Limited correlation between expansin gene expression and elongation growth rate

Abstract

The aim of this work was to study the role of the cell wall protein expansin in elongation growth. Expansins increase cell wall extensibility in vitro and are thought to be involved in cell elongation. Here, we studied the regulation of two tomato (Lycopersicon esculentum cv Moneymaker) expansin genes, LeExp2 and LeExp18, in rapidly expanding tissues. LeExp2 was strongly expressed in the elongation zone of hypocotyls and in the faster growing stem part during gravitropic stimulation. LeExp18 expression did not correlate with elongation growth. Exogenous application of hormones showed a substantial auxin-stimulation of LeExp2 mRNA in etiolated hypocotyls and a weaker auxin-stimulation of LeExp18 mRNA in stem tissue. Analysis of transcript accumulation revealed higher levels of LeExp2 and LeExp18 in light-treated, slow-growing tissue than in dark-treated, rapidly elongating tissue. Expansin protein levels and cell wall extension activities were similar in light- and dark-grown hypocotyl extracts. The results show a strong correlation between expansin gene expression and growth rate, but this correlation is not absolute. We conclude that elongation growth is likely to be controlled by expansin acting in concert with other factors that may limit growth under some physiological conditions.

Figure 1

Schematic overview of the genomic LeExp2 sequence. A, Numbers 1 to 5 represent putative cis-acting elements on the promoter. The exons are depicted as black rectangles, the arrows on the putative auxin-response element (AuxREs) depict the orientation of the elements with respect to the ATG. sp, Signal peptide; cys, Cys-rich region; br, basic region; cbd, cellulose-binding domain (according to Cosgrove, 1997). B, DNA gel-blot analysis of genomic DNA with a probe of 476 bp from the coding region of LeExp2. Genomic DNA was digested with EcoRI (lane 1), EcoRV (lane 2), HindIII (lane 3), XbaI (lane 4), or BamHI (lane 5), respectively. When the blot was stripped and hybridized with a 3′ gene-specific probe, only the bands marked with an asterisk were visible (data not shown). C, The analysis of amino acid similarity was performed with the PAM-table program from WebGenetics (available at http://www.webgenetics.com). The expression patterns of these genes were analyzed either by northern blot or in situ hybridization. D, Numbers 1 to 5 correspond to the diagram in A. The upper DNA strand of the regulatory element shows its core sequence, the lower strand shows the sequence of LeExp2.

Figure 2

Expansin mRNA accumulation in the hypocotyl. A, Tomato hypocotyls were labeled with paint marks every 2 mm, and length increase was measured 24 h later. B, Lanes from left to right contain hypocotyl segments (1 cm) cut from the top, middle, and bottom regions, the hook region, or the total hypocotyl. Five micrograms of total RNA was separated per lane and hybridized with cDNA probes indicated on the right side. The lower panel represents an ethidium bromide gel and visualizes the ribosomal RNAs as a control for equal loading and intactness of the RNA.

Figure 3

Expansin mRNA accumulation during gravitropic stimulation. Control stem segments were split in right and left halves before gravitropic stimulation (0 min). Gravistimulated plants were placed horizontally, and after 30, 150, and 300 min, stem segments were cut in upper (up) and lower (low) halves. Total RNA was isolated, and 10 μg was separated per lane. Hybridizations and controls were the same as in Figure 2.

Figure 4

Effect of plant hormones on hypocotyl segment length elongation and on LeExp2 mRNA accumulation. A, Segments (1 cm) were cut from the apical (top) region of etiolated tomato hypocotyls and incubated in buffer (control) or buffer plus 2,4-D, GA₃, ACC, or BL for 16 h. Segment length was measured after hormone treatment. The error bars represent the sds. B, Northern-blot analysis of apical segments was performed after hormone treatment. Five micrograms of total RNA was separated per lane and hybridized with cDNA probes indicated on the left side. Controls were the same as in Figure 2.

Figure 5

Time course analysis of 2,4-D and effect of auxin and other effectors on LeExp2 mRNA accumulation in wild type and in the dgt mutant. A, Northern-blot analysis was performed after incubation of whole hypocotyls in buffer plus 5 μm 2,4-D for the indicated times. Bars in the top panel represent the 2,4-D-stimulated relative expression of LeExp2 corrected by control levels (−2,4-D). B, Northern-blot analysis was performed after treatments. Whole hypocotyls were incubated in buffer (control) or buffer plus the indicated hormones or effectors. IAA, Indole-3-acetic acid; NAA, naphthalene-acetic acid. C, Northern-blot analysis was performed after auxin treatment. Stem segments from the auxin-insensitive mutant dgt and its corresponding wild type (cv VFN8) were incubated in buffer alone (−) or buffer plus 5 μm 2,4-D (+). Total RNA was isolated, and 10 μg was separated per lane. Hybridizations and controls were the same as in Figure 2.

Figure 6

Localization of LeExp2 mRNA transcript by in situ hybridization. Cross sections of apical segments from dark- grown hypocotyls are shown in A through D, cross sections of apical segments from light-grown stems in E through H. A and E represent control hybridizations with sense probes. B and F, Sections from untreated tissue were hybridized with the antisense probe; C and G, antisense hybridization of 2,4-D-treated tissue. Acridine orange staining is shown in D and H. Bar = 100 μm.

Figure 7

Hypocotyl elongation kinetics and morphology of light- and dark-grown seedlings. A, Hypocotyl length was measured from d 5 to 21 in light- and dark-grown tomato seedlings. The calculated growth rate was six times higher in dark-grown seedlings than in light-grown ones. B, SEM of the apical region in a dark-grown hypocotyl. Bar = 200 μm. C, SEM of the apical region in a light-grown hypocotyl. Bar = 200 μm.

Figure 8

Effect of dark-adaptation and light-induction on LeExp2, Xet, Cel, or gh3 mRNA accumulation in whole hypocotyls. A, One-half of light-grown seedlings remained in a 16- to 8-h light to dark cycle (light grown), and the other one-half was transferred to darkness for 3 d (dark adapted). B, One-half of dark-grown seedlings remained in the dark (dark grown), and the other one-half was light-induced for 24 h (light induced). Hypocotyls were incubated in buffer (C) or buffer plus 5 μm 2,4-D or 1 μm BL, respectively. Total RNA was isolated from hypocotyls, and 10 μg was separated per lane. Hybridizations and controls were the same as in Figure 2.

Figure 9

Effect of light quality on LeExp2 mRNA accumulation in wild type and in phytochrome mutants. A, Dark-grown wild-type hypocotyls were 24-h light-induced or treated with 5-min R, 5-min R and 15-min FR treatment, or 15-min FR only and returned to darkness for 16 h. B, Dark-grown hypocotyls (D) were treated for 5 min with R in wild type (wt), and in the phytochrome mutants aurea (au), tri1, and fri1. The R-treated seedlings were returned to darkness for 16 h. Total RNA was isolated, and 10 μg was separated per lane. Hybridizations and controls were the same as in Figure 2.

Figure 10

Effect of dark-adaptation on LeExp2 and LeExp18 mRNA accumulation in stem segments. One-half of 4-week-old plants remained in a 16- to 8-h light to dark cycle (light grown), and the other one-half was transferred to darkness for 3 d (dark adapted) prior to hormone treatments. Segments of 2-cm length were isolated from the growing region and were incubated in buffer (C), or buffer plus 5 μm 2,4-D or 1 μm BL, respectively. Total RNA was isolated, and 10 μg was separated per lane. Hybridizations and controls were the same as in Figure 2.

Figure 11

Expansin expression and cell wall extensibility of protein extracts from light- and dark-grown whole hypocotyls. A, Expansin protein expression in light- and dark-grown tomato hypocotyls. Ten micrograms of crude cell wall protein extract was loaded per lane and probed with anti-LeExp1 antibody. B, In vitro extension activity induced with light- and dark-grown protein extracts. Equal concentrations of crude cell wall proteins, isolated from light- and dark-grown tomato hypocotyls, were added to the extending material, as indicated, and length increase was recorded. The average expansin activity was 21.5 μm/min (se 3.8 for dark-grown protein extract) and 25.5 μm/min (se 4.9 for light-grown extracts). Values are the means ± se of six measurements.