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. 2004 Jun;135(2):959-68.
doi: 10.1104/pp.104.038711. Epub 2004 Jun 4.

Interaction between wall deposition and cell elongation in dark-grown hypocotyl cells in Arabidopsis

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Interaction between wall deposition and cell elongation in dark-grown hypocotyl cells in Arabidopsis

Guislaine Refrégier et al. Plant Physiol. 2004 Jun.

Abstract

A central problem in plant biology is how cell expansion is coordinated with wall synthesis. We have studied growth and wall deposition in epidermal cells of dark-grown Arabidopsis hypocotyls. Cells elongated in a biphasic pattern, slowly first and rapidly thereafter. The growth acceleration was initiated at the hypocotyl base and propagated acropetally. Using transmission and scanning electron microscopy, we analyzed walls in slowly and rapidly growing cells in 4-d-old dark-grown seedlings. We observed thick walls in slowly growing cells and thin walls in rapidly growing cells, which indicates that the rate of cell wall synthesis was not coupled to the cell elongation rate. The thick walls showed a polylamellated architecture, whereas polysaccharides in thin walls were axially oriented. Interestingly, innermost cellulose microfibrils were transversely oriented in both slowly and rapidly growing cells. This suggested that transversely deposited microfibrils reoriented in deeper layers of the expanding wall. No growth acceleration, only slow growth, was observed in the cellulose synthase mutant cesA6(prc1-1) or in seedlings, which had been treated with the cellulose synthesis inhibitor isoxaben. In these seedlings, innermost microfibrils were transversely oriented and not randomized as has been reported for other cellulose-deficient mutants or following treatment with dichlorobenzonitrile. Interestingly, isoxaben treatment after the initiation of the growth acceleration in the hypocotyl did not affect subsequent cell elongation. Together, these results show that rapid cell elongation, which involves extensive remodeling of the cell wall polymer network, depends on normal cellulose deposition during the slow growth phase.

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Figures

Figure 1.
Figure 1.
Kinetics of hypocotyl cell elongation of wild-type Arabidopsis cv Columbia seedlings grown in the dark. A, Cell length. B, REGR. Third epidermal cell (▪) and 13th epidermal cell (♦) as counted from the collet were measured.
Figure 2.
Figure 2.
Wall architecture of wild-type hypocotyl cells at different stages of elongation in 4-d-old dark-grown seedlings. Ultrathin sections were extracted with methylamine and subsequently stained with PATAg to reveal nonextracted polysaccharides (mainly cellulose). The cartoon on the lower left of each photograph indicates the position of the section with respect to the cell wall orientation. A to D, External epidermal wall. E and F, Radial cortical walls. Transverse (A and E) and longitudinal (B) sections through slowly growing cells at the top of the hypocotyl. Longitudinal (C) and transverse (D and F) section through rapidly growing cells toward the hypocotyl base. Arrowhead in C shows the transverse orientation of the innermost polysaccharide layer in this section, which was slightly tangential to the plasma membrane. cu, cuticle; pl. mb, plasma membrane. Scale bar = 300 nm.
Figure 3.
Figure 3.
Transversely oriented innermost cell wall layers in wild-type hypocotyl cells at different stages of elongation. Field emission scanning electron microscopy of 80-h-old dark-grown hypocotyls. A, Low magnification of the cryosectioned sample. The positions of higher magnifications presented in B, C, and D are shown. The arrowhead marks the approximate location of the growth acceleration zone. Scale bar in A = 300 μm. B and C, Slowly growing cells at the top of the hypocotyl. D, Rapidly growing cell further toward the hypocotyl base. B, Epidermal cell. C and D, Cortical cells. Scale bars in B, C, and D = 500 nm.
Figure 4.
Figure 4.
Kinetics of hypocotyl cell elongation (as compared with wild type) and orientation of innermost cellulose microfibrils in cellulose-deficient prc1-1 seedlings grown in the dark. Cell length (A), REGR (B), and cell width (C). D and E, FESEM of 80 h dark-grown prc1-1 cryosectioned hypocotyl. D, Low magnification (scale bar = 300 μm); the position of the higher magnification presented in E is shown. E, High magnification (same magnification as Fig. 3A). Third epidermal cells as counted from the collet were measured in prc1-1 (black triangle) and compared with wild type (white square; same magnification as Fig. 3, B–D).
Figure 5.
Figure 5.
Effects of cellulose synthesis inhibition by isoxaben on hypocotyl elongation. A, Hypocotyl length of 4-d-old dark-grown seedlings. B, Diagram showing experimental growth conditions for each sample. Seedlings were grown for 4 d continuously in the absence (a) or presence (b) of isoxaben (ix); 4 h without, followed by 92 h with isoxaben (c); 30 h without, followed by 66 h with isoxaben (d); 50 h without, followed by 46 h with isoxaben (e). C, FESEM of innermost wall layer of an epidermal cell of a seedling grown in the presence of isoxaben. Scale bar of the high magnification = 500 nm. Inset shows a low-magnification image of the cryosectioned hypocotyl and the position of the cell in which the presented image was taken. The cartoon in the inset shows the location of the image with respect to the cell wall orientation. Isoxaben concentration in all experiments was 4 nm.
Figure 6.
Figure 6.
Two scenarios for cell wall synthesis and remodeling in elongating dark-grown hypocotyl cells. These scenarios were based on the following observations: embryonic cells are small with thin walls and they elongate slowly upon germination. Slowly growing cells at the top of 4-d-old hypocotyls have a thick polylamellated cell wall, especially the external epidermal walls. Rapidly growing cells around the middle of those hypocotyls have thin walls with primarily axially oriented polysaccharides. Inhibition of CESA6-dependent cellulose synthesis during the slow growth phase in the mutant or through administering isoxaben prevents cells from accelerating their growth. Isoxaben treatment after the initiation of the growth acceleration does not prevent further growth. In the first scenario, both cells at the top and the bottom of the hypocotyls follow the same growth steps: they accumulate a thick polylamellated wall during the slow growth phase (1a). During the rapid growth phase (1b), this wall is extensively remodeled through the action of wall relaxing agents. In the second scenario, cell wall thickness reflects the balance between wall synthesis and wall relaxation. Cell wall synthesis occurs continuously in all cells, an increase in the rate of wall relaxation and hence cell elongation is initiated at the hypocotyls basis at 48 h, whereas cells toward the top accelerate their growth later. As a result, smaller cells accumulate a thicker wall before their growth acceleration (1a), whereas cells toward the base maintain a thin wall before and after their growth acceleration (2). It is still conceivable in this scenario that the thick walls in smaller cells at the hypocotyl top undergo thinning after growth acceleration (1b). In this second scenario, the differential effect of isoxaben on cell elongation before and after growth acceleration is more difficult to explain. Question mark refers to a growth stage that has not been observed directly. The thicker side of the small cells represents the outer epidermal wall.

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