The regulation of cell wall extensibility during shade avoidance: a study using two contrasting ecotypes of Stellaria longipes

Abstract

Shade avoidance in plants involves rapid shoot elongation to grow toward the light. Cell wall-modifying mechanisms are vital regulatory points for control of these elongation responses. Two protein families involved in cell wall modification are expansins and xyloglucan endotransglucosylase/hydrolases. We used an alpine and a prairie ecotype of Stellaria longipes differing in their response to shade to study the regulation of cell wall extensibility in response to low red to far-red ratio (R/FR), an early neighbor detection signal, and dense canopy shade (green shade: low R/FR, blue, and total light intensity). Alpine plants were nonresponsive to low R/FR, while prairie plants elongated rapidly. These responses reflect adaptation to the dense vegetation of the prairie habitat, unlike the alpine plants, which almost never encounter shade. Under green shade, both ecotypes rapidly elongate, showing that alpine plants can react only to a deep shade treatment. Xyloglucan endotransglucosylase/hydrolase activity was strongly regulated by green shade and low blue light conditions but not by low R/FR. Expansin activity, expressed as acid-induced extension, correlated with growth responses to all light changes. Expansin genes cloned from the internodes of the two ecotypes showed differential regulation in response to the light manipulations. This regulation was ecotype and light signal specific and correlated with the growth responses. Our results imply that elongation responses to shade require the regulation of cell wall extensibility via the control of expansin gene expression. Ecotypic differences demonstrate how responses to environmental stimuli are differently regulated to survive a particular habitat.

Figure 1.

The alpine and prairie ecotypes of S. longipes. Shown are representative ramets from the alpine (left) and prairie (right) plants after 1 week in the indicated treatments. Low R/FR 0.25, PAR 140 μmol m⁻² s⁻¹; green shade, PAR 65 μmol m⁻² s⁻¹, R/FR 0.19, and blue light photon fluence rate of 2 μmol m⁻² s⁻¹. Control plants were grown in light with an unaltered spectral composition and a PAR of 140 μmol m⁻² s⁻¹. Each bar on the scale = 1 cm.

Figure 2.

The effects of canopy light signals on the growth rates of alpine and prairie plants. Ramet elongation rate was measured every day for 1 week. Alpine (A and C) and prairie (B and D) plants were grown under low R/FR (A and B; white circles) or green shade (C and D; gray circles) conditions. Controls (black circles) were grown under normal light conditions (spectral composition unaltered). Growth rates were calculated from length measurements of the total ramet height obtained using a digital caliper. Data points represent means of 30 to 35 ramets (mean ± se, n = 30–35). Experiments were carried out twice with similar results. Growth rates under green shading show statistically significant differences relative to controls, at all time points, in both ecotypes. Under low R/FR, only the growth rates for the prairie plants showed statistically significant differences relative to controls at days 3 to 5 (Student's t test, P < 0.05).

Figure 3.

Xyloglucan-degrading activity in response to canopy light signals. Shown is xyloglucan-degrading activity measured in the top internodes of alpine (top) and prairie (bottom) plants grown for 3 d under low R/FR (white bars) and green shade (gray bars) conditions. Controls (black bars) refer to data from plants grown under normal light conditions (unaltered spectral composition). Data points represent means ± se (n = 3); each biological replicate consisted of internodes pooled from different ramets from different pots. Different letters above each bar indicate statistically significant differences (P < 0.05, Tukey's b test). Experiments were repeated twice with similar results.

Figure 4.

Effects of low blue light conditions on the growth rates and xyloglucan-degrading activity in the alpine and prairie ecotypes. A, Representative ramets from the alpine and prairie plants after 1 week in the indicated treatments. Control plants were grown in light with an unaltered spectral composition. Each bar on the scale = 1 cm. B and C, Ramet elongation rate for alpine (B) and prairie (C) plants grown under low blue light (white circles) and control (black circles) conditions for 1 week. Data points represent means ± se (n = 30–35). Ramet length measurements were made using a digital caliper. Growth rate differences between control and low blue light treatments were statistically significant at all time points (P < 0.05, Student's t test). D and E, Xyloglucan-degrading activity measured in the top internodes of alpine (D) and prairie (E) plants grown for 3 d under low blue light conditions. Controls refer to data from plants grown under normal light conditions (unaltered spectral composition). Data points represent means ± se (n = 3); each biological replicate consisted of internodes pooled from different ramets from different pots. Different letters above each bar indicate statistically significant differences (P < 0.05, Tukey's b test). Experiments were repeated twice with similar results. [See online article for color version of this figure.]

Figure 5.

The effects of canopy light signals on the AIE of internodes from alpine and prairie plants. AIE of the topmost internodes of ramets from alpine (A) and prairie (B) plants grown under low R/FR (white bars) and green shade (gray bars) growth conditions for 3 d. Control plants (black bars) were grown under light with an unaltered spectral composition. AIE was measured using a constant-load extensometer with a pulling weight of 20 g and is calculated as the difference in the slopes of lines fitted through 10-min intervals before and after the bending point observed due to a change in pH from 6.8 to 4.5. Data points represent means ± se (n = 8–10). Each biological replicate consisted of the topmost internode from different ramets from different pots. Different letters above each bar indicate statistically significant differences (P < 0.05, Tukey's b test). Experiments were repeated twice with similar results.

Figure 6.

Phylogenetic analysis of α-expansin proteins. The deduced amino acid sequences of S. longipes α-expansins were aligned with highly similar amino acid sequences from the GenBank database using ClustalX software. This alignment was then used to generate a phylogenetic tree using TreeView software (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). The following are the sequences used along with their accession numbers in parentheses: Stellaria longipes, SlEXPA1 to SlEXPA7 (EU840703–EU840714, EU840720, and EU840721); Arabidopsis thaliana, AtEXPA6 (NP_180461), AtEXPA8 (O22874); Cucumis sativus, CsEXPA9 (AAL31480); Petunia hybrida, PhEXPA1 (AAR82849); Populus tremula, PtEXPA1 (AAR09168); Rumex palustris, RpEXPA11 (AAM22625); and Zinnia elegans, ZeEXPA1 (AAF35900).

Figure 7.

Differential regulation of S. longipes α-expansins in response to low R/FR. Relative transcript abundance of S. longipes α-expansins expressed in the internodes of alpine and prairie plants exposed to low R/FR (white circles) and control (unaltered spectral composition; black circles) light conditions for 3 d. Values were measured using real-time RT-PCR with 18S as an internal standard (means ± se; n = 3–4). Statistically significant differences are indicated by asterisks (Student's t test, P < 0.05).

Figure 8.

Differential regulation of S. longipes α-expansins in response to green shading. Relative transcript abundance of S. longipes α-expansins expressed in the internodes of alpine and prairie plants exposed to green shade (gray circles) and control (unaltered spectral composition; black circles) light conditions for 3 d. Values were measured using real-time RT-PCR with 18S as an internal standard (means ± se; n = 3–4). Statistically significant differences are indicated by asterisks (Student's t test, P < 0.05).