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

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.

Brachypodium distachyon UNICULME4 and LAXATUM-A are redundantly required for development.

In cultivated grasses, tillering, leaf, and inflorescence architecture, as well as abscission ability, are major agronomical traits. In barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and brachypodium (Brachypodium distachyon), NOOT-BOP-COCH-LIKE (NBCL) genes are essential regulators of vegetative and reproductive development. Grass species usually possess 2-4 NBCL copies and until now a single study in O. sativa showed that the disruption of all NBCL genes strongly altered O. sativa leaf development. To improve our understanding of the role of NBCL genes in grasses, we extended the study of the two NBCL paralogs BdUNICULME4 (CUL4) and BdLAXATUM-A (LAXA) in the nondomesticated grass B. distachyon. For this, we applied reversed genetics and generated original B. distachyon single and double nbcl mutants by clustered regularly interspaced short palindromic repeats - CRISPR associated protein 9 (CRISPR-Cas9) approaches and genetic crossing between nbcl targeting induced local lesions in genomes (TILLING) mutants. Through the study of original single laxa CRISPR-Cas9 null alleles, we validated functions previously proposed for LAXA in tillering, leaf patterning, inflorescence, and flower development and also unveiled roles for these genes in seed yield. Furthermore, the characterization of cul4laxa double mutants revealed essential functions for nbcl genes in B. distachyon development, especially in the regulation of tillering, stem cell elongation and secondary cell wall composition as well as for the transition toward the reproductive phase. Our results also highlight recurrent antagonist interactions between NBCLs occurring in multiple aspects of B. distachyon development.

BdERECTA controls vasculature patterning and phloem-xylem organization in Brachypodium distachyon.

BACKGROUND: The vascular system of plants consists of two main tissue types, xylem and phloem. These tissues are organized into vascular bundles that are arranged into a complex network running through the plant that is essential for the viability of land plants. Despite their obvious importance, the genes involved in the organization of vascular tissues remain poorly understood in grasses. RESULTS: We studied in detail the vascular network in stems from the model grass Brachypodium distachyon (Brachypodium) and identified a large set of genes differentially expressed in vascular bundles versus parenchyma tissues. To decipher the underlying molecular mechanisms of vascularization in grasses, we conducted a forward genetic screen for abnormal vasculature. We identified a mutation that severely affected the organization of vascular tissues. This mutant displayed defects in anastomosis of the vascular network and uncommon amphivasal vascular bundles. The causal mutation is a premature stop codon in ERECTA, a LRR receptor-like serine/threonine-protein kinase. Mutations in this gene are pleiotropic indicating that it serves multiple roles during plant development. This mutant also displayed changes in cell wall composition, gene expression and hormone homeostasis. CONCLUSION: In summary, ERECTA has a pleiotropic role in Brachypodium. We propose a major role of ERECTA in vasculature anastomosis and vascular tissue organization in Brachypodium.

Spatial and temporal distribution of cell wall polysaccharides during grain development of Brachypodium distachyon.

Brachypodium distachyon (Brachypodium) is now well considered as being a suitable plant model for studying temperate cereal crops. Its cell walls are phylogenetically intermediate between rice and poaceae, with a greater proximity to these latter. By microscopic and biochemical approaches, this work gives an overview of the temporal and spatial distribution of cell wall polysaccharides in the grain of Brachypodium from the end of the cellularization step to the maturation of grain. Variation in arabinoxylan chemical structure and distribution were demonstrated according to development and different grain tissues. In particular, the kinetic of arabinoxylan feruloylation was shown occuring later in the aleurone layers compared to storage endosperm. Mixed linked ß-glucan was detected in whole the tissues of Brachypodium grain even at late stage of development. Cellulose was found in both the storage endosperm and the outer layers. Homogalacturonan and rhamnogalacturonan I epitopes were differentially distributed within the grain tissues. LM5 galactan epitope was restricted to the aleurone layers contrary to LM6 arabinan epitope which was detected in the whole endosperm. A massive deposition of highly methylated homogalacturonans in vesicular bodies was observed underneath the cell wall of the testa t2 layer at early stage of development. At maturity, low-methylated homogalacturonans totally fulfilled the lumen of the t2 cell layer, suggesting pectin remodeling during grain development. Xyloglucans were only detected in the cuticle above the testa early in the development of the grain while feruloylated arabinoxylans were preferentially deposited into the cell wall of t1 layer. Indeed, the circumscribed distribution of some of the cell wall polysaccharides raises questions about their role in grain development and physiology.