Nicholas C. Carpita

The Sorghum bicolor genome and the diversification of grasses

Sorghum, an African grass related to sugar cane and maize, is grown for food, feed, fibre and fuel. We present an initial analysis of the ∼730-megabase Sorghum bicolor (L.) Moench genome, placing ∼98% of genes in their chromosomal context using whole-genome shotgun sequence validated by genetic, physical and syntenic information. Genetic recombination is largely confined to about one-third of the sorghum genome with gene order and density similar to those of rice. Retrotransposon accumulation in recombinationally recalcitrant heterochromatin explains the ∼75% larger genome size of sorghum compared with rice. Although gene and repetitive DNA distributions have been preserved since palaeopolyploidization ∼70 million years ago, most duplicated gene sets lost one member before the sorghum–rice divergence. Concerted evolution makes one duplicated chromosomal segment appear to be only a few million years old. About 24% of genes are grass-specific and 7% are sorghum-specific. Recent gene and microRNA duplications may contribute to sorghum’s drought tolerance.

Measurement of uronic acids without interference from neutral sugars.

Replacement of carbazole with meta-hydroxydiphenyl greatly improves the determination of uronic acids in the presence of neutral sugars by preventing substantially, but not completely, the browning that occurs during the heating of sugars in concentrated sulfuric acid and avoiding the formation of additional interference by the carbazole reagent (Blumenkrantz, N., and Asboe-Hansen, G. (1973) Anal. Biochem. 54, 484-489). However, interference is still substantial when uronic acids are determined in the presence of excess neutral sugar, particularly because of the browning that occurs during the first heating before addition of the diphenyl reagent. The browning can be essentially eliminated by addition of sulfamate to the reaction mixture (Galambos, J. T. (1967) Anal. Biochem. 19, 119-132). Although others have reported that sulfamate and the diphenyl reagent were incompatible, we find that a small amount of sulfamate suppresses color production by a 20-fold excess of some neutral sugars without substantial sacrifice of the sensitive detection of uronic acids by the diphenyl reagent. Sodium tetraborate is required for the detection of D-mannuronic acid and enhances color production by D-glucuronic acid. We propose this modified sulfamate/m-hydroxydiphenyl assay as a rapid and reliable means for the assay of uronic acids, particularly when present in much smaller amounts than neutral sugars.

The MUR3 Gene of Arabidopsis Encodes a Xyloglucan Galactosyltransferase That Is Evolutionarily Related to Animal Exostosins

Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved α-l-fucosyl-(1→2)-β-d-galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants. The MUR3 gene was cloned positionally and shown to encode a xyloglucan galactosyltransferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan. MUR3 belongs to a large family of type-II membrane proteins that is evolutionarily conserved among higher plants. The enzyme shows sequence similarities to the glucuronosyltransferase domain of exostosins, a class of animal glycosyltransferases that catalyze the synthesis of heparan sulfate, a glycosaminoglycan with numerous roles in cell differentiation and development. This finding suggests that components of the plant cell wall and of the animal extracellular matrix are synthesized by evolutionarily related enzymes even though the structures of the corresponding polysaccharides are entirely different from each other.

The mur2 mutant of Arabidopsis thaliana lacks fucosylated xyloglucan because of a lesion in fucosyltransferase AtFUT1

Cell walls of the Arabidopsis mutant mur2 contain less than 2% of the wild-type amount of fucosylated xyloglucan because of a point mutation in the fucosyltransferase AtFUT1. The mur2 mutation eliminates xyloglucan fucosylation in all major plant organs, indicating that Arabidopsis thaliana fucosyltransferase 1 (AtFUT1) accounts for all of the xyloglucan fucosyltransferase activity in Arabidopsis. Despite this alteration in structure, mur2 plants show a normal growth habit and wall strength. In contrast, Arabidopsis mur1 mutants that are defective in the de novo synthesis of l-fucose exhibit a dwarfed growth habit and decreased wall strength [Reiter, W. D., Chapple, C. & Somerville, C. R. (1993) Science 261, 1032–1035]. Because the mur1 mutation affects several cell wall polysaccharides, whereas the mur2 mutation is specific to xyloglucan, the phenotypes of mur1 plants appear to be caused by structural changes in fucosylated pectic components such as rhamnogalacturonan-II. The normal growth habit and wall strength of mur2 plants casts doubt on hypotheses regarding roles of xyloglucan fucosylation in facilitating xyloglucan–cellulose interactions or in modulating growth regulator activity.

Phosphate starvation responses are mediated by sugar signaling in Arabidopsis

Phosphate (Pi) is one of the least available plant nutrients in soils. It is associated with dynamic changes in carbon fluxes and several crucial processes that regulate plant growth and development. Pi levels regulate the expression of large number of genes including those involved in photosynthesis and carbon metabolism. Herein we show that sugar is required for Pi starvation responses including changes in root architecture and expression of phosphate starvation induced (PSI) genes in Arabidopsis. Active photosynthesis or the supplementation of sugar in the medium was essential for the expression of PSI genes under Pi limiting conditions. Expression of these genes was not only induced by sucrose but also detected, albeit at reduced levels, with other metabolizable sugars. Non-metabolizable sugar analogs did not induce the expression of PSI genes. Although sugar input appears to be downstream of initial Pi sensing, it is absolutely required for the completion of the PSI signaling pathway. Altered expression of PSI genes in the hexokinase signaling mutant gin2 indicates that hexokinase-dependent signaling is involved in this process. The study provides evidence for requirement of sugars in PSI signaling and evokes a role for hexokinase in some components of Pi response mechanism.

Maize and sorghum: genetic resources for bioenergy grasses

The highly photosynthetic-efficient C4 grasses, such as switchgrass (Panicum virgatum), Miscanthus (Miscanthusxgiganteus), sorghum (Sorghum bicolor) and maize (Zea mays), are expected to provide abundant and sustainable resources of lignocellulosic biomass for the production of biofuels. A deeper understanding of the synthesis, deposition and hydrolysis of the distinctive cell walls of grasses is crucial to gain genetic control of traits that contribute to biomass yield and quality. With a century of genetic investigations and breeding success, recently completed genome sequences, well-characterized cell wall compositions, and a close evolutionary relationship with future bioenergy perennial grasses, we propose that maize and sorghum are key model systems for gene discovery relating to biomass yield and quality in the bioenergy grasses.

Biosynthesis of plant cell wall polysaccharides.

The cell wall is the principal structural element of plant form. Cellulose, long crystals of several dozen glucan chains, forms the microfibrillar foundation of plant cell walls and is synthesized at the plasma membrane. Except for callose, all other noncellulosic components are secreted to the cell surface and form a porous matrix assembled around the cellulose microfibrils. These diverse noncellulosic polysaccharides and proteins are made in the endomembrane system. Many questions about the biosynthesis and modification within the Golgi apparatus and integration of cell components at the cell surface remain unanswered. The lability of synthetic complexes upon isolation is one reason for slow progress. However, with new methods of membrane isolation and analysis of products in vitro, recent advances have been made in purifying active synthases from plasma membrane and Golgi apparatus. Likely synthase polypeptides have been identified by affinity‐labeling techniques, but we are just beginning to understand the unique features of the coordinated assembly of complex polysaccharides. Nevertheless, such progress renews hope that the first gene of a synthase for a wall polysaccharide from higher plants is within our grasp.— Gibeaut, D. M., Carpita, N. C. Biosynthesis of plant cell wall polysaccharides. FASEB J. 8: 904‐915; 1994.

Designing the deconstruction of plant cell walls.

Cell wall architecture plays a key role in the regulation of plant cell growth and differentiation into specific cell types. Gaining genetic control of the amount, composition, and structure of cell walls in different cell types will impact both the quantity and yield of fermentable sugars from biomass for biofuels production. The recalcitrance of plant biomass to degradation is a function of how polymers crosslink and aggregate within walls. Novel imaging technologies provide an opportunity to probe these higher order structures in their native state. If cell walls are to be efficiently deconstructed enzymatically to release fermentable sugars, then we require a detailed understanding of their structural organization in future bioenergy crops.

Cell-Wall Polysaccharides of Developing Flax Plants

Flax (Linum usitatissimum L.) fibers originate from procambial cells of the protophloem and develop in cortical bundles that encircle the vascular cylinder. We determined the polysaccharide composition of the cell walls from various organs of the developing flax plant, from fiber-rich strips peeled from the stem, and from the xylem. Ammonium oxalate-soluble polysaccharides from all tissues contained 5-linked arabinans with low degrees of branching, rhamnogalacturonans, and polygalacturonic acid. The fiber-rich peels contained, in addition, substantial amounts of a buffer-soluble, 4-linked galactan branched at the 0–2 and 0–3 positions with nonreducing terminal-galactosyl units. The cross-linking glycans from all tissues were (fucogalacto)xyloglucan, typical of type-I cell walls, xylans containing (1->)-[beta]-D-xylosyl units branched exclusively at the xylosyl O-2 with t-(4-O-methyl)-glucosyluronic acid units, and (galacto)glucomannans. Tissues containing predominantly primary cell wall contained a larger proportion of xyloglucan. The xylem cells were composed of about 60% 4-xylans, 32% cellulose, and small amounts of pectin and the other cross-linking polysaccharides. The noncellulosic polysaccharides of flax exhibit an uncommonly low degree of branching compared to similar polysaccharides from other flowering plants. Although the relative abundance of the various noncellulosic polysaccharides varies widely among the different cell types, the linkage structure and degree of branching of several of the noncellulosic polysaccharides are invariant.

Mixed linkage (1→3), (1→4)-β-D-glucans of grasses

Cereal Chem. 81(1):115–127 The mixed-linkage (1 3),(1 4)-D-glucans are unique to the Poales, the taxonomic order that includes the cereal grasses. (1 3), (1 4)-Glucans are the principal molecules associated with cellulose microfibrils during cell growth, and they are enzymatically hydrolyzed to a large extent once growth has ceased. They appear again during the developmental of the endosperm cell wall and maternal tissues surrounding them. The roles of (1 3),(1 4)-glucans in cell wall architecture and in cell growth are beginning to be understood. From biochemical experiments with active synthases in isolated Golgi membranes, the biochemical features and topology of synthesis are found to more closely parallel those of cellulose than those of all other noncellulosic -linked polysaccharides. The genes that encode part of the (1 3),(1 4)-glucan synthases are likely to be among those of the CESA/CSL gene superfamily, but a distinct glycosyl transferase also appears to be integral in the synthetic machinery. Several genes involved in the hydrolysis of (1 3),(1 4)-glucan have been cloned and sequenced, and the pattern of expression is starting to unveil their function in mobilization of -glucan reserve material and in cell growth. The starchy endosperm, a special trait of the Poales, is the fundamental reason that cereals are of such central importance in human nutrition (Langenheim and Thimann 1982). The world harvests over 1 billion tons of cereal grains annually. Rice and wheat alone provide at least half of the calories that humans ingest. The cell walls of the grasses also figure heavily in human and animal nutrition, from the mixed-linkage (1 3),(1 4)glucans that constitute a major portion of the endosperm walls to the vast amounts of xylanand cellulose-rich walls that are consumed by grazing animals. Scientific interest in (1 3),(1 4)glucans is associated partly with problems they cause in brewing and animal feed industries and partly from benefits they offer to human diets (as reviewed by Stone and Clarke 1992). The (1 3),(1 4)-glucans are the wall constituents responsible for the enhanced ability of barley and oat brans to reduce serum cholesterol in hypercholesterolemic individuals (for example, Braaten et al 1994) and to modulate the glycemic index in diabetics (Wood et al 1990). The (1 3),(1 4)-glucans and xylans of the endosperm cell walls in flours are contributing factors to bread quality (Girhammerar et al 1986). Incomplete hydrolysis of the viscous (1 3),(1 4)-glucans during the brewing process is a major production problem and contributes to hazing of beers upon storage (for review see Woodward and Fincher 1983). Because of these practical aspects of (1 3), (1 4)-glucans, they have been the focus of many studies describing their unique structure and their content and dynamics in cereal grains. These studies of (1 3),(1 4)-glucan structure and physiology and the special cell wall of grasses have been covered by several reviews and book chapters, and the readers are directed to these for more extensive coverage of the early literature (Wilkie 1979; Bacic et al 1988; Carpita and Gibeaut 1993; Carpita 1996). Grass species use (1 3),(1 4)-glucans as structural elements of the walls of growing cells and as an endosperm storage material that is hydrolyzed during germination to provide an extra source of carbon during early seedling establishment (Meier and Reid 1982). (1 3),(1 4)-Glucan synthase represents one of the few biosynthetic machines whose activity can be preserved in vitro, and the polymer synthesized is identical in size and structure to that produced by the plants. This feature gives us a unique opportunity to study all of the interacting proteins and enzymes that comprise this system. Although plant biologists have finally identified and characterized a few genes that encode the synthesis machinery, we still don’t understand the biochemical mechanisms of synthesis very well. Identification of a (1 3),(1 4)-glucan synthase genes and determination of the three-dimensional structures of their active sites will ultimately yield important clues to understanding the chemical mechanism of glycosyl transfer, not only of this specific synthase, but also for cellulose synthase and all other plant polysaccharide synthases. In this review, we summarize the features that make cell walls of cereals different from all other flowering plants and give a special focus to the special role that (1 3),(1 4)-glucans play in wall architecture and plant development. We will highlight recent advances in structurefunctional relationships, including the characterization of the enzymes that participate in the hydrolysis of (1 3),(1 4)glucan during germination, reserve mobilization, and cell growth, and the cloning of their genes. Our central effort will be to provide new insights and speculations into the mechanisms of synthesis at the Golgi apparatus and the progress toward identification of the genes that encode the synthetic machinery. Special Cell Wall of Grasses and Cereals All plant cell walls are composites of at least three independent but coextensive, interwoven networks of polymers. The cellulose and cross-linking glycan framework embedded in a matrix of pectic substances form two of them, and these networks are eventually cross-linked into a firm, inextensible structure by structural proteins or polyphenolic substances (McCann and Roberts 1991; Carpita and Gibeaut 1993). A vast majority of flowering plants possess a type I wall in which the principal cellulose cross-linking glycan is xyloglucan and as much as 35% of the wall mass is pectin (Carpita and Gibeaut 1993). The grasses and cereals possess a type II cell wall (Carpita and Gibeaut 1993; Carpita 1996), and at least two important evolutional changes resulted in their vastly different cell wall 1 Secao de Fisiologia e Bioquimica de Plantas, Instituto de Botânica CP 4005 CEP 01061-970, Sao Paulo, SP Brazil. 2 Department of Botany and Plant Pathology, Purdue University West Lafayette, IN 47907–1155. 3 Present address: UMR CNRS-UPS 5546, Pole de Biotechnologie Vegetale, BP 17, Auzeville, F-31326 Castanet Tolosan, France. 4 Present address: Department of Plant Biology, 228 Plant Science Building, Cornell University, Ithaca, NY 14853. 5 Corresponding author. Phone: +1-765-494-4653. Fax: +1-765-494-0393. Email: carpita@purdue.edu Publication no. C-2003-1216-01R.