research-cwb

A defining feature of plants is the cell wall. The wall affects all aspects of a plant’s life: growth, development, morphology, and response to the environment. Plant cell walls consist mainly of cellulose microfibrils embedded in a matrix of hemicellulose and pectin polysaccharides. Cellulose is the same in all plants and all tissues: linear chains of beta-1,4-linked glucose. On the other hand, hemicelluloses are chemically more complex, being composed of many different sugars in many different, often branched, linkages.

Plant cell walls have major impacts on humans. Plant cell walls are an important part of the environment of humans and other organisms. A better understanding of wall biosynthesis could contribute to the improvement of food, fiber, and fuel in many ways. For example, the production of paper requires the removal of tightly bound hemicelluloses from wood cellulose by chemical processes that are a major source of environmental pollution. The intractability of the arabinoxylan hemicellulose of cereal walls has been cited as a major limitation in the enzymatic conversion of stover (the plant debris remaining after harvest) to fermentable sugars for ethanol production. The cell walls of cereals are a major component in the diets of animals and people, and some, such as mixed-linked glucan (MLG) of cereals, are a major component of "soluble fiber", high intake of which has ben proven to have beneficial health effects. On the other hand, MLG is a nuisance to brewers because it causes cloudiness in beer. There are many opportunities for the improvement of agricultural plants if the genes involved in hemicellulose biosynthesis could be identified.

Cellulose is the same everywhere but hemicelluloses aren't. Whereas cellulose is present in all plants and all plant cell walls, the hemicelluloses show striking differences between plant species and between cell types and tissues within a plant. Of particular interest to us are the hemicelluloses of grasses (family Poaceace; edible grasses are known as cereals, such as wheat, corn, barley, oats, sorghum, rye, and rice). The hemicelluloses of grasses are distinct from those of dicots (broad-leaved plants).

A major biosynthetic difference between cellulose and hemicelluloses is that cellulose is biosynthesized at the plasma membrane of plant cells, whereas hemicelluloses are biosynthesized in the Golgi apparatus. Hemicelluloses but not cellulose must travel through the secretory pathway to get to the wall.

Hemicellulose biosynthesis must be more complicated than cellulose biosynthesis. We predict that hundreds of genes are involved in hemicellulose biosynthesis. These genes include those that encode biosynthesis of the component sugars (UDP-glucose, UDP-arabinose, UDP-xylose, etc.), proteins to import the hemicellulose precursors into the Golgi, glycosyl synthases to make the backbones, glycosyl transferases to add the branches to the backbones, proteins for export, and finally proteins for cross linking and modification within the cell wall.

We are using genomics and biochemical approaches to study the biosynthesis of hemicelluloses in representative dicots (Arabidopsis, nasturtium, and cotton) and cereals (rice and maize). This work is funded by a grant from the NSF Plant Genomics Program to Ken Keegstra (MSU), Jonathan Walton (MSU), Curtis Wilkerson (MSU), and Natasha Raikhel (UC Riverside).

The CSL genes of rice

The structure of cereal cell walls. Cereals, including rice, maize, wheat, millet, sorghum, rye, and barley, are the major food source for humans. Cereal stover, composed mainly of cell walls, represents a huge quantity of renewable biomass that is currently underutilized as a source of fuel, chemicals, and food.

The primary walls of grasses (family Poaceae) are distinct not just from those of dicotyledenous plants but even from those of other monocotyledons. The major hemicellulosic component of the primary walls of grass seedlings is glucuronoarabinoxylan (GAX), composed of a beta-1,4-D-xylan backbone substituted with terminal alpha-L-arabinofuranose (mainly at the O-3 position) and, to a lesser extent, alpha-D-glucuronose (mainly at the O-2 position).

Cereals also contain a unique hemicellulose known as mixed-linked glucan (MLG), a linear glucan containing both beta-1,3- and beta-1,4- linkages. Next to GAX, MLG is the predominant hemicellulose in a variety of cereal tissue types.

Root cap "slime" is an abundant polymer in root caps. Slime polysaccharides from maize contain a high content of fucose (32%) and galactose (21%).

Formerly, cereals were though to have only low levels of xyloglucan, which is the major hemicellulose in dicots. However, cereals do contain significant levels of xyloglucan (Hayashi, 1989, Annu. Rev. Plant Physiol. 40:139). The structure of cereal xyloglucan differs in being less substituted. That is, whereas in dicot xyloglucan almost every glucose molecule is substituted with xylose and/or galactose and fucose, the xyloglucan of cereals is sparsely substituted, and with only xylose. One effect of a lower level of substitution is decreased solubility in water.

The CESA and CSL genes. Recent progress has led to the identification of genes encoding cellulose synthases (CESA genes). Plants also contain a large number of genes that are related to the CESA genes. These "cellulose synthase-like" (CSL) genes have been found in all plants and those from Arabidopsis have been classified into six sub-families (Richmond and Somerville, 2000). It has been proposed that Csl proteins are also involved in the biosynthesis of non-cellulosic polysaccharides, that is, hemicelluloses.

The CSL genes of rice. We have analyzed the available CSL and CESA genes from cereals, including rice, maize, sorghum, and wheat. We have concluded that there are 9 (maybe 10) CESA genes and 34 CSL genes in rice. Click here for the latest updates to the rice CSL gene family. (This was updated February, 2006).

Protein coding regions were predicted using GenMark, SoftBerry, and by manual alignments with each other and with the predicted protein sequences of the Arabidopsis CSL genes.

A pdf copy of our paper is available here: Hazen et al. It is now obsolete due to the availability of the full genome sequence and reanalysis.We have made a new tree showing the relationship of all rice CES and CSL proteins. See the tree: rice CES CSL tree.

Conclusions derived from the tree (all still valid as of February, 2006):

1. Rice has a distinct clade, which we call family CSLF. Members of this family are related to CSLD and CESA but form a separate branch. One reason that we think it is appropriate to put the CSLF genes into a separate sub-family is that their protein products are all significantly shorter than either the CESA or the CSLD proteins. Recent evidence from Geoff Fincher’s lab indicates that the CSLF proteins encode beta1,3;1,4 mixed-linked glucan synthases. See the tree: rice CES CSL tree.

2. Rice has no genes that cluster with CSLB genes of Arabidopsis, but instead has a distinct family that we call CSLH.
3. Rice does not appear to contain any CSLG members. This family has been found in most other dicots and no other cereals to date (see Richmond’s web page at: http://cellwall.stanford.edu).

Approaches to studying hemicellulose biosynthesis in grasses

We are taking two approaches: genetic and biochemical.

1. Natural variation to study the genetics of hemicellulose biosynthesis in grasses

2. Enzymology of glucan biosynthesis in maize

(revised February, 2006)