TRICHOME BIREFRINGENCE and its homolog AT5G01360 encode plant-specific DUF231 proteins required for cellulose biosynthesis in Arabidopsis

Abstract

The Arabidopsis (Arabidopsis thaliana) trichome birefringence (tbr) mutant has severely reduced crystalline cellulose in trichomes, but the molecular nature of TBR was unknown. We determined TBR to belong to the plant-specific DUF231 domain gene family comprising 46 members of unknown function in Arabidopsis. The genes harbor another plant-specific domain, called the TBL domain, which contains a conserved GDSL motif known from some esterases/lipases. TBR and TBR-like3 (TBL3) are transcriptionally coordinated with primary and secondary CELLULOSE SYNTHASE (CESA) genes, respectively. The tbr and tbl3 mutants hold lower levels of crystalline cellulose and have altered pectin composition in trichomes and stems, respectively, tissues generally thought to contain mainly secondary wall crystalline cellulose. In contrast, primary wall cellulose levels remain unchanged in both mutants as measured in etiolated tbr and tbl3 hypocotyls, while the amount of esterified pectins is reduced and pectin methylesterase activity is increased in this tissue. Furthermore, etiolated tbr hypocotyls have reduced length with swollen epidermal cells, a phenotype characteristic for primary cesa mutants or the wild type treated with cellulose synthesis inhibitors. Taken together, we show that two TBL genes contribute to the synthesis and deposition of secondary wall cellulose, presumably by influencing the esterification state of pectic polymers.

Figure 1.

Lack of trichome birefringence in tbr mutants. A and B, Birefringence phenotypes of a wild-type Col-0 leaf (A) and a tbr mutant leaf (B). C to F, Appearance of individual wild-type and tbr mutant trichomes under polarized (C and D) and nonpolarized (E and F) white light.

Figure 2.

Hypocotyl phenotypes of etiolated tbr mutants. A, Aspects of 4-d-old etiolated wild-type and tbr mutant seedlings. The length distribution among tbr seedlings is indicated by percentage numbers. Numbers at the bottom [13.98 ± 1.37 and 7.78 ± 2.48 (n = 42)] indicate mean values (in millimeters) of hypocotyl length in wild-type and tbr populations. B to D, Cell-swelling phenotype occasionally observed at the top of tbr mutant hypocotyls (B and C) and the invariant, regular wild-type phenotype (D). E, Crystalline cellulose determination as measured by the Updegraff (1969) protocol. Mean values ± se (n = 3) are given. DW, Dry weight; WT, wild type.

Figure 3.

Altered cellulose and noncellulosic sugar composition in tbr mutant trichomes. Individual sugars and uronic acids (UA) are expressed as a percentage of the total noncellulosic cell wall sugar. Cellulose-derived Glc (right side) is expressed as a percentage of dry weight (DW). Typical results (means ± se; n = 4) from one of several experiments are shown. Significant changes as deduced from Student's t test (P ≤ 0.05) are marked with asterisks. WT, Wild type.

Figure 4.

Identification of TBR. A, View of the approximately 5-kb region responsible for complementation of the tbr phenotype. The structure of the one annotated gene (AT5G06700) in that region is given. Exons are depicted as white boxes, and untranslated regions of AT5G06700 (i.e. TBR) are shown as gray boxes. The arrow indicates the direction of transcription. The point mutation in the tbr-1 allele (G→A) leads to a predicted Gly-to-Glu exchange at position 427 of the encoded protein. B, Knockdown of TBR by RNAi (the three photographs to the right show leaves of different RNAi lines) leads to strongly reduced trichome birefringence as compared with the wild type (the photograph to the left).

Figure 5.

Unrooted, bootstrapped tree of the TBL gene family in Arabidopsis. MAFFT (version 5) was used to create an alignment of the protein sequences available at TAIR. Highly variable, gapped N-terminal regions were removed, and a maximum likelihood phylogeny was generated with RAxML model PROTCATWAG (Stamatakis, 2006) and bootstrapped (n = 1,000 trials) to generate the final tree. Bootstrap values greater than 70 are shown. Segmental/tandem gene pairs are highlighted in blue, and genes mentioned in the text are shown in red.

Figure 6.

GUS expression of TBR. A, Staining of a 9-d-old seedling transformed with GUS driven by the TBR promoter. B, Magnified view of stained leaf trichomes of a 9-d-old seedling. C to E, Staining of stem trichomes of seedlings transformed with TBR:GUS (C), TBL1:GUS (D), or TBL2:GUS (E). F to I, Typical staining of 3-week-old (F), 4-week-old (G), 5-week-old (H), and 6-week-old (I) TBR:GUS transgenic plants.

Figure 7.

Biochemical cell wall phenotypes of tbl3 T-DNA mutants. A, Cellulose-derived Glc is expressed as percentage of dry weight (DW) and shows the typical result from one of several experiments (means ± se; n = 5). B, Individual sugars and uronic acids (UA) of wild-type and tbl3 stems are expressed as percentage of the total noncellulosic cell wall sugar (means ± se; n = 4). Significant changes as deduced from Student's t test (P ≤ 0.05) are marked with asterisks.

Figure 8.

FTIR spectra and PME activity in tbr and tbl3 mutants. A, FTIR spectrum from 4-d-old, dark-grown tbr (dotted line) and tbl3 (solid line) mutant hypocotyls. Student's t test values for the comparison between the wild type and tbr or tbl3 (x axis) are plotted against the wave numbers (y axis). Horizontal gray lines refer to the significance threshold (P = 0.95). Several highly significant maxima can be assigned to pectin ester linkages and alterations in cell wall crystallinity. B, Measurement of PME activity isolated from etiolated seedlings. The mean values of PME activity are expressed as release of methanol in nmol g⁻¹ fresh weight (FW) derived from wild-type (WT), tbr, and tbl3 seedlings. Mean values ± se are shown (n = 5). Significant changes (P ≤ 0.01) as deduced from Student's t test are marked with asterisks.