With continued consumption of fossil fuels, humankind faces a growing challenge of finding renewable sources of energy. Although plant derived biomass contains appreciable energy, to serve as a fuel for transportation it must be chemically or biologically liquefied, a conversion that is the subject of strenuous efforts to make economical. Success will presumably require not only advances in chemistry such as better catalysts, but also improved plants to serve as feedstock. As an ideal input, grasses are receiving considerable attention because certain species grow to great density, are perennial, and can require little if any fertilizer or irrigation . But their very size, longevity, and genome complexity makes these species difficult subjects to study and breed.
For studying grasses, whether as sources for biofuel or for any other grass-specific question, an emerging model is Brachypodium distachyon. This species is closely related to cereals and temperate grasses, and is about the same size as Arabidopsis thaliana and has about the same generation time. Studies on B. distachyon benefit from rapidly developing community resources, such as a completely sequenced genome, mutant collections (both chemically derived and sequence-indexed DNA insertions), and efficient protocols for transformation and crossing [2–5]. Of particular note for biofuels research, B. distachyon has cell walls that are similar compositionally to that of other grasses, like wheat (Tritium aestavum), barley (Hordeum vulgare), and Miscanthus[6–8]. Insofar as the cell wall constitutes almost the entirety of the input for converting biomass to biofuel, this similarity, along with the genetic attributes of this small grass, emphasize its suitability as a model for grass-related, biomass crop research.
Within the cell wall, as a target for optimization, cellulose is pre-eminent. Cellulose is the most abundant of any single wall component and is made exclusively of glucose, a tractable and energy rich molecule. Cellulose comprises long polymers of (1–4) β-linked glucose that are synthesized at the plasma membrane and associate laterally into a microfibril. Because of the configuration of the glucose residues, hydrogen bonds form at great density both within and between chains, a density that gives cellulose an elastic modulus rivaling that of steel but makes the structure impervious to degradation whether chemical or enzymatic.
Within the plasma membrane, the structure synthesizing cellulose is called a “terminal complex” [9–11]. In land plants and related green algae, the terminal complex as seen in the electron microscope comprises six subunits with hexagonal symmetry and is called a “rosette” . The primary constituents of the rosette are CELLULOSE SYNTHASE A (CESA) proteins. These proteins belong to processive glycosyltransferase family 2 and are thought to be the catalytic subunits for polymerizing the glucose chain. In angiosperms, CESAs usually comprise a small gene family with around ten members [13, 14].
Identification of CESA proteins and characterization of their function has greatly benefited from the facile genetics of A. thaliana. From this work, it emerged that certain CESA proteins synthesize the primary cell wall whereas others synthesize the secondary cell wall [15, 16]. Furthermore, it appeared that a given cell must express three distinct CESA proteins to produce cellulose at optimal levels. For the secondary cell wall, a screen based on collapsed xylem cells led to the identification of several irregular xylem (irx) lines, three of which, irx5, irx3, and irx1, harbor lesions in AtCESA4, 7, and 8, respectively [17–19]. Supporting the hypothesis of non-redundancy, these genes are expressed at similar levels in similar cell types, and the null mutants have indistinguishable phenotypes, including weak stems, collapsed xylem, and thin secondary cell walls that are deficient in cellulose.
Identification of a trio of CESA genes primarily responsible for synthesizing cellulose in secondary cell walls has been supported by work in other systems, including grasses. First, AtCESA4, 7, and 8 are represented in some angiosperm species by a single sequence each, and orthologs are more closely related than homologs (i.e., CESA4s of various species resemble each other more closely than do CESA4, 7, and 8 of a single species) [13, 20, 21]. Second, in grasses, a mutant of barley, fragile stem 2, with brittle stems and low cellulose content in the mature plant was attributed to a lesion in the barley ortholog of AtCESA4 . In addition, “brittle culm” mutants in rice (Oryza sativa) have been mapped to the three orthologs of AtCEA4, 7 and 8 and again these mutants have similar phenotypes, including modest dwarfism, thinner and weaker culms, and reduced cellulose content . However, it is not understood why three distinct proteins are needed nor is it known which of the loss-of-function phenotypes result directly from the lost protein and which result as a consequence of cumulative effects.
Here we describe the CESA gene family in B. distachyon with the aid of gene expression profiling and phylogeny. Furthermore a detailed analysis of the candidate secondary CESAs were performed by functionally characterizing mutants generated with specific artificial microRNA constructs.