Mixed-Linkage Glucan Is the Main Carbohydrate Source and Starch Is an Alternative Source during Brachypodium Grain Germination

Abstract

Seeds of the model grass Brachypodium distachyon are unusual because they contain very little starch and high levels of mixed-linkage glucan (MLG) accumulated in thick cell walls. It was suggested that MLG might supplement starch as a storage carbohydrate and may be mobilised during germination. In this work, we observed massive degradation of MLG during germination in both endosperm and nucellar epidermis. The enzymes responsible for the MLG degradation were identified in germinated grains and characterized using heterologous expression. By using mutants targeting MLG biosynthesis genes, we showed that the expression level of genes coding for MLG and starch-degrading enzymes was modified in the germinated grains of knocked-out cslf6 mutants depleted in MLG but with higher starch content. Our results suggest a substrate-dependent regulation of the storage sugars during germination. These overall results demonstrated the function of MLG as the main carbohydrate source during germination of Brachypodium grain. More astonishingly, cslf6 Brachypodium mutants are able to adapt their metabolism to the lack of MLG by modifying the energy source for germination and the expression of genes dedicated for its use.

Keywords: Brachypodium; cell wall; germination; grain; lichenase; mixed-linkage glucan (MLG).

Figure 1

Histology of Brachypodium grains at 0, 65, and 96 h after the beginning of germination. Images (A–C): longitudinal cross-sections of grains stained with Toluidine blue. Magnifications indicate the nucellus epidermis. Images (D–F): endosperm cells stained with iodine and Toluidine blue. Starch granules are coloured purple by iodine while the other cellular components appear turquoise and brown with Toluidine blue. Arrows indicate starch granules. Scale bar = 100 µm. en: endosperm; pe: pericarp; ne: nucellar epidermis; em: embryo; sc: scutellum.

Figure 2

Biochemical analysis of germinated grains (A). Composition of soluble sugars obtained from Brachypodium wild type grains during the germination process (hours counted after the imbibition step). (B). HPAEC analysis of tri- and tetramers (BG3 and BG4, respectively) released from MLG after lichenase treatment of the AIR of germinated WT grains. (C). HPSEC analysis of hemicelluloses extracted by alkaline treatment from grain at various germination times and after xylanase digestion to detect the MLG profile, or xylanase + lichenase digestion to degrade both arabinoxylans and MLG.

Figure 3

Immunolabeling of germinated grains (0, 65, and 96 h after imbibition) using antibodies specific to MLG. Scale bar = 50 µm. g: germ; pe: pericarp; en: endosperm; ne: nucellar epidermis.

Figure 4

(A). Lichenase activity measured from protein extracts of germinated grains (without germ) and germs. Lichenase activity was determined by the Nelson method and barley MLG was used as substrate. (B). Transcript levels (qRT-PCR) of Brachypodium genes encoding putative lichenases during the germination (0, 8, 16, 24, and 48 h of germination) in grains and germs. Error bars represent standard deviation of the mean (n = 3).

Figure 5

Enzymatic activity of the recombinant enzymes encoded by Bradi2g22224 and Bradi2g22226. Enzymes were incubated with 10 mg/mL of substrate during one night for substrate specificity determination (A,E) or 2 h for other kinetics based on substrate concentration (B,F), temperature (C,G), and pH (D,H). MLG: barley mixed linkage glucan; AX: wheat arabinoxylans; XG: tamarin xyloglucan; CMC: carboxymethylcellulose.

Figure 6

Morphological characterization of mutant grains. (A): Average weights of wild type and mutant grains (mean of 10 grains, n = 10). (B): Stereomicroscopic images of wild type and homozygous cslf6 mutants showing the size and external aspect of grain. Scale bar = 1 mm. (C): Cross-sections of wild type and mutant grains (15 days of flowering) stained with Toluidine blue. Scale bar = 200 µm. (D): Calcofluor staining of wild type and mutant grains (15 days after flowering). Scale bar = 50 µm. cslf6-ho: homozygous cslf6 mutant; cslf6-az: azygous cslf6 mutant.

Figure 7

(A–D): Fluorescence immunolabeling of MLG in a grain cross-section of WT, cslh1, azygous (az), and homozygous (ho) cslf6 mutants (15 days after flowering). The homozygous cslf6 (cslf6-ho) does not show any fluorescence signal (D). (E): Differential interference contrast of the cslf6-ho mutant. Scale bar = 50 µm. (F): MLG content in the endosperm and pericarp of WT and mutant grains (15 days after flowering) expressed as a percentage of AIR (w/w).

Figure 8

Localization of starch in grains of cslf6 and cslh1 mutants and controls (WT and azygous cslf6). (A–D): Bright-field images of grain cross-sections stained with iodine. Scale bar: 50 µm. pe: pericarp; ne: nucellar epidermis; en: endosperm; t: testa. (E): Total amount of starch expressed as a percentage of AIR (w/w). Arrows indicate starch granules. cslf6-ho: homozygous cslf6 mutant; cslf6-az: azygous cslf6 mutant.

Figure 9

Development of germinated lines. (A). Germination rate at 24 and 48 h of germination of cslf6 and cslh1 mutants and controls (WT and azygous cslf6). (B). Size of radicles and hypocotyls of a cslf6 mutant compared to controls (WT and azygous cslf6). n = 100 grains. cslf6-ho: homozygous cslf6 mutant; cslf6-az: azygous cslf6 mutant.

Figure 10

Catabolism of reserve sugars in homozygous cslf6 mutant. (A–F): Histology of cslf6 grains at 0 h (images (A,B)), 65 h (images (C,D)), and 96 h (images (E,F)) of germination. (A,C,E): Images of longitudinal grain cross-sections stained with toluidine blue. (B,D,F): Higher magnification images of the sections corresponding to the dotted boxes on the images (A,C,E), respectively, and stained with both Toluidine blue and iodine. Starch granules are coloured purple by iodine. Scale bar = 100 µm. en: endosperm; al: aleurone; pe: pericarp; t: testa; ne: nucellar epidermis; em: embryo; sc: scutellum. (G): Lichenase activity from wild type and cslf6 germinated seeds. (H): Transcript levels of Bradi2g22222, Bradi2g22224, and Bradi2g22226 genes in WT and cslf6 grains at 0, 24, 48, and 96 h of germination. (I): α-amylase activity in WT and cslf6 mutant grains at 0, 48, and 96 h of germination. (J): α-amylase transcript level in WT and cslf6 mutant grains at 24 and 96 h of germination. Error bars represent standard deviation of the mean (n = 3).