PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis

Abstract

Mutants at the PROCUSTE1 (PRC1) locus show decreased cell elongation, specifically in roots and dark-grown hypocotyls. Cell elongation defects are correlated with a cellulose deficiency and the presence of gapped walls. Map-based cloning of PRC1 reveals that it encodes a member (CesA6) of the cellulose synthase catalytic subunit family, of which at least nine other members exist in Arabidopsis. Mutations in another family member, RSW1 (CesA1), cause similar cell wall defects in all cell types, including those in hypocotyls and roots, suggesting that cellulose synthesis in these organs requires the coordinated expression of at least two distinct cellulose synthase isoforms.

Figure 1.

prc1 Mutants Show Reduced Elongation of Roots and Dark-Grown Hypocotyls. (A) Dark-grown hypocotyl lengths of wild-type (ecotypes Columbia [Col-0] and Wassilewskija [WS]) seedlings and 11 prc1 alleles. Seedlings were grown for 7 days in total darkness on sucrose-free agar medium. (B) Root lengths of wild-type seedlings and 10 prc1 alleles. Seedlings were grown for 7 days in 16-hr-light/8-hr-dark cycles on agar medium containing 4.5% sucrose. prc1-1 through prc1-9 and prc1-11 are Col-0 alleles; prc1-10 and prc1-12 are Ws ecotype. Error bars indicate ±sd.

Figure 2.

Dark-Grown prc1 Hypocotyls Contain Incomplete Cell Walls. (A) and (B) Calcofluor-stained hypocotyl cross-sections of 4-day-old dark-grown wild-type (A) and prc1-1 (B) seedlings. The top arrow indicates an incomplete wall and the bottom arrow indicates an abnormally filled cell in a prc1-1 hypocotyl section. (C) Congo Red–stained section of a prc1-1 hypocotyl cell in which the incomplete cell wall stubs are connected by a thin structure (arrow). (D) Calcofluor-stained section through a prc1-1 immature embryo. No incomplete wall or cell filled with stained material was observed. ; ; .

Figure 3.

prc1 Dark-Grown Hypocotyls Display Abnormal Cell Wall Structures. (A) to (C) Transmission electron micrographs of cross-sections from dark-grown hypocotyls (4 days old) of prc1-1 mutant ([A] and [B]) and wild-type (C) seedlings in the cortical cell layer. Arrow in (A) indicates an abnormal wall portion shown at a higher magnification in (B). (D) and (E) Scanning electron micrographs of prc1-1 dark-grown hypocotyls. Arrowheads indicate protruding stubs of incomplete cell walls in the epidermal cell layer. co, cortex cell; cw, cell wall; cy, cytoplasm; ep, epidermal cell; va, vacuole. ; ; ; .

Figure 4.

prc1 Dark-Grown Hypocotyl Cell Walls Are Cellulose Deficient. (A) FTIR analysis of 4-day-old dark-grown hypocotyls. PC analysis was performed by using 20 FTIR spectra from wild-type and two different prc1 alleles. PC1 explained 72% of the variance between spectra from wild-type alleles and spectra from prc1 alleles. Col0, Columbia. (B) The PC1 loading showed positive peaks characteristic of cellulose in the fingerprint region (1162, 1108, 1061, and 1038 cm⁻¹, respectively), indicating that prc1 dark-grown hypocotyls were deficient in cellulose relative to the wild type. Two other major peaks (1652, and 1548 cm⁻¹, respectively) appeared negatively in this PC-loading. These peaks corresponded to amide groups of protein, suggesting an enrichment for protein in prc1 walls relative to the wild type.

Figure 5.

Chemical Analysis Confirms Cellulose Deficiency in Dark-Grown prc1 Seedlings. (A) Walls were purified and extracted successively with 0.1 M KOH, yielding a pectin-rich fraction (fraction 1), and 4 M KOH, yielding a hemicellulose-rich fraction (fraction 2). The residual fraction was enriched for a glucan corresponding to cellulose (see text). prc1-1 walls were deficient for the residual fraction relative to the wild type, indicating a deficiency in cellulose for this mutant. Col0, Columbia. (B) Incorporation of ¹⁴C-glucose (14C) in the acid-resistant cellulosic fraction in wild-type and prc1 alleles. Each value represents the mean of five independent measurements. CW, cell wall. (C) to (E) Sugar composition of fraction 1 (C), fraction 2 (D), and the residue (E). Only fraction 1 reproducibly showed qualitative differences in sugar composition. r, rhamnose; f, fucose; a, arabinose; x, xylose; m, mannose; ga, galactose; gl, glucose; u, uronic acid. Error bars indicate ±sd.

Figure 6.

The PRC1 Gene Is Isolated Through Map-Based Cloning. (A) Physical map of the region of chromosome (chr.) 5 containing the PRC1 gene. The prc1-1 mutation was mapped to the bottom of chromosome 5, south of molecular marker LFY. New markers positioned the prc1-1 mutation between markers Mub3.p2B and 2F2-E12. The number of recombinant chromosomes is indicated in parentheses. Sequence analysis of candidate genes between these two markers showed that six prc1 mutant alleles carried a point mutation or a deletion in the CesA6 gene carried by P1 clone MVP7. The PRC1 (CesA6) gene comprises 13 exons (filled boxes) and 12 introns. The prc1 mutations that were identified are indicated. (B) Predicted topology of the PRC1 protein. The protein is predicted to contain eight membrane-spanning helices (indicated by the eight gray bars). The putative Zn binding domain, the U1, U2, U3, and U4 domains, the plant conserved region (PCR), and the HVR are predicted to face the cytosol (Delmer, 1999). The positions of the mutations are indicated. N, N terminus; C, C terminus.

Figure 7.

prc1-8 and rsw1-10 Have Similar, and Additive, Hypocotyl Phenotypes. Seedlings were grown for 4 days in the dark. Left to right: wild type (wt), prc1-8, rsw1-10, and double mutant. The prc1-8 and rsw1-10 mutants showed a similar decrease in hypocotyl length compared with wild-type seedlings, whereas the double mutant showed an even shorter hypocotyl. .

Figure 8.

No Gene Dosage Effect Is Observed for CesA1 and CesA6. Hypocotyl lengths of dark-grown prc1-8 seedlings, mutant for CesA6, rsw1-10 seedlings, mutant for CesA1, and the progeny of a plant homozygous for rsw1-10 and heterozygous for prc1-8 were measured. In the segregating population (SP), a discrete 1:3 segregation of the additive phenotype was observed. No gene dosage effect was observed. Indeed, an intermediate hypocotyl length for half of the seedlings, those corresponding to prc1-8 heterozygotes, would be expected if the dosage of the CesA6 gene were critical for hypocotyl growth.

Figure 9.

Members of the CesA Family Exhibit Functional Specialization. Shown is a phylogenetic tree based on the HVR (see text and Figure 5) of protein sequences of Arabidopsis and cotton CesA family members. Alignment data are from bootstrap values sampled 100 times and used to construct the consensus tree shown. Numbers are bootstrap values and indicate the number of trees in which the proteins to the right of the bootstrap values clustered together. Gh EST-AI727450 was included as an outlier. The five other cotton genes did not cluster together but instead clustered with one or two Arabidopsis sequences, suggesting that the CesA amino acid sequences diverged before the divergence between Brassicaceae and Malvaceae; that, in turn, suggests a functional specialization of most CesA isoforms. At, Arabidopsis thaliana; Gh, Gossypium hirsutum.

Figure 10.

PRC1 and RSW1 Have Similar Transcript Profiles. Total RNA of different samples was hybridized with labeled probes corresponding to a variable sequence at the N-terminal end of CesA proteins. (A) PRC1 probe. (B) RSW1 probe. (C) Ethidium bromide–stained agarose gel of the same samples. Lane 1, 1-day-old germinating seeds; lane 2, 4-day-old dark-grown seedlings; lane 3, 4-day-old light-grown seedlings; lane 4, elongating inflorescence stem sections; lane 5, mature leaves; lane 6, roots; lane 7, mature flowers.