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First published online January 27, 2006; 10.1104/pp.105.073007 Plant Physiology 140:972-983 (2006) © 2006 American Society of Plant Biologists GOLD HULL AND INTERNODE2 Encodes a Primarily Multifunctional Cinnamyl-Alcohol Dehydrogenase in Rice1State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China (K.Z., Z.H., Y.W., M.L., L.H., D.Z., C.C., Z.C.); State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, People's Republic of China (Q.Q., D.Z.); Department of Agronomy, Yangzhou University, Yangzhou 225009, People's Republic of China (M.G.); and Graduate School of the Chinese Academy of Sciences, Beijing 100049, People's Republic of China (K.Z., Z.H., L.H., D.Z.)
Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis.
In addition to cellulose, lignin is the second major biopolymer component of the plant cell wall. Lignin cross-links with carbohydrates, such as cellulose and hemicellulose, to play an important role of the cell walls, enhancing the rigidity, conferring resistance to pathogens and mechanical stress, and enabling solute transport in the xylem (Brill et al., 1999  ; Chabannes et al., 2001; Jones et al., 2001). Lignin is a complex phenolic polymer that is mainly derived from the oxidative polymerization of p-coumaryl, coniferyl, and sinapyl alcohol precursors as the principal monomers, and the monomers are defined as p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units. The types and amount of lignin units vary depending on plant species, tissues, cell types, and cell wall layers and are influenced by developmental and environmental cues (Campbell and Sederoff, 1996; Donaldson, 2001).
Apart from its critical role in plant development, lignin has great economical and environmental value. Considerable monetary and environmental costs are incurred by industry in removing lignin from cellulose during production of pulp and paper (Hu et al., 1999
Cinnamyl-alcohol dehydrogenase (CAD) was one of the first enzymes studied in lignin biosynthetic pathway, and it catalyzes the final reduction of the hydroxyl-cinnamaldehydes to the corresponding alcohols (Mansell et al., 1974
The rice gold hull and internode (gh) mutant was first described as early as 1917 (http://www.gramene.org/db/mutant/search_mutant?id=GR:0060365). It exhibits a reddish-brown pigmentation in the hull and internode, instead of in the midrib. The hull and internode become golden yellow at maturation, so the mutant is named as gh rice. Based on the morphological and genetic descriptions, a series of gh mutates (gh1, gh2, gh3, and gh4) have been identified. Among them, gh1 was located on chromosome 5 (Nagao and Takahashi, 1963
Morphological Characterization of the gh2 Mutant We evaluated the morphology of the gh2 mutant and compared it with the wild-type variety, Zhefu802. gh2 exhibits an obvious reddish-brown pigment in the panicle (hull), internode, and basal leaf sheath at the heading stage (Fig. 1, A and B ). The reddish-brown pigments gradually become intensive starting from the basal internode to the apical internode. During plant maturing, seed coloration becomes darker and finally changes to golden yellow at maturation (Fig. 1C). Except the reddish-brown coloration in the above specific tissues, the gh2 mutant plants show similar development with the wild type under normal cultivated condition.
The GH2 Gene Encodes a CAD
The gh2 gene has been genetically mapped to the region around 20 cM on chromosome 2 by the primary trisomics (Sanchez and Khush, 1994
The identity of GH2 was confirmed by genetic complementation experiments. The plasmid pCGH, containing the entire open reading frame (ORF), 2,397-bp upstream region and 2,460-bp downstream region, and pCGHS, containing a partial coding region of the ORF (Fig. 2D), were introduced into the recessive mutants from the F3 generation of the mapping population, then 12 and eight independent transgenic lines were obtained from the two constructs, respectively. All 12 lines of pCGH showed complete complementation of the gh2 phenotype, whereas all eight lines of pCGHS failed to rescue the gh2 mutant. Additionally, the mutation in the gh2 produces an Aat II site in the genomic DNA, which can be used as a CAPS marker to determine the gh2 mutant background in the complementation test (Fig. 2E). Therefore, we conclude that the candidate gene controls the gh2 phenotype in rice.
To determine the evolutionary relationship between GH2 and CAD family members from the rice and Arabidopsis CAD families as well as other representative identified CAD homologs, an unrooted tree was built using the neighbor-joining method based on full-length protein sequences (Fig. 3 ). The result indicates that all the CAD protein sequences are divided into two subfamilies: subA (GH2, sugarcane [Saccharum officinarum] CAD, maize CAD, aspen PtCAD, eucalyptus [Eucalyptus globules] CAD, tobacco [Nicotiana tabacum] CAD1, tobacco CAD2, Lucerne [Medicago sativa] MsaCad2, Arabidopsis CADC, Arabidopsis CADD, pine [Pinus taeda] CAD, and spruce [Picea abies] CAD) and subB (Lucerne MsaCad1, aspen PtSAD, OsCAD1, OsCAD3, OsCAD4, OsCAD5, OsCAD6, OsCAD7, OsCAD8A-D, OsCAD9, AtCAD1, AtCAD2, AtCAD3, AtCAD6, AtCAD7, AtCAD8, and AtCAD9).
SubA members are evolutionally conserved CAD genes in gymnosperm and angiosperm, which has been regarded as the true CAD clade before angiosperm and gymnosperm split (Raes et al., 2003 ; Peter and Neale, 2004). In the complete CAD family of Arabidopsis, two CAD members (AtCADC and AtCADD) belong to subA subfamily. Only double mutation of the two subA CAD genes (AtCADC and AtCADD) can cause the lignin-deficient phenotype. Although rice has 12 CAD members, only GH2 belongs to the subA CAD subfamily. The fact that the point mutation of GH2 in rice has led to the lignin-deficient phenotype demonstrates that GH2 is the only primary enzyme that catalyzes the last step of lignin precursor biosynthesis. SubB is an angiosperm-specific subfamily, which can be regarded as the duplication of subA, and the members are likely to gain new functions in evolution. So far, Lucerne MsaCAD1 and Arabidopsis AtCAD7 in SubB have been proven to participate in plant defense, aspen PtSAD has been proven to catalyze sinapyl alcohol biosynthesis (Kiedrowski et al., 1992; Brill et al., 1999; Li et al., 2001), but the functions of rice subB subfamily remain to be elucidated. Based on the role of subB members in other plants, we predict that the subB CAD members in rice may function in enhancing the cell wall for plant defense or act in assistant roles of lignification.
Since the mutation is a point mutation, GH2 gene expression was detected in the wild-type plants only at the heading stage (Fig. 4B ). We detected GH2 gene transcription in all seven tissues, including panicles, hulls, blades, midribs, leaf sheaths, internodes, and young roots, with a 217-bp 3' untranscriptional sequence as the specific probe. The results showed GH2 gene was expressed in the lignified tissues, including panicles, hulls, internodes, young roots, and leaf sheaths. In the leaves (blade and midrib), which are slightly lignified, the GH2 transcripts cannot be obviously detected by northern blot. Furthermore, GH2 gene expressed stronger in the first internode than in the second internode, suggesting that the gene expressed gradually stronger starting from the basal internode to the apical internode (data not shown). Consequently, the GH2 expression pattern is closely related to the rice lignification pattern and the localization of reddish-brown pigments.
To study the catalytic ability of native GH2 in rice, CAD activity and SAD activity of the total proteins were assayed in all tissues of wild-type and gh2 plants at the heading stage. Strong CAD activity and weak SAD activity were detected in all the tissues of the wild type (Fig. 4A). In the gh2 sheaths, roots, panicles, hulls, and internodes in which GH2 gene was strongly or relatively strongly expressed, the CAD activity was drastically reduced and the SAD activity was not detectable, whereas, in the leaves (blade and midrib) in which GH2 gene was weakly expressed, the CAD activity and SAD activity were slightly reduced. The reductions are consistent with the expression pattern of GH2 described above (Fig. 4B). Thus, the result shows that GH2 protein has CAD activity and SAD activity, and the CAD activity and SAD activity were drastically reduced in the gh2 plants. In the leaves in which the GH2 gene is slightly expressed, the total proteins of the gh2 mutant still exhibit high CAD activity and weak SAD activity. Additionally, the reduction ratios of CAD activity in the different tissues are unequal. So we speculate it is possible that additional CAD isoenzymes with CAD activity also unevenly exist in different tissues of the plants. Most importantly, the fact that the SAD activity was completely abolished due to the defective GH2 protein in panicles, hulls, internodes, and sheaths shows that no other SAD homolog with obvious SAD activity exists in these tissues.
To evaluate the way in which the G to A nucleotide substitution impaired enzymatic function in the gh2 mutant plant, we expressed the wild-type protein GH2 and the point-mutated protein gh2 (G185D) in Escherichia coli. The recombinant proteins were purified by glutathione-Sepharose 4B affinity chromatography and cleaved by PreScission Protease on column (Fig. 5 ). The enzyme activities of the cleaved recombinant proteins were assayed toward the substrates coniferaldehyde and sinapaldehyde. GH2 exhibited strong CAD and SAD activities, while gh2 completely lost the CAD and SAD activities (Fig. 6 ; Table II ). The result shows the point mutation G185D completely abolished the enzyme activities of GH2 for substrates coniferaldehyde and sinapaldehyde. Furthermore, the result confirms that native GH2 also completely lost the enzyme activities, and additional CAD isoenzymes with CAD activity exist in different tissues of rice.
To further know the substrate specificity of GH2 in vitro, we study the kinetics of the purified recombinant GH2 toward the substrates coniferaldehyde and sinapaldehyde by HPLC analysis. The overall catalytic properties were revealed by Km, Vmax, Kcat, and Kenz (calculated by dividing Kcat by Km) values (Table II) calculated on the basis of the Lineweaver-Burk plots (Fig. 7 ). The Km value of GH2 for sinapaldehyde is 4.7-fold higher than that for coniferaldehyde, showing GH2 has higher affinity for coniferaldehyde than sinapylaldehyde. The Vmax value of GH2 for sinapylaldehyde is 2.6-fold higher than that for coniferaldehyde, indicating that the GH2 exhibits higher catalytic ability for coniferaldehyde than sinapylaldehyde. Based on the above, the specific constant Kenz (Kcat/Km) for coniferylaldehyde is 1.8-fold higher than that for sinapylaldehyde. The results suggest that coniferaldehyde and sinapaldehyde both are the favored substrates of GH2, but coniferaldehyde is the main substrate and sinapaldehyde is the secondary substrate.
Phloroglucinol and Mäule Staining of the Wild Type and gh2 Mutant
Phloroglucinol, a reagent traditionally used to detect cinnamyl aldehyde and lignin (Speer, 1987
The lignin composition of the wild-type and gh2 plants was also estimated by Mäule regent, which can stain G residues yellow and S residues red (Meyer et al., 1998 ). In the Mäule staining, the fresh hand-cut internode sections of wild type were stained reddish yellow in the Co region and purely yellow in the VB region, suggesting that the rice Co region contains abundant G monomers and a small amount of S monomer while the VB region mainly contains G monomers (Fig. 8C). In the corresponding tissues of the gh2 plants, the Co region and VB region were stained black-yellow by Mäule reagent (Fig. 8F). The phenomenon showed that the lignin monomers of the Co and VB regions were both altered in the gh2 mutant for the deficient GH2 protein, but the reason the tissues were stained black-yellow is unknown. The lignin properties of the gh2 mutants will be studied to explain the phenomenon.
To further detect the lignin deficiency of the gh2 mutant plants, lignin content and composition were estimated in the wild-type and gh2 plants. Cell wall residue (CWR) was prepared from the dried mature hulls and internodes. Klason lignin analysis of the CWR showed that the total lignin content of the gh2 plants was reduced by 5% to 6% compared with the wild-type plants (Table III
). The result demonstrates that GH2 is a critical enzyme that regulates lignin content in rice. Lignin composition of the wild-type and gh2 plants was also detected in the hulls and internodes by the derivatization followed by reductive cleavage (DFRC) method. The results show rice lignin consists of the three kinds of basal monomers, the H, G, and S monomers (Table III). In wild-type rice, G monomer is the major monomer and the G:S ratio is 60.66 in the hulls and 6.05 in the internodes, which is consistent with the Mäule staining (Fig. 8C). In the gh2, the three monomers are all reduced by approximately 35% in the hulls and approximately 25% in the internodes compared with the wild type. Although the reduction ratios are different in different tissues (hull and internode), the reductions of H, G, and S monomers in the same tissue are almost similar in degree. The results show that the GH2 enzyme takes the role of synthesizing H, G, and S monomer precursors in the hulls and internodes. The unequal reductions in hulls and internodes may be caused by the different lignification pattern between the hulls and internodes. Such analogous differences have been observed among Arabidopsis floral stems, roots, and hypocotyls (Sibout et al., 2003
The gh2 Plant Is Defective in Lignin Biosynthesis
gh mutants have long been used as morphological markers and classified into coloration mutant categories (Zeng et al., 2003 Comparing the maize CAD mutant bm1 with the rice gh2 mutant in our study, the phenotype difference between bm1 and gh2 is that maize bm1 mutant plants have reddish-brown pigments in the leaf midrib and stalk pith, whereas rice gh2 mutant plants have the pigments in the hull, internode, and basal leaf sheath. In truth, the reddish-brown pigments in the two mutants are all associated with the high-lignified tissues. When the expression pattern of CAD genes was investigated, maize CAD gene was expressed strongly in leaves but GH2 gene was slightly expressed in the same tissue (Fig. 4B). So it is likely that the phenotype difference may be caused by the expression pattern or loss-of-function extent of the CAD genes between bm1 and gh2. The identification of the gh phenotype provides an important clue for finding lignin mutants in grass crops such as wheat (Triticum aestivum) and barley (Hordeum vulgare).
Rice is the first crop plant whose genome has been sequenced (International Rice Genome Sequencing Project, 2005
We conclude GH2 is the primary CAD that acts as a multifunctional CAD enzyme to synthesize coniferyl alcohol and sinapyl alcohol precursors in lignified tissues of rice for the following reasons. First, in the rice CAD family, GH2 is the only one CAD belonging to the subA subfamily, which is widely proven to participate in constitutive lignin biosynthesis (Fig. 3). Second, when the GH2 gene was mutated, the CAD and SAD activities of both the native and purified recombinant GH2 protein were drastically reduced relative to the wild type (Figs. 4A and 6). Third, the kinetics properties of recombinant GH2 on the substrates of coniferaldehyde and sinapaldehyde show GH2 exhibits strong catalytic ability toward coniferaldehyde and sinapaldehyde (Table II). Finally, the G monomers and S monomers were reduced to a similar degree in the gh2 mutant plants compared with the wild type (Table III).
The difference between gymnosperm and angiosperm lignin is that gymnosperm plants contain mainly G monomers and monogenic CAD gene, whereas angiosperm plants consist of G monomers, S monomers, and one CAD family (Galliano et al., 1993
The available data lead to a paradox that questions whether there is a specific SAD in angiosperm plants. We think the conflicting data may be interpreted by elucidating the source of the S monomers. The proportion of S monomers in the total lignin monomers changes greatly among various kinds of species (Sederoff et al., 1999
Rice is an important crop that feeds more than half of the world's population. Effective utilization of rice by-products will definitely increase the income of rice producers. Rice residue has the potential to be used for bio-oil extraction by pyrolysis, for mushroom growth, and even for dietary fiber preparation (Kong et al., 2005
Materials
The original rice (Oryza sativa) gh2 mutant (RGS no. 6) was kindly provided by Dr. Khush at the International Rice Research Institute, The Philippines. The gh2 gene was transferred to Zhefu802 (spp. indica) by 10 rounds of backcrosses with Zhefu802 (Zeng et al., 2003
The primary locus of GH2 locus was determined by newly developed marker P1 and reported STS marker S21668. To fine map the gh2, seven STS markers and two CAPS markers (Table I) were developed on comparisons of original or CAPS length between indica var. 9311 and japonica var. Nipponbare according to the data published in http://www.ncbi.nlm.nih.gov.
A 8.44-kb genomic DNA fragment containing the entire GH2 coding region, the 2,397-bp upstream sequence, and the 2,460-bp downstream sequence was inserted into the binary vector pCAMBIA1300 to generate the transformation plasmid pCGH for complementation test. A control plasmid, pCGHS, containing truncated GH2 gene also was constructed according to the strategy described previously (Li et al., 2003a
The full-length CAD protein sequences were retrieved from GenBank and used for phylogenetic analyses according to the methods described by Li et al. (2003b)
Total RNA was extracted from tissues of wild-type plants at the heading stage with TRIZOL reagent (Invitrogen). Twenty micrograms of total RNA per lane was electrophoresed on 1% agarose-formaldehyde gels and capillary blotted onto Hybond-N+ membranes (Amersham). The 217-bp 3'-terminal untranscriptional fragment of the GH2 gene was labeled with
Plant total proteins were extracted as the method described by Halpin et al. (1998)
Both GH2 and gh2 full-length cDNAs were isolated by reverse transcriptase-PCR from the total RNA from gh2 and wild-type plant roots with RT-PCR system (Invitrogen) using primer 1 (5'-CTTCTTGTTCTTGTTCTCTT-3') and primer 2 (5'-GCCGAATTTATACCGGAAAG-3'). The PCR products were inserted into pGEM-T vectors and sequenced to obtain the correct clones pGEMGH2 and pGEMgh2. The ORF sequences of GH2 and gh2 were amplified from cDNA clones pGEMGH2 and pGEMgh2 using primer 3 (5'-GCGGGATCCATGGGCAGCCTCGCCGCCGA-3') and primer 4 (5'-GCGGAATTCCTAGCGGACGTCGTTGCGCT-3'). The primers incorporated a BamH site at the N-terminal end and an EcoRI site at the C-terminal end of the ORF. The PCR products were digested and cloned into the corresponding site of pGEX-6P-1 to generate pGEXGH2 and pGEXgh2, sequenced to assure GH2 and gh2 sequence, and then introduced into E. coli BL21 for protein expression. The bacteria containing pGEXGH2 or pGEXgh2 were cultured overnight at 37°C, diluted to 400 times, and grown at 37°C for about 3 h to an OD600 of 0.6, then transferred to 18°C for 16 h after adding isopropyl-
For the substrate-specific activity test, the enzyme reaction mixture contained 100 mM Tris-HCl, pH 8.8, buffer, 200 µM NADPH, 200 µM aldehyde substrate in a final volume of 250 µL, and 1.2 µg of purified recombinant GH2 or gh2 protein were used. The reactions were for 10 min at 30°C and stopped by adding glacial acetic acid (10 µL). For the kinetic analyses, the enzyme reaction mixture contained 100 mM Tris-HCl, pH 8.8, buffer, 200 µM NADPH, 0.5 µg purified recombinant protein, and varying concentrations (10–200 µM) of coniferaldehyde (Aldrich, High Purity) or sinapaldehyde (Apin Chemicals, High Purity) in a final volume of 250 µL. Enzymatic reactions were initiated by cinnamyl aldehyde addition and, after 4 min incubation at 30°C, were stopped by adding glacial acetic acid (10 µL). All the reactions were carried out with NADPH-free mixture as the control. An aliquot (50 µL) of each assay mixture was subjected to reverse-phase HPLC analysis with UV detection for the product identification. Authentic coniferyl alcohol (Aldrich, High Purity) and sinapyl alcohol (Aldrich, technical grade) were used as the standards for qualifying and quantifying the hydroxy-cinnamyl alcohol products. The kinetics values, Km, Vmax, Kcat, and Kenz, were defined as described by Kim et al. (2004)
Histochemical Staining
Lignin Content and Composition Analysis Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number DQ234272 (GH2).
We thank Dr. John Ralph (University of Wisconsin, Madison, WI) for supplying authentic standards for DFRC analysis, Dr. Khush (International Rice Research Institute, The Philippines) for providing the original gh2 mutant, Dr. Mu Zhang (Institute of Genetics and Developmetal Biology, Chinese Academy of Sciences) for assistance in gas chromatography-mass spectrometry analysis. We also thank the two anonymous reviewers and the editor for critical comments on the manuscript. Received October 17, 2005; returned for revision January 17, 2006; accepted January 17, 2006.
1 This work was supported by the Ministry of Sciences and Technology of China (grant no. 2005CB120805), the Chinese Academy of Sciences, and the National Natural Science Foundation of China (grant nos. 30325008 and 30530070). 
2 These authors contributed equally to the paper. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Zhukuan Cheng (zkcheng{at}genetics.ac.cn). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.073007. * Corresponding author; e-mail zkcheng{at}genetics.ac.cn; fax 0086–10–6487,3428.
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ABSTRACT
RESULTS
, coniferaldehyde; 
, sinapaldehyde. The SE bars were obtained from triplicate assays.
-32p-dCTP using the primer-a-gene labeling system (Promega) as the specific probe, and ethidium bromide staining of the 28S ribosomal RNA band was used as the loading control. The membrane was incubated in standard prehybridization solution at 65°C for 6 h and then hybridized with the 
-D-thiogalactopyranoside to 0.5 mM. The cells were collected, and lysed by moderate ultrasonic. The recombinant GH2 and gh2 protein were purified by glutathione-Sepharose 4B affinity chromatography and cleaved by PreScission Protease on a column according to the manufacturer's instructions (Pharmacia Biotech). The purified proteins were quantified by the bicinchoninic acid method and used for the enzyme kinetics analysis. 
340 nm, 10% to 47% acetonitrile; coniferyl alcohol, 
