylan ([B], [D], [F], [H], [J], [L], [N], and [P]) were visualized by S4B staining and immunolabeling using LM10 antibody, respectively. Cells in cotyledons are shown. Bars = 10 µm. (Q) Time course RT-qPCR analysis of secondary cell wall-related genes. Total RNA was extracted every 6 h from the 6-day-old seedlings of the vector control VP16-GR (vector control), wild-type VND7-VP16-GR (wild-type), and baculites VND7-VP16-GR (baculites) after treatment with DEX. UBQ10 was used for the internal control. Results are means ±sd (n = 3).

Figure 5.

Patterned Deposition of Xylan and Lignin Is Directed by the Cortical Microtubule Array. (A) to (D) Cortical microtubules were visualized during VND7-VP16-GR-induced protoxylem differentiation using the UBQ10pro::GFP-TUB6 reporter line in wild-type ([A] and [B]) and baculites ([C] and [D]) backgrounds, treated with only DEX (Mock; [A] and [C]) or both DEX and the microtubule-depolymerizing drug, oryzalin (Oryzalin; [B] and [D]). Cells in cotyledons are shown. (E) to (P) Six-day-old seedlings with VND7-VP16-GR in wild type ([E] to [J]) or baculites ([K] to [P]) were treated with only DEX (Mock; [E] to [G] and [K] to [M]) or both DEX and oryzalin (Oryzalin; [H] to [J] and [N] to [P]). Cellulose ([E], [H], [K], and [N]) and xylan ([F], [I], [L], and [O]) were visualized by S4B staining and immunolabeling using LM10 antibody, respectively. Merged view of cellulose and xylan are shown in (G), (J), (M), and (P). Cells in cotyledons are shown. (Q) to (T) Lignin was labeled by the fluorescently tagged monolignol analogs, γ-nitrobenzofuran (NBD)-tagged coniferyl alcohol (CA) (Tobimatsu et al., 2013; Schuetz et al., 2014). Cells in hypocotyls are shown. Bars = 10 µm ([A] to [P]), 30 µm ([Q] to [T]).