Rice is a model organism for studying the mechanism of cell wall biosynthesis and remolding in Gramineae. Mechanical strength is an important agronomy trait of rice (Oryza sativa L.) plants that affects crop lodging and grain yield. As a prominent physical property of cell walls, mechanical strength reflects upon the structure of different wall polymers and how they interact. Studies on the mechanisms that regulate the mechanical strength therefore consequently results in uncovering the genes functioning in cell wall biosynthesis and remodeling. Our group focuses on the study of isolation of brittle culm (bc) mutants and characterization of their corresponding genes. To date, several bc mutants have been reported. The identified genes have covered several pathways of cell wall biosynthesis, revealing many secrets of monocot cell wall biosynthesis. Here, we review the progress achieved in this research field and also highlight the perspectives in expectancy. All of those lend new insights into mechanisms of cell wall formation and are helpful for harnessing the waste rice straws for biofuel production.
As the most abundant renewable resources on the earth, cell wall materials are of a great practical importance, which could be summarized with “four Fs” of food, feed, fiber, and fuel (McCann and Rose 2010). However, only 2% of the plant cell wall-based biomass is currently utilized by humans (Pauly and Keegstra 2008), due to the natural recalcitrance and no thorough understanding of molecular mechanisms in cell wall formation and remodeling.
The plant cell wall is a rigid and dynamic network system that is mainly composed of polysaccharides, although the secondary wall also includes aromatic substances (lignin). The polysaccharides in plant cell walls consist of cellulose, hemicelluloses and pectins. To our knowledge, thousands of genes are dedicated to cell wall biogenesis and modifications (Carpita et al. 2001), only a few of which have been characterized. A powerful approach to probe the functions of individual components of cell walls is through the identification and characterization of mutants (Reiter et al. 1993). Several famous series of mutants defective in stem strength have been studied in Arabidopsis and rice. Characterization of irregular xylem (irx) and fragile fiber (fra) mutants in Arabidopsis have revealed that the genes involved in cellulose biogenesis, lignin metabolism, xylan formation, and cytoskeleton maintenance are essential for cell wall properties and mechanical strength (Turner and Somerville 1997; Taylor et al. 1999, 2000; Burk et al. 2001; Zhong et al. 2002; Brown et al. 2005, 2007).
Rice is an ideal model organism for studying cell wall biosynthesis and remolding in Poaceae (Carpita and Gibeaut 1993), in which the type II walls are present. The type II walls are different from the type I walls on either types or cross-linkings of polysaccharides (Carpita 1996; Vogel 2008; Hou and Li 2010). Thus, monocot plants may have varied mechanism from dicots in the control of mechanical property. Mechanical strength of rice plants is also an important agronomic trait that affects lodging and stress resistance. In recent years, studies on the brittle culm (bc) mutants have identified several genes responsible for secondary wall biosynthesis (Table 1), which are comparable and distinctive from those reported in Arabidopsis (Li et al. 2003; Tanaka et al. 2003; Zhang et al. 2009; Zhou et al. 2009). Here, we highlight the progress made in the efforts to understand the formation and regulation of the monocot cell wall network through characterization of rice brittle culm mutants.
|OsCESA4||cesa4a bc7 bc11||Os01g54620||Brittleness||AtCESA8||(Tanaka et al. 2003; Yan et al. 2007; Zhang et al. 2009)|
|OsCESA7||cesa7a||Os10g32980||Brittleness||AtCESA4||(Tanaka et al. 2003)|
|OsCESA9||cesa9a||Os09g25490||Brittleness||AtCESA7||(Tanaka et al. 2003)|
|OsKinesin-4||bc12||Os09g02650||Brittleness, altered microfibril orientation||FRA1||(Zhang et al. 2010)|
|OsDRP2B||bc3||Os02g50550||Brittleness||AtDRP2B||(Xiong et al. 2010a,b; Hirano et al. 2010)|
|BC10||bc10||Os05g07790||Brittleness after heading||AT5G14550||(Zhou et al. 2009)|
|BC1||bc1||Os03g30250||Brittleness||AtCOBL4||(Li et al. 2003)|
Cellulose Synthase Catalytic Subunit, Proteins Catalyzing Cellulose Synthesis
Cellulose, the most abundant polysaccharide in plant cell walls, is unbranched β-1,4-glucan, which is packed into microfibrils while depositing into walls. In higher plants, cellulose is synthesized at the plasma membrane (PM) by cellulose synthases (CESA) complexes (CSC) using uridine diphosphate (UDP)-glucose as substrates (Somerville 2006). Most CESA proteins are highly conserved and share several common features, including 900–1 100 amino acids in length and possessing multi-spanned membrane domains and conserved catalytic motifs (Pear et al. 1996; Mutwil et al. 2008). During screening for collapsed xylem (irx) and fragile fiber (fra) mutants in Arabidopsis, three CESAs for cell wall biosynthesis in mechanical tissues were identified and the relevant morphological alterations were also reported. Therefore, according to their functions reported in Arabidopsis, CESAs are divided into two classes: the primary cell wall-related and the secondary cell wall-related CESAs; this knowledge is still limited in rice (Turner and Somerville 1997; Taylor et al. 2003; Zhong et al. 2003).
The rice CESAs responsible for secondary cell wall formation has been characterized through the isolation of Tos17 insertional mutants of three CESAs homologous to those in Arabidopsis (Tanaka et al. 2003). The major phenotypes of these mutants are brittleness, along with the dwarfism and withering of leaf apex, due to a significant reduction in cellulose content. Several alleles for oscesa4 were found, in which the mutations occur at different sites of CESA4 protein and consequently result in distinct phenotypes. brittle culm7 (bc7t), an allele identified from a japonica variety, Zhonghua-11, has a 7 bp-deletion mutation at the tenth exon of OsCESA4 and shows indistinguishable phenotypes from the wild-type plants except brittleness (Yan et al. 2007; Wei et al. 2008). Different from bc7, which only has ∼10% decrease in the cellulose content, bc11, another allele of OsCESA4 mutation in Nipponbare, displays 60% reduction in cellulose content and a twofold compensatory increase in arabinoxylan (Zhang et al. 2009). The compensatory effects are different from those observed in Arabidopsis cesa7 (mur10) mutant (Bosca et al. 2006; Taylor et al. 2000). Immunolabeling analysis with antibodies against cell wall polysaccharides further ascribed these overall alterations to both secondary walls and primary walls. bc11 results from a missense mutation occurring at the end of the fifth transmembrane domain. This mutation does not alter the CESA4 expression at the transcriptional and translational level, but alters the abundances of CESA4 at the PM, highlighting an interesting correlation between the distribution of CESA proteins and cellulose biosynthesis. bc11-d, mutation occurred in a conserved amino acid in the second cytoplasmic domain of OsCESA4, causes a semidominant phenotype in brittleness and the reduction of cellulose content (B. Zhang, F. Li and Y. Zhou, unpubl. data, 2007), reminiscent of the fra5 mutants in Arabidopsis (Zhong et al. 2003).
Therefore, characterization of bc mutants in rice has identified comparative CESAs for secondary wall cellulose biosynthesis as in Arabidopsis (Zhang et al. 2009), indicating a similar performance of CESAs in both species.
Vesicle Trafficking is Necessary for Cell Wall Biosynthesis
Cell wall is a polysaccharide-rich extracellular matrix. Except that cellulose is synthesized at the PM and secreted outside directly, most noncellulosic wall components are considered being produced in the Golgi apparatus and transported to the walls through secretory pathways. In addition, some proteins essential for cell wall biosynthesis and remodeling, such as CESAs and KORRIGAN1 (KOR1), have multiple localization patterns, indicating that these proteins are translocated through intracellular trafficking (Robert et al., 2005; Paredez et al., 2006; Johansen et al. 2006). Therefore, vesicle trafficking is a key point to regulate cell wall formation (Wightman and Turner 2010). Direct evidence of CESA trafficking was obtained from in vivo viewing the movement of fluorescence-labeled CESA6 along cortical microtubules (Paredez et al., 2006). Later, small CESA-containing compartments (SmaCCs) and microtubule associated cellulose synthase compartments (MASCs) have been observed (Crowell et al. 2009; Gutierrez et al. 2009), clarifying the role of vesicle trafficking in cellulose biosynthesis. Because the trafficking is generally cytoskeleton associated, the factors that regulate cytoskeleton dynamics often affect cell wall biosynthesis.
In Arabidopsis, three actin and two microtubule organization-related proteins have been identified through isolation of fra mutants (Burk et al. 2001; Zhong et al. 2002, 2004, 2005a; Hu et al. 2003). Compared with these findings in Arabidopsis, the documented cytoskeleton genes involved in cell wall biosynthesis are fewer in rice. Mutation in DGL1 (dwarf and gladius leaf1), the rice homolog of AtKTN1/FRA2, displays dwarfism due to aberrant cortical microtubule. However, the cell wall alteration in dgl1 has not been reported (Komorisono et al. 2005). BC12, an ortholog of AtKIF-4/FRA1 (Zhong et al. 2002), is a rice motor protein being comprehensively characterized (Zhang et al. 2010). Similar to fra1 phenotypes, bc12 mutation results in brittleness and dwarfism. The abnormal deposition of cellulose microfibrils has also been observed in bc12. However, its increased contents in arabinoxylan and lignin have not been found in fra1. BC12 possesses an authentic nuclear localization signal (NLS), which is absent in FRA1. The NLS positions BC12 protein in both the nucleus and cytoplasm and confers it an additional role in cell cycle progression. BC12 therefore performs a diverse function from Arabidopsis FRA1.
Dynamin and dynamin-related proteins (DRPs) are large guanosine-5'-triphosphatases (GTPases) that participate in tubulation and vesiculation of membrane systems (Takei et al. 2005). Considering their importance in membrane dynamics, the DRPs are proposed to participate in cell wall metabolism. AtDRP1A is a key protein involved in cell plate formation (Collings et al. 2008). Cellulose deficiency found in a null mutant of DRP1A causes radial swollen roots and was identified as having primary cell wall defects. We currently reported BC3, a gene encoding the classic dynamin OsDRP2B, functioning in cellulose biosynthesis (Xiong et al. 2010a, 2010b). Fluorescence-tagged OsDRP2B is targeted to the trans-Golgi network (TGN) and clathrin-mediated vesicles, placing OsDRP2B in membrane trafficking pathways. Cellulose is the only deficient component in the mutant walls (Hirano et al. 2010; Xiong et al. 2010a). The altered abundance of OsCESA4 at the PM in bc3 and OsDRP2B overexpressing transgenic plants suggest that this protein directly or indirectly affects the delivery of CESAs and plays an essential role for the control of mechanical strength in rice plants. This study furthers our understanding of the roles of DRPs in cell wall biosynthesis (Xiong et al. 2010a,b). Although none of the Arabidopsis DRPs has been identified so far from the stem strength-deficient mutants, characterization of a fra mutant, fra7, has identified AtSac1 that encodes PtdIns(3,5)P2-specific phosphoinositide phosphatase (Zhong et al. 2005a). Phosphoinositide phosphatases are well-known key factors affecting the distribution of individual vesicle compartments (Thole and Nielsen 2008). All of these findings only revealed the tip of iceberg of how endomembrane organization and trafficking regulate cell wall biosynthesis.
Proteins Modulating Cell Wall Biosynthesis
Another set of genes identified from Arabidopsis irx and fra mutants are members of glycosyltransferase (GT) family 43 and 47, which are involved in xylan synthesis. Mutations in IRX7/FRA8, IRX8, IRX9, and IRX14 all disturb the xylan structure and abundance, furthermore affecting cellulose formation (Zhong et al. 2005b; Brown et al. 2007; Pena et al. 2007; Persson et al. 2007). Therefore, a coordinated regulation of xylan and cellulose biosynthesis may exist in Arabidopsis. Although xylan is a major hemicellulose in rice plants, no gene responsible for xylan synthesis has been cloned from the rice bc mutants. This interesting phenomenon implies that monocot wall may have a distinct co-regulatory mechanism for the biosynthesis of xylan. This might explain why the opposite synthesis of cellulose and xylan was found in rice bc11 rather than in Arabidopsis irx8 and irx9 (Brown et al. 2007; Persson et al. 2007; Zhang et al. 2009).
A putative GT critical for cellulose biosynthesis and mechanical strength has been identified through the characterization of bc10 mutant (Zhou et al. 2009). BC10 is a Golgi-localized type II membrane protein containing a domain of unknown function 266 (DUF266). Although its enzymatic activity is still unclear, it was identified as a putative GT belonging to a new GT family/subfamily. Further studies are needed for undoubtedly understanding its function in cell wall biosynthesis. Besides this GT, the bc mutants also contributed to identification of one critical protein essential for cellulose biosynthesis. BC1, the first cloned BC gene via map-based cloning approach, encodes a COBRA-like protein (Li et al. 2003). Before that, Arabidopsis COBRA (COB) has been reported for control of anisotropic expansion in many developing tissues (Schindelman et al. 2001). COB and COB-like proteins are therefore clustered into an important family involved in cell wall biosynthesis (Roudier et al. 2002; Li et al. 2003). Mutation in a COB-like gene, IRX6, causes a reduced level of cellulose in secondary walls (Brown et al. 2005), providing additional support for this function. Furthermore, AtCOBs were found coexpressed with CESA genes (Brown et al. 2005; Persson et al. 2005). Although COB and BC1 have been detected in cell wall (Roudier et al. 2005 and L. Liu, J. Li and Y. Zhou, unpubl. data, 2009), the mechanism of this action remains to be elucidated.
Highlights and Future Perspectives
Rice and Arabidopsis are model organisms for studying biosynthesis and remolding of two types of cell wall, respectively. As an easily identified phenotype, screening for plants with altered mechanical strength is the most effective way for isolation of wall-related mutants. Here, we highlight the common and discrepant features of the genes identified from stem strength-deficient mutants of two species, indicating that monocot plants may have varied and overlapped regulatory mechanisms from the dicots in the control of mechanical strength. Cellulose is the major component of the secondary cell wall. It is thus reasonable that the common effect of these mutations, no matter as a direct or indirect effect, is the cellulose content or deposition pattern. In addition, the reduction level of cellulose is highly correlated with abnormality of visible phenotypes (Kokubo et al. 1989).
Plant cell wall biosynthesis and modification are tremendously complex and require the involvement of an intricate series of steps in an orderly manner. Since BC1 was cloned in 2003, several bc mutants and the corresponding genes have been characterized (Table 1). Although the documented genes are still limited, they have been involved in diverse aspects of cell wall biosynthesis, including cellulose biosynthesis, matrix polysaccharide/glycoprotein formation, cytoskeleton conformation, and vesicle trafficking (Figure 1). The potential links between them could be revealed soon. For example, the abundance of BC11/OsCESA4 on the PM appears to be regulated by BC3/OsDRP2B through membrane trafficking (Xiong et al. 2010a). However, our knowledge about monocot cell wall biosynthesis is still very preliminary. The corresponding genes of several described classic bc mutants (bc2, 4, 5, 6) still remain to be cloned (Kinoshita 1995). bc5, a node-specific brittle mutant, is one example. The phenotypic characterization of bc5 indicated its important role in cell wall biosynthesis in a specific organ (Aohara et al. 2009). As more bc mutants being isolated and characterized in the near future, it is in prospect that light would be shined into the ‘black box’ and more secrets for monocot cell wall biosynthesis will be uncovered.
(Co-Editor: Hai-Chun Jing)
These works were continuously supported by grants from the National Natural Science Foundation of China (30370753, 30470112 and 30870141) and the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-G-033).