The control of storage xyloglucan mobilization in cotyledons of Hymenaea courbaril

Abstract

Hymenaea courbaril is a leguminous tree species from the neotropical rain forests. Its cotyledons are largely enriched with a storage cell wall polysaccharide (xyloglucan). Studies of cell wall storage polymers have been focused mostly on the mechanisms of their disassembly, whereas the control of their mobilization and the relationship between their metabolism and seedling development is not well understood. Here, we show that xyloglucan mobilization is strictly controlled by the development of first leaves of the seedling, with the start of its degradation occurring after the beginning of eophyll (first leaves) expansion. During the period of storage mobilization, an increase in the levels of xyloglucan hydrolases, starch, and free sugars were observed in the cotyledons. Xyloglucan mobilization was inhibited by shoot excision, darkness, and by treatment with the auxin-transport inhibitor N-1-naphthylphthalamic acid. Analyses of endogenous indole-3-acetic acid in the cotyledons revealed that its increase in concentration is followed by the rise in xyloglucan hydrolase activities, indicating that auxin is directly related to xyloglucan mobilization. Cotyledons detached during xyloglucan mobilization and treated with 2,4-dichlorophenoxyacetic acid showed a similar mobilization rate as in attached cotyledons. This hormonal control is probably essential for the ecophysiological performance of this species in their natural environment since it is the main factor responsible for promoting synchronism between shoot growth and reserve degradation. This is likely to increase the efficiency of carbon reserves utilization by the growing seedling in the understorey light conditions of the rain forest.

Figure 1.

Dry masses of detached and attached cotyledons (A), whole seedlings (B), the shoot above the cotyledons insertions (C), and total leaf area of seedlings of H. courbaril (D). Seedlings with cotyledons grown under control conditions (control), in the darkness, or with the shoot excised above the cotyledons insertions at 34 d (excised). Cotyledons were detached at 19, 34, and 41 d and kept in water in the darkness. Germination (G), emergence of seedlings (E), and fall of cotyledons (F). Bars represent sd of the mean of five replicates.

Figure 2.

Contents of xyloglucan and starch in detached cotyledons (A and B, respectively; legend in A) and in cotyledons attached to the seedlings (C and D; legend in C) of H. courbaril. The attached cotyledons were from seedlings grown under control conditions (control), in the darkness. or with shoot excised above the cotyledons insertions at 34 d (excised). Cotyledons were detached at 19, 34, and 41 d and kept in water in the darkness or in 10⁻⁶ m 2,4-D (41 d only). Bars represent sd of the mean of three composed replicates.

Figure 3.

Specific activities of xyloglucan hydrolases in attached (A, C, and E; legends at E) and detached cotyledons (B, D, and F; legends at F) of H. courbaril during seedling development. The attached cotyledons used were from seedlings grown under control conditions (control), in the darkness. or with shoot excised above the cotyledons insertions (shoot excised). The detached cotyledons were taken from developing seedlings at 19, 34. and 41 d and kept in water in the darkness or in 10⁻⁶ m 2,4-D (41 d only). A and B, XEH. C and D, β-glucosidase. E and F, β-galactosidase. The activities detected in attached cotyledons (control) were added to each graph as a reference. Bars represent sd of the mean of three composed replicates.

Figure 4.

Specific activities of xyloglucan hydrolases in attached cotyledons of seedlings of H. courbaril grown as a control without excision treatment (control) and intact seedlings treated with N-1-naphthylphthalamic acid at 200 μm (NPA); and in shoot excised plants (shoot excised), shoot excised seedlings with light-protected cotyledons (shoot excised LPC) and shoot apex excised seedlings (top shoot excised). A, XEH. B, α-xylosidase. C, β-glucosidase. D, β-galactosidase. The activities detected in attached cotyledons (control) were added to each graph as a reference. Bars represent sd of the mean of three composed replicates.

Figure 5.

Concentration of endogenous IAA determined by ELISA (A; Peres et al., 1997) and by GC-SIM-MS (B; Chen et al., 1988). In A, the IAA was measured in the attached cotyledons from intact seedlings of H. courbaril growing in light, darkness, or with shoot excised above the cotyledons insertions (shoot excised) at 34 d. The only isolated cotyledons used as a reference in this case were the ones detached at 34 d and subsequently kept in the darkness. The plant materials used for auxin analyses were selected according to availability of tissue and to maximize the differences observed during the period of analysis. In B, the IAA was also measured in attached cotyledons of intact seedlings of H. courbaril grown in the light and in intact seedlings treated with NPA at 200 μm (NPA 200 μm). In the last technique, the IAA was also evaluated in shoot excised seedlings at 34 d (shoot excised), in shoot excised seedlings with light-protected cotyledons (shoot excised LPC), and in seedlings with the shoot apex excised (top shoot excised). Bars represent sd of the mean of three composed replicates.

Figure 6.

Representation of the synchronism between events taking place during xyloglucan mobilization and shoot development in developing seedlings of H. courbaril. Auxin (IAA) is produced in expanding leaves (eophyll and first metaphyll) and transported to cotyledons by polar transport, which can be stimulated by red light (1) and inhibited by NPA. In the cotyledons, the IAA may act on the modulation of expression of the genes coding for cell wall hydrolases (2); on ⁺H-pump activity reducing the apoplastic pH (3); or on the establishment of vascular system (4). Following the xyloglucan (XG) degradation (5) in storage cell walls of cotyledons by the concerted action of the hydrolases, monosaccharides (Ms) are transported to the cytoplasm, where they are metabolized (6) to Suc and starch. The Suc produced is driven mainly toward the growing shoot (7). In the shoot, light stimulates (8) growth, using Suc as a carbon backbone, which results in IAA availability to the cotyledons and consequently stimulation of xyloglucan mobilization.

Figure 7.

Schematic representation of the two experiments performed. See “Materials and Methods” for details.