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First published online April 8, 2005; 10.1104/pp.105.059337 Plant Physiology 138:393-401 (2005) © 2005 American Society of Plant Biologists A Stilbene Synthase Gene (SbSTS1) Is Involved in Host and Nonhost Defense Responses in Sorghum1Departments of Botany (C.K.Y.Y., W.K.Y., C.L.) and Chemistry (I.K.C.), University of Hong Kong, Hong Kong, China; Leibniz-Institut für Pflanzenbiochemie, 06120 Halle/Saale, Germany (K.S., J.S.); and Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907 (R.L.N.)
A chalcone synthase (CHS)-like gene, SbCHS8, with high expressed sequence tag abundance in a pathogen-induced cDNA library, was identified previously in sorghum (Sorghum bicolor). Genomic Southern analysis revealed that SbCHS8 represents a single-copy gene. SbCHS8 expression was induced in sorghum mesocotyls following inoculation with Cochliobolus heterotrophus and Colletotrichum sublineolum, corresponding to nonhost and host defense responses, respectively. However, the induction was delayed by approximately 24 h when compared to the expression of at least one of the other SbCHS genes. In addition, SbCHS8 expression was not induced by light and did not occur in a tissue-specific manner. SbCHS8, together with SbCHS2, was overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) tt4 (transparent testa) mutants defective in CHS activities. SbCHS2 rescued the ability of these mutants to accumulate flavonoids in seed coats and seedlings. In contrast, SbCHS8 failed to complement the mutation, suggesting that the encoded enzyme does not function as a CHS. To elucidate their biochemical functions, recombinant proteins were assayed with different phenylpropanoid-Coenzyme A esters. Flavanones and stilbenes were detected in the reaction products of SbCHS2 and SbCHS8, respectively. Taken together, our data demonstrated that SbCHS2 encodes a typical CHS that synthesizes naringenin chalcone, which is necessary for the formation of different flavonoid metabolites. On the other hand, SbCHS8, now retermed SbSTS1, encodes an enzyme with stilbene synthase activity, suggesting that sorghum accumulates stilbene-derived defense metabolites in addition to the well-characterized 3-deoxyanthocyanidin phytoalexins.
Sorghum (Sorghum bicolor) is well known for its adaptability to adverse environments such as hot and dry conditions. The plant is also a rich source of distinct natural products. For example, sorghum seedlings accumulate high levels of dhurrin, a cyanogenic glycoside derived from Tyr (Busk and Møller, 2002  ). To preclude competition for resources, sorghum roots exude sorgoleone and derivatives, a group of hydrophobic p-benzoquinone compounds, which inhibit electron transfer in PSII (Czarnota et al., 2001). In response to pathogen infection, sorghum synthesizes a unique class of flavonoid phytoalexins, the 3-deoxyanthocyanidins, as an essential component in the plant's active defense mechanisms (Lo et al., 1999).
Chalcone synthase (CHS) catalyzes the first committed step in flavonoid biosynthesis. The enzyme is the prototype of the plant type III polyketide synthase (PKS) family, including the closely related stilbene synthases (STSs), pyrone synthases, acridone synthases, valerophenone synthases, and benzalacetone synthases (Springob et al., 2003
We have previously described a family of 8 CHS genes, SbCHS1 to SbCHS8, in sorghum (Lo et al., 2002 ). SbCHS1 to SbCHS7 (AF152548–AF152554) are highly conserved (at least 97.5% sequence identity at amino acid level) and closely related to the maize (Zea mays) C2 and Whp genes encoding CHS enzymes. SbCHS8 (AY069951), on the other hand, is only 81% to 82% identical to SbCHS1 to SbCHS7 at the amino acid level and appears to be more distantly related as revealed by phylogenetic analysis (Lo et al., 2002). These findings suggested that SbCHS8 was duplicated from the ancestral form of SbCHS1 to SbCHS7 and diverged in protein coding sequence. In in silico analysis, SbCHS8 was found to have significantly higher expressed sequence tag (EST) abundance in a pathogen-induced library (Lo et al., 2002). This EST library was prepared from 2-week-old seedlings 48 h after inoculation with the anthracnose pathogen Colletotrichum sublineolum (University of Georgia). Accumulation of 3-deoxyanthocyanidin was consistently detected in sorghum tissues inoculated with this fungal pathogen (Snyder and Nicholson, 1990; Lo et al., 1999), leading to our speculation that SbCHS8 is involved in the biosynthesis pathway (Lo et al., 2002).
In this study, we used the well-established mesocotyl inoculation system (Hipskind et al., 1996
Genomic Southern Analysis of SbCHS Genes For genomic Southern analysis, total DNA samples from 3 different sorghum cultivars (BTx623, Sc748-5, and DK46) were digested to completion with EcoRI, HindIII, or XbaI. A SbCHS8-specific PCR fragment containing part of the coding sequence and a 3'-untranslated region was used as a hybridization probe. Results indicated that SbCHS8 is a single-copy gene and there are no RFLPs among the different cultivars examined (Fig. 2A). In contrast, a number of signals with varying intensities and sizes were detected when the digested DNA samples were hybridized with a CHS universal probe (Fig. 2B), which was derived from a conserved region in the SbCHS1 to SbCHS7 coding sequences. RFLPs were observed among the different cultivars following HindIII digestion. For example, Sc748-5 displayed a hybridization pattern distinct from the other 2 cultivars (Fig. 2B).
Northern Analysis of SbCHS Gene Expression
Sorghum cultivar DK46 accumulates anthocyanin pigments in mesocotyls of etiolated seedlings upon light induction (Lo and Nicholson, 1998
To study the expression of SbCHS8 during defense responses, etiolated seedlings of DK46 were inoculated with either Cochliobolus heterotrophus, a maize pathogen but nonpathogenic to sorghum, or C. sublineolum, the causal agent of sorghum anthracnose. The inoculated seedlings were either kept in the dark or placed under constant light. Total RNA samples from various time points were analyzed by northern hybridizations. Transcripts of SbCHS genes, including SbCHS8, were detected in all the inoculation conditions examined (Fig. 3, C–H). However, pathogen-induced accumulation of SbCHS8 transcripts was delayed compared to transcripts detected by the universal SbCHS probe. For example, transcripts of SbCHS8 were not detected until 24 h after inoculation with C. heterotrophus under dark conditions, while transcripts of at least 1 of the other SbCHS genes were detected within 3 h (Fig. 3D). Similarly, SbCHS8 gene expression was not observed until 72 h after inoculation with C. sublineolum under dark conditions, while the expression of at least 1 of the other SbCHS genes was observed within 36 h (Fig. 3F). Although SbCHS8 is not light inducible, the pathogen-induced gene expression appeared to be enhanced under light. Thus, transcripts of SbCHS8 were detected 12 h earlier in C. heterotrophus-inoculated plants and 24 h earlier in C. sublineolum-inoculated plants under light compared to the respective infected plants kept in the dark (Fig. 3, C–F).
The expression of SbCHS8 was also examined in two sorghum inbred lines, BTx623 and Sc748-5, with differential physiological and biochemical responses to the anthracnose pathogen C. sublineolum (Lo et al., 1999
The complementation of Arabidopsis transparent testa (tt) mutants by maize genes demonstrated the convenience of this system for establishing the function of uncharacterized coding sequences with homology to flavonoid structural genes (Dong et al., 2001
Transgenic tt4 mutants expressing SbCHS2 produced T1 seeds with brown pigmentation characteristic of wild-type seeds (Fig. 4A), indicating the accumulation of tannins in seed coats. In addition, these transgenic seedlings showed anthocyanin pigments in cotyledons and hypocotyls when germinated in medium devoid of nitrogen sources, a sensitive condition previously employed to induce the anthocyanin biosynthesis pathway in Arabidopsis (Hsieh et al., 1998
To further characterize the flavonoids synthesized by the transgenic Arabidopsis tt4 mutants, HPLC experiments were performed using acid-hydrolyzed methanol extracts prepared from 14-d-old seedlings. Expression of SbCHS2 in transgenic tt4 plants resulted in the accumulation of the flavonols quercetin and kaempferol, which were not present in the extracts prepared from nontransformed mutants (Fig. 4B). The flavonoid profile, monitored at A360, of these transgenic plants was near identical to that of the wild-type plant, Landsberg erecta (Ler), confirming the complete complementation of tt4 mutation by SbCHS2. In contrast, accumulation of these flavonols was not detected in the SbCHS8-expressing tt4 plants, further suggesting that this sorghum enzyme does not function as a CHS in planta.
SbCHS2 and SbCHS8 were overexpressed in Escherichia coli and purified by immobilized metal-affinity chromatography to generate electrophoretically homogeneous recombinant proteins (Fig. 5A). Purified protein samples were incubated with 14C-malonyl CoA and different phenylpropanoid-CoA esters. Recombinant proteins of Cassia alata CHS (CalCHS1; Samappito et al., 2002
To unambiguously identify the reaction products, recombinant proteins were incubated with unlabeled malonyl-CoA and starter CoA esters in scaled-up reactions. The product mixtures obtained in these experiments were analyzed by combined liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) mode using the reactions leading to key ions. Under positive ESI conditions, flavanones were detected by reactions leading to a key ion at m/z 153 (trihydroxybenzoyl moiety) as well as the respective phenylpropanoyl cations: cinnamoyl cation at m/z 131 and p-coumaroyl cation at m/z 147 (Fig. 5C; Samappito et al., 2002 ). The mass spectral behavior of stilbenes under negative ESI conditions is characterized by the loss of ketene units. Resveratrol was confirmed by measuring the 2 key reactions, m/z 227 [M-H]– to m/z 185 [M-H–CH2CO]– and m/z 227 [M-H]– to m/z 143 [M-H-2CH2CO]–, respectively (Fig. 5C; Stecher et al., 2001; Samappito et al., 2003). Similarly, pinosylvin was identified by the reaction m/z 211 [M-H]– to m/z 169 [M-H-CH2CO]– (Fig. 5C). Taken together, our results clearly demonstrated that SbCHS8 encodes an enzyme with STS activity.
SbCHS8 was initially annotated as a CHS-like gene having high EST abundance in a cDNA library prepared from infected sorghum plants with the accumulation of 3-deoxyanthocyanidin phytoalexins (Lo et al., 2002 ). However, we demonstrated SbCHS8 is not involved in flavonoid biosynthesis in planta as it failed to complement the tt4 mutation in Arabidopsis (Fig. 4). Instead, the recombinant SbCHS8 protein synthesized pinosylvin and resveratrol as major products in vitro using cinnamoyl-CoA and p-coumaroyl-CoA as starter molecules, respectively (Fig. 5), and the sorghum gene was, therefore, retermed SbSTS1.
To our knowledge, SbSTS1 represents the first example of a STS gene in monocots. The gene is not constitutively expressed but inducible following fungal inoculation. Related enzymes performing STS-like cyclizations (e.g. bibenzyl synthases) have been isolated from a Phalaenopsis orchid (Preisig-Müller et al., 1995
The expression of STS genes is often induced by a variety of abiotic and biotic stresses, such as elicitor treatment, pathogen inoculation, wounding, UV irradiation, and postharvest wilting procedures (Preisig-Müller et al., 1999
An intriguing question remains regarding the identities of the sorghum defense metabolites derived from SbSTS1 enzyme activities. In members of the Poaceae, resveratrol has been isolated from endophyte-infected grasses such as fescue, ryegrass, barley, sleepygrass, and bluegrass (Powell et al., 1994
Sorghum Growth Conditions and Fungal Inoculations
All sorghum (Sorghum bicolor) seeds and fungal strains used in this study were described previously (Lo and Nicholson, 1998
Genomic DNA samples were extracted from 4-week-old sorghum plants. Leaf tissues (1 g) were ground to a fine powder in liquid nitrogen and transferred to microfuge tubes containing the DNA extraction buffer (100 mM Tris-HCl, pH 8.0; 50 mM EDTA, pH 8.0; 500 mM NaCl; 10 mM mercaptoethanol). Twenty percent (w/v) SDS (1 mL) was added to each tube and the mixtures were incubated at 65°C for 10 min. Five molar potassium acetate (5 mL) was then added and the tubes were incubated at 4°C for 20 min. The final mixtures were centrifuged at 4,000 rpm for 20 min and the supernatants were transferred into tubes containing 10 mL of isopropanol. After incubation at –20°C for 30 min, DNA samples were centrifuged at 14,000 rpm for 20 min. The pellets were washed in 70% ethanol, air-dried, and resuspended in 0.5 mL of Tris-EDTA buffer (50 mM Tris-HCl, pH 8.0; 10 mM EDTA, pH 8.0). DNA samples (20 µg) were digested to completion with selected restriction enzymes. The digested DNA was separated by electrophoresis on a 0.8% agarose gel, depurinated, denatured, and blotted in 20x SSC (3 M NaCl, 0.3 M sodium citrate) by downward capillary transfer for at least 16 h onto a GeneScreen Plus nylon membrane (Perkin-Elmer, Boston), then covalently cross-linked to the membrane with a UVP CL-1000 UV cross-linker (UVP, Cambridge, UK).
Sorghum tissues (1 g) were ground into a fine powder with liquid nitrogen and extracted with 1 mL of Trizol reagent (Invitrogen, Carlsbad, CA) in microfuge tubes. Chloroform (200 µL) was added to each tube and the resulting mixtures were centrifuged at 14,000 rpm for 10 min. The supernatants were transferred to new tubes containing 500 µL of isopropanol and 60 µL of 3 M sodium acetate. The mixtures were then centrifuged at 14,000 rpm for 10 min. The pellets were washed with 70% ethanol, air-dried, and resuspended in 30 µL of RNase-free water. Fifteen micrograms of total RNA from each sample were denatured and fractionated on a 1% formaldehyde agarose (FA) gel in 1x FA buffer, pH 7.0 (20 mM MOPS; 5 mM sodium acetate; 1 mM EDTA) and transferred to nylon membranes as described above. Equal loading of RNA on gels was confirmed by ethidium bromide staining.
Individual membranes were prehybridized in hybridization buffer (1 M sodium chloride; 0.1% dextran sulfate; 1% SDS; 100 µg mL–1 salmon sperm DNA) for 1 h at 65°C. The membranes were then hybridized in the same buffer containing different denatured 32P-labeled DNA probes for at least 16 h at the same temperature. The hybridized membranes were washed twice in 2x SSC, 0.1% SDS for 20 min at 65°C, and twice in 0.2x SSC, 0.1% SDS for 20 min at 65°C. High-stringency washes in 0.1x SSC at 65°C were performed when necessary. After washing, the membranes were exposed to FUJI 100NIF x-ray films (Fuji Photo, Tokyo) with intensifying screens at –80°C.
PCR fragments were generated for use as probes in the hybridization experiments. The CHS8 probe (394 bp) was amplified from a full-length cDNA clone (Lo et al., 2002
Full-length SbCHS2 and SbCHS8 cDNA fragments were each cloned into the BamHI and XhoI sites of 103c-SK (E. Lam, Rutgers University, New Brunswick, NJ), an overexpression vector containing the cauliflower mosaic virus 35S promoter, and the nopaline synthase 3' terminator. The resulting plasmids were cloned into the EcoRI and HindIII sites of the binary vector pCAMBIA 1300 (CAMBIA, Canberra, Australia) to generate pCAM1300-SbCHS2 and pCAM1300-SbCHS8 for Arabidopsis (Arabidopsis thaliana) transformation.
The Arabidopsis tt4 mutants (CS8605) were obtained from the Arabidopsis Biological Resource Center (ABRC; The Ohio State University, Columbus, OH). They are of the Ler genetic background and have a yellow seed coat color. The plant expression vectors were transformed into the mutants by the floral-dip method (Clough and Bent, 1998
T1 and T2 lines expressing SbCHS2 or SbCHS8 were grown on Murashige and Skoog agar containing 3% (v/v) Suc and hygromycin (25 µg mL–1). Plant materials (0.5–1.0 g) were collected from 10- to 14-d-old seedlings and ground to a fine powder in liquid nitrogen. Methanol (300 µL) containing 1% (v/v) HCl was then added to the tissue powder. Acid hydrolysis was carried by addition of an equal volume of 2 N HCl, followed by incubation at 70°C for 1 h. The hydrolyzed samples were evaporated to dryness under nitrogen and resuspended in 100 µL of acidified methanol. Final sample preparations (20 µL) were injected onto a HP 1100 series HPLC system (Agilent Technologies, Palo Alto, CA) equipped with a Nucleosil 100-5 C18 column (5 µm, 250 x 4 mm; Agilent Technologies). Chromatographic separation was performed using a solvent system of 1% acetic acid (v/v; A) and acetonitrile (B), with a linear gradient of 20% to 60% B over 25 min. Flow rate was maintained at 0.6 mL min–1 and the elution was monitored by a diode-array detector (200–600 nm). Flavonol standards were obtained from Sigma.
To express the sorghum proteins in E. coli, cDNAs were cloned into the NdeI and BamHI sites of the pET14b vector (Novagen, San Diego) containing a hexahistidine N-terminal fusion tag. To engineer the restriction sites in the inserts, PCR amplifications were performed using gene-specific primers: SbCHS2-F (5'-AGTCATATGGCCGGCGCGACTGTGACC-3') and SbCHS2-R (5'-AGTGGATCCTCAGGCGGTGATGGCCGC-3'); SbCHS8-F (5'-AGTCATATGACGACTGGGAAGGTAACA-3'); and SbCHS8-R (5'-GATGGATCCTCATGCAGCCACTGTGGT-3') with the corresponding full-length cDNA clones as templates and the enzyme Pfu polymerase (Promega, Madison, WI). The resulting plasmids were each transformed into E. coli BL21-CodonPlus (DE3)-RIL cells (Stratagene, La Jolla, CA). After a 20-h induction with 0.4 mM isopropyl-1-thio-
The standard enzyme assays contained 100 mM HEPES buffer, pH 7.0, 20 µM starter CoA, 15 µM [2-14C]malonyl-CoA (24,000 dpm), and 1.0 µg protein in a 50-µL reaction. Starter CoAs (cinnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA), prepared essentially as described (Stöckigt and Zenk, 1975
We thank Dr. Lee Pratt (University of Georgia) for providing the sorghum cDNA clones and Dr. Eric Lam (Rutgers University) for the use of his laboratory facilities at the University of Hong Kong as well as his suggestions on the manuscript. We are also indebted to Verona Dietl (IPB) for expression and purification of the recombinant proteins, Dagmar Knöfel (IPB) for providing the starter CoAs, and Christine Kuhnt (IPB) for recording the LC-ESI-SRM measurements. Received January 4, 2005; returned for revision February 1, 2005; accepted February 1, 2005.
1 This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region, China (grant no. HKU 7349/03M). C.K.Y.Y. was supported by a research postgraduate studentship from The University of Hong Kong.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.059337. * Corresponding author; e-mail clivelo{at}hkucc.hku.hk; fax 852–2858–3477.
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-D-galactopyranoside at 28°C, the recombinant proteins were purified from the bacterial cultures following procedures essentially as described previously (Samappito et al., 2002
