KOBITO1 encodes a novel plasma membrane protein necessary for normal synthesis of cellulose during cell expansion in Arabidopsis

Abstract

The cell wall is the major limiting factor for plant growth. Wall extension is thought to result from the loosening of its structure. However, it is not known how this is coordinated with wall synthesis. We have identified two novel allelic cellulose-deficient dwarf mutants, kobito1-1 and kobito1-2 (kob1-1 and kob1-2). The cellulose deficiency was confirmed by the direct observation of microfibrils in most recent wall layers of elongating root cells. In contrast to the wild type, which showed transversely oriented parallel microfibrils, kob1 microfibrils were randomized and occluded by a layer of pectic material. No such changes were observed in another dwarf mutant, pom1, suggesting that the cellulose defect in kob1 is not an indirect result of the reduced cell elongation. Interestingly, in the meristematic zone of kob1 roots, microfibrils appeared unaltered compared with the wild type, suggesting a role for KOB1 preferentially in rapidly elongating cells. KOB1 was cloned and encodes a novel, highly conserved, plant-specific protein that is plasma membrane bound, as shown with a green fluorescent protein-KOB1 fusion protein. KOB1 mRNA was present in all organs investigated, and its overexpression did not cause visible phenotypic changes. KOB1 may be part of the cellulose synthesis machinery in elongating cells, or it may play a role in the coordination between cell elongation and cellulose synthesis.

Figure 1.

Phenotype of kob1. (A) to (D) Adult plants grown for 6 weeks in a greenhouse. (A) From left to right: Col0, Ws, kob1-2 (Col0 background), and kob1-1 (Ws background). (B) Detail of kob1-2. (C) Detail of kob1-1. (D) Siliques of kob1-1. Mutant kob1 plants are severely dwarfed and sterile. (E) Seven-day-old dark-grown wild-type (left) and kob1-1 (right) seedlings (two seedlings each). (F) Seven-day-old light-grown wild-type (left) and kob1-1 (right) seedlings (two seedlings each).

Figure 2.

Cellular Phenotype of kob1-1 Hypocotyls and Roots. (A) and (B) Transverse sections halfway along hypocotyls of 5-day-old dark-grown wild-type (A) and kob1-1 (B) seedlings after Calcofluor staining. Mutant hypocotyls show unaltered anatomy, but all cells show increased radial expansion compared with the wild type. Mutant cell walls are interrupted frequently (arrowheads in [B]). wt, wild type. Bar in (B) = 50 μm for (A) and (B). (C) to (F) Confocal optical median sections, stained in vivo with FM1-43, of 7-day-old light-grown wild-type ([C] and [D]) and kob1-1 ([E] and [F]) roots. Root meristem ([C] and [E]), elongation zone (D), and mature part (F) are shown. Note the dramatically reduced elongation zone in kob1-1, as shown by the premature appearance of root hairs (arrowheads). Bar in (C) = 50 μm for (C) to (F). (G) Phloroglucinol staining of 4-day-old dark-grown kob1-1 (left) and wild-type (right) seedlings showing red staining of ectopic lignin in the mutant root. (H) and (I) Sirofluor staining of 4-day-old dark-grown wild-type (H) and kob1-1 (I) seedlings showing light blue staining of ectopic callose in the mutant.

Figure 3.

Cell Wall Alterations in Dark-Grown kob1-1. (A) Neutral sugar and galacturonic acid contents of TFA-hydrolyzable cell wall fractions of wild-type (Ws) and kob1-1 seedlings. Mutant cell walls show increased pectin content, as shown by the higher galacturonic acid levels compared with the wild-type control. (B) and (C) Matrix-assisted laser desorption ionization–time of flight mass spectra of the fragments produced from wild-type (B) and kob1-1 (C) cell walls. No significant differences in the composition of xyloglucan fragments released by endo-β-1,4-glucanase treatment was observed in the mutant compared with the wild type. XXXG, XXLG, XXFG, and XLFG refer to the xyloglucan fragments according to the nomenclature of Fry et al. (1993). The acetylated versions of the fragments XXLG, XXFG, and XLFG (from left to right) are indicated by asterisks. (D) and (E) Cellulose content (D) and incorporation in the cellulosic fraction of ¹⁴C-Glc (percentage of total cell wall [CW] material) (E). Cell walls of kob1-1 show reduced relative cellulose content compared with the wild type, and both kob1 alleles incorporate less ¹⁴C-Glc into the cellulosic fraction. Error bars indicate sd values; n = 3 ([A] and [D]) and n = 5 (E).

Figure 4.

Microfibril Organization Is Altered in Elongating Cells of kob1-1. FESEM of cryosectioned roots of the wild type (Ws) ([A], [D], [F], and [H]), kob1-1 ([B], [E], [G], [I], and [K]), and pom1 ([C] and [J]) at low magnification (×100) ([A] to [C]) and ×25,000 magnification of hypochlorite-treated samples ([D] to [G] and [J]) and ×50,000 magnification of hypochlorite-pectolyase–treated samples ([H] and [I]). Microfibrils are parallel and oriented transversely to the elongation axis in cells in the division zone (D) and the elongation zone (F) in the wild type. In kob1-1, microfibrils are indistinguishable from those of the wild type in the division zone (E); however, in the elongation zone (G), only amorphous material can be seen. This is primarily pectic material, because pectolyase treatment of the same cells reveals microfibrils in kob1-1 (I). These remaining microfibrils are disordered, as opposed to the ordered microfibrils seen in the wild type treated in the same way (H). This microfibril defect is unlikely an indirect effect of reduced cell elongation in kob1-1, because in pom1, a mutant with a comparable cell elongation defect in the root (C), microfibrils are indistinguishable from those of the wild type (J). In contrast to the findings in elongating cells, in secondary thickenings in xylem cells of kob1-1 roots, microfibrils show a normal parallel orientation (K). Bars = 100 μm in (A) to (C), 1 μm in (D) to (G) and (J), and 500 nm in (H) and (I).

Figure 5.

Cloning of KOB1. (A) Overview of the intron-exon organization of KOB1 and positions of mutations in kob1-1 and kob1-2. (B) PCR with the primers indicated by arrows in (A) on cDNA from wild-type seedlings (lane 1) and heterozygous kob1-2 seedlings (lane 2) and on genomic DNA (gDNA) as a control (lane 3). Note the aberrant splicing of the third intron in kob1-2 seedlings leading to two distinct products, one still containing the third intron and a second lacking the fourth exon. WT, wild type. (C) Sequence alignment of KOB1, KOB2, KOB3, and KOBRICE, a homolog in rice.

Figure 6.

Plasma Membrane Localization of GFP-KOB1 in Elongated Cells. Fluorescence of GFP-KOB1 ([A] and [D]) and the plasma membrane dye FM4-64 (B) in the root of the GFP-KOB1 transformant. Bars = 50 μm. (A) to (C) Elongated cells. (C) shows a merged image. (D) Cell division zone of the root. GFP-KOB1 is present in the plasma membrane in elongated cells. By contrast, within the cell division zone, GFP fluorescence is present in intracellular structures and not in the plasma membrane.