Transcriptional regulation of genes involved in the pathways of ...

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Apr 4, 2007 - Gopalan Selvaraj Æ Yangdou Wei Æ John King. Received: 3 October ... gesting that the generation of sufficient methyl units at an early stage ...
Plant Mol Biol (2007) 64:305–318 DOI 10.1007/s11103-007-9155-x

Transcriptional regulation of genes involved in the pathways of biosynthesis and supply of methyl units in response to powdery mildew attack and abiotic stresses in wheat Nazmul H. Bhuiyan Æ Weiping Liu Æ Guosheng Liu Æ Gopalan Selvaraj Æ Yangdou Wei Æ John King

Received: 3 October 2006 / Accepted: 20 February 2007 / Published online: 4 April 2007  Springer Science+Business Media B.V. 2007

Abstract From a library of 3,000 expression sequence tags (ESTs), derived from the epidermis of a diploid wheat (Triticum monococcum) inoculated with Blumeria graminis f. sp. tritici (Bgt), we cloned 23 cDNAs representing 12 genes that are involved in the pathways of biosynthesis and supply of methyl units. We studied the transcription of these genes to investigate how the methyl units are generated and regulated in response to Bgt infection and abiotic stresses in wheat. Expression of 5, 10-methylene-tetrahydrofolate reductase, methionine synthase, S-adenosylmethionine synthetase, and S-adenosylhomocystein hydrolase transcripts were highly induced at an early stage of infection. This induction was specific to the epidermis and linked to host cell wall apposition (CWA) formation, suggesting that the pathways for generation of methyl units are transcriptionally activated for the host defense response. Levels of S-adenosylmethionine decarboxylase, caffeic acid 3-O-methyltransferase, 1-aminocyclopropane-1-carboxylate oxidase mRNA, but not phosphoethanolamine N-methyltransferase and nicotianamine synthase mRNA, were up-regulated after infection and showed similar expression patterns to genes involved in the pathways of generation of methyl units,

The authors Nazmul H. Bhuiyan and Weiping Liu contributed equally to this work. N. H. Bhuiyan  W. Liu  G. Liu  Y. Wei  J. King (&) Department of Biology, University of Saskatchewan, Saskatoon, SK, Canada e-mail: [email protected] G. Selvaraj Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, SK, Canada

revealing possible routes of methyl transfer towards polyamine, lignin and ethylene biosynthesis rather than glycine betaine and nicotianamine in response to Bgt attack. After imposing various abiotic stresses, genes involved in the pathways of generation and supply of methyl units were also up-regulated differentially, suggesting that the generation of sufficient methyl units at an early stage might be crucial to the mitigation of multiple stresses. Keywords C1 metabolism  Gene expression pattern  Stress resistance  Transmethylation Abbreviations THFD/C 5, 10-Methylene-tetrahydrofolate dehydrogenase/5, 10-methenyl-tetrahydrofolate cyclohydrolase MTHFR 5, 10-Methylene-tetrahydrofolate reductase MetSyn Cobalamin-independent methionine synthase SAMS S-adenosylmethionine synthetase SAHH S-adenosylhomocystein hydrolase SHMT Serine hydroxy methyltransferase SAMDC S-adenosyl methionine decarboxylase COMT Caffeic acid 3-O-methyltransferase ACCO 1-Aminocyclopropane-1-carboxylate oxidase PEAMT Phosphoethanolamine N-methyltransferase NAS Nicotianamine synthase SAM S-adenosyl-L-methionine PGT Primary germ tube AGT Appressorial germ tube PR Pathogenesis related DAB 3, 3¢-Diaminobenzidine CWA Cell wall apposition

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Introduction The synthesis of numerous biological compounds and the regulation of many metabolic processes require the addition or removal of one-carbon units (C1 metabolism). In plants, C1 units are needed to synthesize proteins, nucleic acids, pantothenate, and a great variety of methylated molecules. C1 pathways are particularly active in tissues that produce methylated compounds such as lignin, alkaloids, and betaine (Hanson and Roje 2001). Methionine is an essential determinant of one-carbon metabolism. It is the precursor of S-adenosyl-Met (SAM), which, in turn, serves as the major methyl-group donor for numerous highly specific methyl-transferase reactions in all organisms. These reactions involve a large variety of acceptor molecules, such as phenylpropanoid derivatives, cyclic fatty acids, proteins, polysaccharides, and nucleic acids (Moffatt and Weretilnyk 2001). Plants produce a multitude of secondary products in response to pathogen attack or various unfavorable conditions that include one or more methyl groups added during their biosynthesis by methyltransferases, many of which use SAM as the methyl-group donor. Thus, SAM is required for the biosynthesis of phenylpropanoid compound with broad biological functions, including structural constituents of the cell wall (Higuchi 1981; Lewis and Yamamoto 1990; Campbell and Sederoff 1996). SAM is synthesized from methionine and ATP by SAM synthetase (SAMS) in the methyl cycle. The SAMS gene is considered to be a housekeeping gene because SAM is an essential substance for living cells as a methyl group donor and as a precursor of ethylene, polyamines, and nicotianamine (Tabor and Tabor 1984; Moffatt and Weretilnyk 2001). It is also induced under various conditions that reflect a need for SAM. SAMS in elicitor-treated cells of alfalfa is co-induced with caffeic acid 3-O-methyltransferase (COMT), which is a key enzyme of lignin biosynthesis, and the expression pattern of SAMS is similar to that of COMT at various developmental stages in different organs of alfalfa (Gowri et al. 1991). The SAMS mRNA levels in tomato, Catharanthus roseus and Atriplex are also increased by salt stress (Espartero et al. 1994; Schro¨der et al. 1997; Tabuchi et al. 2005). Powdery mildew disease of wheat is caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici (Bgt) which colonizes epidermal cells of the host by penetrating directly through the cell wall and forming a feeding haustorium inside by invagination of the plasmalemma. After 3–6 h post-inoculation (hpi), conidia of Bgt germinate on host leaves to produce a primary germ tube (PGT). Contact between the PGT and the leaf surface stimulates appressorial germ tube (AGT) formation, 6– 12 hpi; then the AGT elongates to differentiate into a

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mature appressorium, 12–24 hpi. A penetration peg is produced from the appressorium, which then penetrates the host epidermal cell wall and produces a haustorium at 24– 48 hpi. The haustorium is formed inside the living plant cell and enables the fungus to obtain nutrients from its host (Bushnell 2002). At least two genetically distinct pathways determine resistance in barley to powdery mildew. The first one is dependent on the presence of dominantly acting resistance genes and confers incompatibility only to those fungal isolates carrying corresponding avirulence genes. This race-specific resistance is commonly associated with a host cell death reaction at attempted infection sites (hypersensitive reaction; HR). The second pathway confers broad spectrum resistance to all tested isolates of the fungus and is stimulated by the absence of the Mlo wild type gene, a negative regulator of pathogen defense (Von Ro¨penack et al. 1998). The function of this pathway is also dependent on at least two further genes, Ror1 and Ror2 (Collins et al. 2003). Incompatibility mediated by recessive mlo resistance alleles is generally not associated with the occurrence of HR. The only visible cellular response is the formation of a sub-cellular cell wall apposition (CWA), termed as papilla, directly subtending the fungal appressorium (Aist and Israel 1977). Fungal penetration attempts are almost invariably arrested in CWAs in mlo-controlled resistance (Von Ro¨penack et al. 1998). CWAs form in both susceptible and resistant hosts, as well as non-hosts, and therefore constitute a basal or ancient form of resistance, halting the early stages of fungal ingress into host cells. In CWAs, the accumulation of callose, insoluble silicon, hydrolytic enzymes, thionins, guanidine compounds, cross-linked proteins and superoxide anion have been described (Hu¨ckelhoven and Kogel 2003). Plants respond to pathogen attack by deploying several defense reactions. Some rely on the activation of preformed components, whereas others depend on changes in transcriptional activity. A number of defense reactions have been observed in wheat and barley attacked by the powdery mildew fungus. These include localized production of active oxygen species, CWA formation at the site of attempted penetration by the fungus, accumulation of phenolic antifungal compounds (Trujillo et al. 2004; Hu¨ckelhoven and Kogel 2003), and induction of defense related genes (Zierold et al. 2005; Collinge et al. 2002). Inhibitor studies have demonstrated that callose and phenylpropanoids are directly involved in defense against infection. The recent finding of localized H2O2 production in CWAs suggests that oxidative cross-linking of cell-wall components, including cell-wall associated proteins, may play a role in CWA-based resistance (Hu¨ckelhoven and Kogel 2003). Involvement of polyamine (Cowley and

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Walters 2002), ethylene (Chen et al. 2003) and lignin accumulation in cereals (Bushnell 2002) due to the infection of biotrophic pathogens have also been described. Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie phenotypic responses. Early attempts to documents global changes in, for example, defense-associated gene expression were limited by the difficulty of identifying the significant genes and their products. Improvements of technology such as the generation of expressed sequence tag (EST) clones allow the elucidation of the whole set of transcripts present at a given time point in a cell, tissue, or organism (Scheideler et al. 2002). Although experiments on one or two enzymes involved in the methyl cycle in response to abiotic or biotic stresses have been performed previously (Moffatt and Weretilnyk 2001; Scheideler et al. 2002; Tabuchi et al. 2005; Moreno et al. 2004), transcriptional regulation of the complete set of genes related to the biosynthesis and supply of methyl units has not been investigated. In this study, we have identified a full set of pathogen-induced genes involved in the pathways of generation and supply of methyl units from a cDNA library derived from Bgt-infected diploid wheat epidermis and demonstrated transcriptional regulation and mRNA expression patterns of those genes in response to pathogen attack and abiotic stresses. Moreover, the possible routes for supplying methyl units during Bgt infection and abiotic stresses are discussed. Given the central role of the pathways for methyl unit generation in plant form and function, an understanding of the transcriptional dynamics of the relevant genes as a plant encounters various stresses will help us to understand its defense responses in general.

plate to expose the abaxial epidermis for inoculation with Bgt (Wei et al. 1998). The inoculation density was about 100–200 conidia mm–2 achieved by shaking powdery mildewed Conway plants. Tm441 and Tm453 plants were then incubated under the same conditions. To ensure that only the most vigorous and youngest conidia were used in experiments, leaves of Conway plants bearing colonies were shaken to remove old mildew conidia 48 h before inoculation. Tm441 and Tm453 leaves infected with Bgt at 0, 1, 3, 6, 12, 24, 48, 72, 96, 120 and 144 hpi were collected into 50 ml centrifuge tubes, immediately frozen in liquid nitrogen and then stored at –80C. Non-inoculated Tm441 and Tm453 leaves were used as controls.

Materials and methods

cDNA library construction

Plant and fungal materials

Abaxial epidermis from infected primary leaves was carefully stripped off at 24 hpi. The inoculum density was approximately 100 conidia mm–2, calculated to challenge each host cell with at least one spore (Wei et al. 1998). Some residual mesophyll contamination of the epidermal tissues was inevitable, especially at 48 hpi, but we roughly estimate a maximum of 5% contamination overall, and this has not greatly impaired the specificity of the library (Liu et al. 2005). Epidermal strips were flash frozen in liquid nitrogen and then stored at –80C until further use. Total RNA was extracted as described by Liu et al. (2005). Poly (A)+ RNA was isolated by the PolyATract mRNA isolation system according to the manufacturer’s instruction (Promega Corp., Madison, WI, USA). DNA sequencing was performed by the Plant Biotechnology Institute (Saskatoon) DNA technology unit.

Individual Triticum monococcum L. susceptible (Tm441, accession number TG13182) and CWA-based resistant (Tm453, accession number TG13192) lines were used throughout. Plant growth and maintenance of the powdery mildew (Blumeria graminis f. sp. tritici) isolate were performed as previously described (Liu et al. 2005). Powdery mildew inoculations Ten seeds each of Tm441 and Tm453 were sown in single rows in 36 pots for each accession line, and then grown in the greenhouse with a cycle of 16 h light and 8 h dark for 10 days. The 10-day-old, fully expanded, primary leaves of Tm441 and Tm453 were placed on a horizontal plexiglass

Abiotic stress treatments Abiotic stress treatments (wounding, cold, drought and NaCl) also were done with 10-day-old primary leaves of the Tm441 plants and stressed leaves were harvested at 1, 6, 12 and 24 h after treatment for total RNA extraction. For wounding treatment, primary leaves of Tm441 plants were placed on a horizontal plexiglass plate and gently rubbed with powdered silicon carbide until slightly damaged. Leaves from unwounded plants were used as controls. For drought treatment, Tm441 plants, which had been grown in perlite, were harvested and the medium gently washed away. These plants were then transferred to trays inlaid with paper toweling. Cold stress was performed by transferring Tm441 plants to a dark chamber at 4C. Control plants were grown at 25C. For sodium chloride treatment, 10-day-old plants of Tm441, which had been grown in perlite, were harvested and the medium gently washed away. The plants were floated in 200 mM NaCl solution. Plants floated in water were used as control.

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Northern blot analysis

Table 1 List of primers used for semi-quantitative reverse transcriptase polymerase chain reaction

Isolation of total RNA by phenol/chloroform extraction and northern blot was performed as described by Liu et al. (2005). Samples (20 lg) were separated on a 1.2% denaturing agarose gel, stained with 0.02% methylene blue, photographed to allow for comparison of RNA loading, transferred onto a GeneScreen Plus Hybridization Transfer membrane (NEN Life Science Products, Inc., Boston), UV cross-linked or baked at 80C, and hybridized at 65C in Quickhyb solution (Stratagene) with [32P] dCTP radiolabeled probes consisting of the entire coding region of the genes. Following hybridization, membranes were washed twice at room temperature in 2· SSC (1· SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS) for 15 min and once in 0.1· SSC, 0.1% SDS at 65C for 30 min, then exposed on X-ray film (Sterlin diagnostics, Newark, DE, USA). When needed, membranes were stripped in boiling 0.1· SSC, 0.1% SDS solution for 30 min before re-hybridization. All hybridizations were repeated at least twice.

Oligo sequences (5¢–3¢)

Amplified gene

F-CTACCCCAATGGCGCAAATC

TmTHFD/C

R-CACGCAGCAAATCAGCTGAC F-AATGGCCGGACCGTCTTCTC

TmMTHFR

R-TCTCATCGAAATTAGATCTC F-ACCGTCTTCTGGTCCAAGATG

TmMetSyn

R-AGCACGGCTTACTGCGCCTTG F-GGACACTTCACCAAGCGTC

TmSAMS2

R-AATGCCATGCTCTTAGGCAG F-AATGCCATGCTCTTAGGCAG

TmSAMS1

R-CATACCCACCTCTCAAGCAG F-GGAAAAGCACACCTCCTCAC R-AGGCTGACCAAGCTCACCAAG

TmSAHH

F-CACCACCCACCATGGCCATG

TmSHMT

R-AGCAGTTCTTAGTTCTTGTAC F-CTCGAACTGCAACAATGGCTG

TmSAMDC

R-AGATTATCGCATCACTCGCAC F-ATCCCGTTCAACAAGGCGTAC

TmCOMT

R-GTCGTGGAGGATCCACTTCAT

Semi-quantitative reverse transcriptase polymerase chain reaction

F-GGACAATGTGCAGTACAAATC F-ATGGTTGTTCCGGTGATCGAC

Semi-quantitative RT-PCR was performed as described by Liu et al. (2005) with some modifications. Total RNA was isolated from the Bgt-infected epidermal and mesophyll cells from the susceptible line. cDNA syntheses were performed using superscript reverse transcriptase (Invitrogen Life Technologies, Burlington, ON, Canada). mRNA was isolated from total RNA by the PolyATract mRNA isolation system (Promega Corp., Madison, WI, USA). Gene specific primers were generated based on areas of low sequence homology, among which most primers were located at 5¢ or 3¢ untranslated regions (Table 1). To ensure that equal amounts of mRNA were used, a positive control encoding the TmGAPD gene was normalized using primers GAPDF and GAPDR. PCR amplifications were performed using Taq polymerase (Amersham Biotech, Piscataway, NJ, USA) under the following conditions: 94C for 20 s, 52–62C for 30 s, and 72C for 60 s for 30 cycles in a thermal Eppendorf Mastercycler (Hamburg, Germany). RT-PCR for all clones was repeated for at least twice. Standard errors were calculated and indicated as size bars. Band intensity was measured by spot densitometry using AlphalmagerTM 2200 documentation and analysis system (Alpha Innotech, SanLeandro, CA, USA). Microscopy study DAB (3, 3¢-diaminobenzidine) uptake was carried out as described by Thordal-Christensen et al. (1997). Epidermis was stripped from both susceptible and resistant line at

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TmACCO

R-GTGGTGCAGTCGGCATCCTC TmPEAMT

R-TCCTGGAACAGCAGGATGAAC F-GGCGCGCCTGGTGGAGCACCTCGGG

TmNAS

R-GCCTGGGCTGCGCCTGCGAGCTGGGGAC F-GGCTCCGATCAAGATCGGCATC

TmGAPD

R-GGAGCAAGGCAGTTAGTGGTGC

24 h after Bgt inoculation. The storage of leaf segments, the staining of fungal structures and the microscopy were done as described by Hu¨ckelhoven et al. (1999). To visualize callose, leaves were cleared in 95% ethanol, stained with aniline blue, and examined by a fluorescence microscope. To detect autofluorescent compounds, epidermis was cleared in 95% ethanol, equilibrated in a solution of lactic acid, glycerol, and water (1:1:1), mounted, and examined by a confocal microscope LSM510 (Zeiss, Oberkochen, Germany). Number of interaction site was counted from two individual experiments. Standard errors were calculated and indicated as size bars.

Results Isolation of genes involved in the pathways of biosynthesis of methyl units from Bgt-infected epidermis cDNA library To identify wheat genes involved in the pathways of generation of methyl units (Fig. 1), an epidermal cDNA library

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Fig. 1 The pathways of biosynthesis of methyl units. THFD/C: 5, 10methylene-tetrahydrofolate dehydrogenase/5, 10-methenyl-tetrahydrofolate cyclohydrolase; MTHFR, 5, 10-methylene-tetrahydrofolate reductase; MetSyn, cobalamin-independent methionine synthase; SAMS, S-adenosylmethionine synthetase; SAHH, S-adenosylhomocystein hydrolase; SHMT, serine hydroxy methyltransferase

from T. monococcum leaves (Tm441 line, a susceptible wheat line) at 24–48 h after powdery mildew infection served as a source; currently, ~3,000 ESTs from the library have been sequenced. Among these ESTs, we identified

amino acid (aa) sequences deduced from two cDNA clones showing high similarity with THFD/C (5, 10-methylenetetrahydrofolate dehydrogenase/5, 10-methenyl-tetrahydrofolate cyclohydrolase), one clone with MTHFR (5, 10methylene-tetrahydrofolate reductase), two clones each with MetSyn (cobalamin-independent methionine synthase), SAMS (S-adenosylmethionine synthetase), and SAHH (S-adenosylhomocystein hydrolase) and three clones with SHMT (serine hydroxy methyltransferase). One representative clone was selected for further molecular characterization for each gene except SAMS. Two cDNA clones were identified for SAMS, one full length and another partial; both were used for transcriptional analysis. Nucleotide and protein sequences of all genes were analyzed by comparison with databases using the BLAST program (Table 2). Among 12 cDNAs identified, four of them were full-length sequences and the rest of them, partial. Based on the sequence analysis the full-length clones were named TmTHFD/C, TmMTHFR, TmSAMS1 and TmSHMT. TmTHFD/C contained a 876-bp ORF that putatively coded for a 292 aa with a calculated molecular mass of 30.8 KDa with a pI of 7.77. The putative protein shared 88.7% aa sequence homology with THFD/C of Oryza sativa (AAG48834). TmMTHFR encoded 582 aa with a calculated molecular mass of 64.9 KDa and pI of 5.86 and the protein showed high similarity (86.6%) with MTFHR of Zea mays (AAD51733). TmSAMS1 had a 1,182-bp ORF that putatively encoded 394 aa with molecular mass of 42.8 KDa and pI of 5.61. TmSAMS1 shared high identity (96.2%) with SAMS of Hordeum vulgare (P50299). TmSHMT contained a 1,530-bp ORF that putatively codes for a 510 aa with a estimated molecular mass of 56.1 KDa

Table 2 List of expressed sequence tags isolated from Blumeria graminis f. sp. tritici-infected wheat epidermis cDNA library Gene name

Genebank accession noa

Copy

pIb

Closest ortholog (identity %)

TmTHFD/C

DQ862826

2

TmMTHFR

DQ862830

1

292

7.77

Oryza sativa (88.7)

582

5.86

TmMetSyn

DQ862829

2

228c

Zea mays (86.6)



Hordeum vulgare (100)

TmSAMS1

DQ862831

1

394 c

5.61

H. vulgare (96.2)

AA

TmSAMS2

DQ862832

1



H. vulgare (97.2)

TmSAHH

DQ862833

2

285

42c



Triticum aestivum (82)

TmSHMT

DQ862827

3

510

8.18

O. sativa (93.1)

TmSAMDC

DQ862828

7

388

4.98

T. aestivum (97.2)

TmCOMT

DQ862834

1

117c



T. aestivum (88)

c

TmPEAMT

DQ862836

1

59



T. aestivum (85)

TmACCO

DQ862835

1

206c



Zea mays (85)

TmNAS

DQ167190

1

56c



H. vulgare (96)

a

mRNAs isolated from epidermis library of T. monococcum line Tm441

b

Isoelectronic point (pI) for mature protein was estimated by Swiss-Prot/TrEMBL

c

Partial sequences

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with a pI of 8.18 and the protein showed the highest putative identity to SHMT of O. sativa (AAP44712). Deduced amino acid sequence analysis of full-length clones showed the lack of transit peptide at the N-terminal regions of the TmTHFD/C, TmMTHFR and TmSAMS1, suggesting that these proteins are localized in cytosol. A mitochondrial transit peptide in the N-terminal region of Tm-SHMT indicates that this protein is targeted to mitochondria. Characterization of other clones was given in Table 2. Phylogenetic analysis showed that all clones are more closely related to monocot plants than dicot (data not shown). Bgt induced expression of genes involved in the pathways of biosynthesis of methyl units To investigate the expression of genes involved in the pathways of generation of methyl units during the process of wheat–Bgt interaction, TmTHFD/C, TmMTHFR, TmMetSyn, TmSAMS1, TmSAMS2, TmSAHH, and TmSHMT were monitored at the transcript level by northern blot analysis over a 144-h period in the susceptible and resistant wheat lines (Fig. 2A). The gel blot data show that TmMTHFR, TmMetSyn, TmSAMS1, TmSAMS2, and TmSAHH all have similar patterns of expression in response to infection, despite differing transcriptional intensities. These five genes were highly induced at 6 h post-inoculation (hpi), followed by a slight decrease in transcript level at 12 hpi and then a sharp increase at 24 hpi in the susceptible line. The two expression peaks at 6 and 24 hpi coincided with the attempted penetration time points of PGT and AGT (Collinge et al. 2002),

Fig. 2 Northern blot analysis of genes involved in the pathways for generation of methyl units (A) and PR genes (B) in response to Blumeria graminis f. sp. tritici infection. Total RNA was isolated at 0–144 h post-inoculation from the 10day-old primary leaves inoculated with B. graminis f. sp. tritici conidia. Un-inoculated leaves were used as controls (CK). Transcript levels of genes were analyzed with 32P-labeled cDNA probes for TmTHFD/C, TmMTHFR, TmMetSyn, TmSAMS1, TmSAMS2, TmSAHH, TmSHMT, TmPR1b, TmPR2, and TmPR5. Total RNA was loaded at 20 lg per lane and equal loading was monitored by methylene blue staining of ribosomal RNA

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respectively. Transcripts of pathogenesis-related (PR) genes (Fig. 2B) showed induction at 6 hpi and continued up to 144 hpi except PR2 in the susceptible line, a pattern completely different from those of genes involved in the pathways of generation of methyl units. On the other hand, during the incompatible interaction in the resistant line, TmMTHFR, TmMetSyn, TmSAMS1, TmSAMS2, and TmSAHH showed a strong induction at 24 hpi (Fig. 2A). Unlike the compatible interaction, there was no observable expression peak of these genes at 6 hpi in the resistant line except moderately in the case of TmMetSyn and TmSAHH. TmTHFD/C and TmSHMT were not activated in either line suggesting that transcriptional regulations of these two genes are not directly involved in response to Bgt attack. Transcripts of PR genes began accumulating at 12 hpi and continued up to 48 hpi in the incompatible interaction (Fig. 2B), a pattern different from that of the genes involved in the pathways of generation of methyl units. Epidermis but not mesophyll shows enhanced expression of genes involved in the pathways of biosynthesis of methyl units in response to Bgt attack The epidermis provides the major line of defense against infection by the surface-growing powdery mildew fungus. To investigate further whether the expression patterns of the genes involved in the pathways of generation of methyl units differ between epidermis and other cell types, samples from susceptible line epidermis and mesophyll tissues were collected at 0 and 24 hpi and were amplified by RTPCR using gene-specific primers (Fig. 3). Distinct

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with the northern blot analysis, semi-quantitative RT-PCR demonstrated that the maximal expression intensity of TmMTHFR, TmMetSyn, TmSAMS1, and TmSAHH after pathogen attack was in epidermis, suggesting that an elevated level of SAM is produced in epidermis to supply methyl units to the generation of defense metabolites. Upregulation of genes involved in the pathways of biosynthesis of methyl units coincides with the formation of effective CWAs (papilla) after Bgt attack

Fig. 3 Semi-quantitative RT-PCR analysis of tissue specific expression of seven genes involved in the pathways for generation of methyl units in response to Blumeria graminis f. sp. tritici infection. Total RNA was isolated from epidermal (E) and mesophyll (M) tissues 0 and 24 h post-inoculation on line Tm441 and reverse transcribed to cDNA. The expression of a glyceraldehydes 3-phosphate dehydrogenase gene (TmGAPD) was used as a control of mRNA normalization. Band intensity was measured from two individual experiments

expression patterns were found in the semi-quantitative RT-PCR analysis. The expression of TmMTHFR but not that of TmTHFD/C was induced in the epidermis. Induction of TmMetSyn was observed in both epidermis and mesophyll tissues after pathogen infection. Interestingly, transcripts of TmSAMS2 and TmSAMS1 showed distinctly different expression at 24 hpi, TmSAMS2 being highly expressed specifically in mesophyll and TmSAMS1 in epidermis tissue. The TmSAHH transcript was highly activated only in epidermis after infection. The level of mRNA for TmSHMT was unchanged before or after inoculation in either tissue that coincides with northern blot. Consistent

We conducted cytological studies for evaluating H2O2 production, callose deposition and autofluorescence response beneath the interaction sites of PGT and AGT in susceptible and resistant wheat lines in order to observe the epidermal host defense response after Bgt attack. As shown in Fig. 4A, B, CWA regions were stained with DAB at 24 hpi in both lines to demonstrating H2O2 production. Large CWAs (Fig. 4J) with enhanced DAB staining (Fig. 4B) were observed in resistant plants at 24 hpi around appressorial germ tube (AGT) penetration sites, which coincides with the strong expression of methyl unit biosynthesis genes (Fig. 2A). In contrast, small CWAs with weak DAB staining were observed in the susceptible line at the time of AGT penetration (Fig. 4A). In addition, the numbers of DAB-stained CWAs in the AGT penetration site were also less in susceptible line compared to resistant line (Fig. 4C). However, a significantly reduced number of DAB-stained CWAs was observed in PGT penetration sites in the resistant line (Fig. 4C). We have also observed the autofluorescence response and callose formation in the interaction sites in both lines. The accumulation pattern of autofluorogenic material (Fig. 4D–F) in CWAs beneath PGT or AGT penetration site was similar to the pattern of H2O2 production (Fig. 4A–C) in susceptible and resistant lines. Callose deposition in CWA areas was also very similar to the accumulation of H2O2 and autofluorogenic material except that a reduced number of callose formations were observed in the resistant line beneath the AGT site (Fig. 4G–I). However, strong callose deposition was seen in the resistant line coinciding with a high accumulation of H2O2 and high level of autofluorescence beneath the AGT site. These demonstrated that increased formation of H2O2, autofluorogenic compound and callose in CWA areas are correlated to host resistance and with the upregulation of methyl unit biosynthesis genes. Influence of abiotic stresses on the expression of genes involved in the pathways of biosynthesis of methyl units Substantial interactions or cross-talk among the biotic and abiotic stress pathways in plants have been reported (Cheong et al. 2002). A compatible osmolyte such as

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Fig. 4 Host response to Blumeria graminis f. sp. tritici infection. (A– C) DAB-stained CWAs are shown in compatible (A) and incompatible (B) interactions. Ten days old wheat primary leaves of Tm441 and Tm453 were inoculated with fungal inoculum. After 1 h of inoculation, leaves were cut off and placed in a solution of 1 mg ml–1 DAB and collected for microscopic analysis at 24 hpi. (C) Bar graphs showing the percentage of germinated conidia with DAB-stained CWAs in the PGT and AGT penetration site in both lines after 24-h post-inoculation. (D–F) Autofluorescence of CWA areas are shown in compatible (D) and incompatible (E) lines beneath PGT and AGT

penetration sites. (F) Percentage of germinated conidia showing autofluorescence near penetration sites of PGT and AGT in both lines. (G–I) Callose formations in CWA areas are shown in compatible (G) and incompatible (H) lines and percentage of germinated conidia showing callose formation in beneath PGT and AGT sites in both lines. (J) Size of CWA was measured beneath PGT and AGT sites in both lines at 24 h post-infection. Beneath PGT and AGT, CWAs are indicated by arrowheads. Scale bar correspond to 10 lm. Each column represents 200 interaction sites

glycine betaine (in the case of wheat) is synthesized in response to abiotic stresses where SAM acts as a methyl donor for their respective methyltransferase enzyme activities. SAM is a direct substrate for polyamine biosynthesis. Therefore, it appeared valuable to test whether the genes involved in the pathways of generation of methyl units, isolated from the Bgt-induced EST library also responded to abiotic stresses. The transcriptional changes of the same set of genes followed during pathogen infection

were also investigated in response to salinity, dehydration, cold, and wounding stresses. The genes involved in the pathways of generation of methyl units showed differential reactions to abiotic stresses (Fig. 5). The key gene of the methyl cycle, TmSAMS1, responded highly to all types of abiotic treatment applied, the highest expression of this gene occurring in response to NaCl. TmSAMS2 was strongly induced by drought at 6 h and by cold at 24 h, indicating that

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Fig. 5 Northern blot analysis of genes involved in the pathways for generation of methyl units in response to abiotic stresses. Total RNA was isolated at 1, 6, 12, 24 h post-treatment from the 10-day-old primary leaves of Triticum monococcum subjected to NaCl, cold, dehydration, and wounding. Transcript levels of genes were analyzed

with 32P-labeled cDNA probes for TmTHFD/C, TmMTHFR, TmMetSyn, TmSAMS1, TmSAMS2, TmSAHH and TmSHMT. Total RNA was loaded at 20 lg per lane and equal loading was monitored by methylene blue staining of ribosomal RNA

TmSAMS1 and TmSAMS2 have different functional roles in response to abiotic stresses. Expression of TmMetSyn was most highly induced by wounding at 6 h and by cold at 12 h. A moderate expression of TmSAHH occurred in response to different abiotic stresses. Like the Bgt-infected transcriptional expression pattern, TmTHFD/C and TmSHMT showed either consistent or down-regulated expression under the abiotic stresses applied in this study.

biosynthetic pathways were found in our wheat epidermis EST library where SAM is utilized as a substrate or as a cofactor for methyl donation. Among the 3,000 expressed sequence tags (ESTs), amino acid sequences deduced from seven cDNA clones showing high similarity with S-adenosyl methionine decarboxylase (SAMDC), and one each with caffeic acid 3-O-methyltransferase (COMT), 1-aminocyclopropane-1-carboxylate oxidase (ACCO), phosphoethanolamine N-methyltransferase (PEAMT), and nicotianamine synthase (NAS), were identified (Table 2). These enzymes are involved in several different metabolic pathways: SAMDC with polyamine, COMT with lignin, ACCO with ethylene, PEAMT with glycine betaine and NAS with nicotianamine biosynthesis. We performed northern blot analyses to determine which gene/s is highly activated in response to Bgt attack. As shown in Fig. 6, TmSAMDC and TmCOMT encoding

Transcripts of TmSAMDC, TmCOMT and TmACCO, but not TmPEAMT and TmNAS, are up-regulated after Bgt infection To investigate the regulation of possible methylationdependent host reactions during Bgt infection, we selected a set of targeted metabolic genes for further expression analysis. Five genes for different defense metabolite

Fig. 6 Northern blot analysis of TmSAMDC, TmCOMT, TmACCO, TmPEAMT, and TmNAS genes in response to Blumeria graminis f. sp. tritici infection. Total RNA was isolated at 0–144 h post-inoculation from the 10-day-old primary leaves inoculated with B. graminis f. sp. tritici conidia. Un-inoculated leaves were used as controls (CK).

Transcript levels of metabolic genes were analyzed with 32P-labeled cDNA probes for TmSAMDC, TmCOMT, TmACCO, TmPEAMT, and TmNAS. Total RNA was loaded at 20 lg per lane and equal loading was monitored by methylene blue staining of ribosomal RNA

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key enzymes of polyamine and lignin biosynthesis, respectively, were highly activated at 6 and 24 hpi in the susceptible line and there was a strong signal at 24 hpi in the resistant line, expression patterns similar to those of genes involved in the pathway of generation of methyl units (Fig. 2). This suggests the utilization of SAM in polyamine and lignin synthesis after pathogen infection. Expression of TmACCO, a key enzyme of ethylene biosynthesis, was induced at 3 h and continued up to 24 hpi in the susceptible line and at 24 hpi in the resistant line, suggesting that SAM is also used in ethylene synthesis after Bgt infection. Expression of TmPEAMT and TmNAS was down regulated in both lines, suggesting that glycine betaine and nicotianamine are not involved in response to Bgt attack. Tissue specific expression of TmSAMDC, TmCOMT, TmACCO, TmPEAMT and TmNAS after Bgt infection To investigate further whether the expression of these five defense-related metabolic genes differs between epidermis and mesophyll tissues, semi-quantitative RT-PCR analysis was carried out using control and 24 hpi samples (Fig. 7). A very clear upregulation of TmSAMDC and TmCOMT transcripts was observed in both epidermis and mesophyll tissues at 24 hpi, consistent with northern blot signals. TmACCO, was also modestly expressed in both types of tissue at the same time point. We have also examined two more genes, which play a key role in glycine betaine (TmPEAMT) and nicotianamine (TmNAS) biosynthesis pathways. The transcript of TmPEAMT did not show any change in either type of tissue before or after infection. A clear down-regulation of TmNAS transcription was observed in both epidermis and mesophyll tissues after Bgt infection. Transcripts of TmSAMDC, TmCOMT, TmACCO, TmPEAMT and TmNAS expressed differentially under abiotic stresses Pathogen-induced defense genes may have a role also in abiotic stresses regulated by different signaling molecules. We used wound, drought, cold and salinity treatments of the susceptible wheat line to investigate more about the cross-talk of the above five defense genes under abiotic stresses. The time course of gel blot analysis (Fig. 8) showed that both TmSAMDC and TmPEAMT were induced by drought and salinity stresses, although the intensity of transcripts and induction times were different. The transcript level of TmPEAMT was very strong compared to TmSAMDC, and expression was earlier. TmPEAMT was also induced by cold treatment. Expression of TmACCO was highly induced by wounding but less so by cold and

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Fig. 7 Semi-quantitative RT-PCR analysis of TmSAMDC, TmCOMT, TmACCO, TmPEAMT, and TmNAS genes in response to Blumeria graminis f. sp. tritici infection. Total RNA was isolated from epidermal (E) and mesophyll (M) tissues 0 (control) and 24 h postinoculation on line Tm441 and reverse transcribed to cDNA. The expression of a glyceraldehyde-3-phosphate dehydrogenase gene (TmGAPD) was used as a control of mRNA normalization. Band intensity was measured from two individual experiments

salinity. The TmCOMT transcript was expressed only weakly under wounding, cold, drought and salinity stresses. The level of TmNAS mRNA was moderately induced by wounding, drought and NaCl.

Discussion In this study, we have shown that transcription of genes (TmMTHFR, TmMetSyn, TmSAMS1, TmSAMS2, and TmSAHH) involved in the pathways of biosynthesis of methyl units is up-regulated at the early stage of powdery mildew attack or by abiotic stresses. We hypothesize that this upregulation is linked and leads to an enhanced flux of SAM. SAM, in turn, is used for methylation in different defense metabolic pathways as indicated by the high

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Fig. 8 Northern blot analysis of TmSAMDC, TmCOMT, TmACCO, TmPEAMT, and TmNAS genes in response to abiotic stresses. Total RNA was isolated at 1, 6, 12, 24 h post-treatment from the 10-day-old primary leaves of Triticum monococcum subjected to NaCl, cold, dehydration, and wounding. Transcript levels of genes were analyzed

with 32P-labeled cDNA probes for TmSAMDC, TmCOMT, TmACCO, TmPEAMT, and TmNAS. Total RNA was loaded at 20 lg per lane and equal loading was monitored by methylene blue staining of ribosomal RNA

degree of expression of TmSAMDC, TmCOMT and TmACCO genes in response to Bgt attack or of TmPEAMT, TmSAMDC, TmACCO and TmCOMT in response to abiotic stresses. An EST library generated from specific conditions is a valuable tool for analyzing the involvement of metabolic genes at a genomic level. The EST library used in this study was established from diploid wheat leaf epidermis 24–48 h after B. graminis f. sp. tritici inoculation. Bgt only infects epidermal cells; host plant resistance depends largely on enhanced cell wall appositions or papillae, a localized strengthening of the epidermal cell wall in response to pathogen attack. Thus, a library constructed from this tissue provides a unique snapshot of host defense gene expression with minimal background interference from other tissues (Liu et al. 2005). Analysis of the epidermis library in our study revealed 12 expressed genes, represented by 23 ESTs, involved in the pathways of biosynthesis and supply of methyl units (Table 2). The expression peaks of the genes involved in the pathways of generation of methyl units after pathogen attack coincided with attempted PGT penetration at 6 hpi and AGT penetration at 24 hpi in the susceptible line. An epidermal cell responds to the PGT by forming a small CWA directly subtending PGT contact sites at 6 hpi and a bigger CWA at sites of appressorium contact at 24 hpi (Collinge et al. 2002). Thus, it can be hypothesized that genes involved in the pathways of biosynthesis of methyl units are linked to CWA formation in the host defense response. Transcripts of genes involved in the pathways of generation of methyl units were accumulated in a pattern that was completely different from those of pathogenesisrelated (PR), or peroxidase class III (Liu et al. 2005) or quinone reductase genes (Greenshields et al. 2005). Hence,

we suggest that genes involved in the pathways of generation of methyl units are more closely related to CWAmediated host defense responses than to a more general defense response. In the compatible interaction, the transcripts of TmMTHFR, TmMetSyn, TmSAMS1, TmSAMS2, and TmSAHH are coordinately regulated but the expression pattern is different from that in the incompatible interaction. The incompatible interaction did not show any significant upregulation of these genes at 6 hpi in the resistant line unlike in the susceptible line. In a cytological study, we found a significantly reduced number of DAB-stained sites as well as callose depositions at PGT penetration sites in the incompatible interaction, which may provide a clue as to why a difference exists between susceptible and resistant plants at this time point. However, further work is necessary to answer this question precisely. A significantly smaller number of DAB-stained papillae were reported at the interaction sites in a susceptible barley line compared to a resistant line after powdery mildew attack (Hu¨ckelhoven et al. 1999). In the resistant line, transcripts of the above five genes were strongly accumulated at 24 hpi, which coincides with the formation of a large CWA underneath the AGT penetration peg; this might block the further penetration of the pathogen. In contrast, a relatively weak expression occurred of genes involved in the pathways of generation of methyl units compared to the resistant line at 24 hpi, which coincides with the formation of small CWAs underneath the AGT penetration peg, suggesting that this level of gene expression might not be sufficient to block the further penetration of the AGT in the susceptible line. It was previously described that host cell defense in the incompatible interaction takes the form of a large CWA produced

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at 24 hpi in response to AGT penetration in barley (Zeyen et al. 2002). Transcript abundance of different defense genes was also observed in resistant barley line at this time point compared to susceptible one after powdery mildew infection (Zierold et al. 2005). From cytological study, we found large CWAs with highly intensified DAB staining at the AGT penetration site in the resistant line compared to the susceptible line at 24 hpi. An enhanced DAB staining surrounding the AGT penetration site revealed greater H2O2 production, which was proposed to be acting as a strong signal for the host defense gene activation in CWAs areas of powdery mildew infected barley (Hu¨ckelhoven et al. 1999). Our gene expression data together with cytological data led us to hypothesize that high activation of defense-related genes at 24 hpi, but not 6 hpi, is crucial for host defense against Bgt infection. However, it is not clear whether H2O2 generation regulates the expression of methyl unit biosynthesis genes at the Bgt interaction site or not. Localized H2O2 generation also causes reinforcement of the cell wall through oxidative cross-linking because H2O2 is essential for the formation of lignin polymer precursors (Mellersh et al. 2002). In this study, the coincidence of DAB staining with autofluorescence response in penetration sites indicates that H2O2 accumulation in CWA area is associated with lignin or phenolic compound accumulation. Localized H2O2 generation was shown to act as a signal to trigger the phenolic compound accumulation in CWA that leads to fungal penetration resistant in cowpea (Mellersh et al. 2002). CWAs or papillae are thought to block penetration of pathogens if they are deposited rapidly and contain antifungal phenolic substances in sufficient quantities (Bushnell 2002). Using mlo-barley (resistant to barley powdery mildew penetration), Von Ro¨penack et al. (1998) demonstrated that, unlike normal cell walls, mature CWA areas cannot be digested with cellulases, and retain their autofluorescence after cellulase treatment. Ride and Barber (1987) found that, in penetration-resistant wheat, removing lignin from the CWA areas also removed autofluorescence and allowed cellulose digestion. Thus, cellulose fibers in CWA regions are tightly bonded to phenolics, making them impervious to cellulase enzymes and presumably very difficult for Bgt to penetrate. Therefore, it can be hypothesized that high lignin or phenolic substance accumulation in the CWA inhibits the penetration of a germ tube resulting in disease resistant in the resistant wheat line. An active transcription of the genes involved in the pathways of generation of methyl units is required for an elevation of SAM, which, in turn, is used in the methylation process to achieve lignin or phenolic substance accumulation in CWA areas because SAM is the methyl donor for specific steps in the biosynthesis of those metabolites (Lewis and Yamamoto 1990; Campbell and Sederoff 1996).

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It has been reported that in cultured parsley and alfalfa cells, the expression of SAMS, the key gene of the methyl cycle, was up-regulated by treatment with fungal elicitor or yeast cell wall, respectively (Kawalleck et al. 1992; Gowri et al. 1991). In the present study, gel blot analysis showed that both TmSAMS1 and TmSAMS2 were induced upon Bgt infection; however, semi-quantitative RT-PCR demonstrated that TmSAMS1 has a more abundant transcript than does TmSAMS2; thus, TmSAMS1 probably accounts for the majority of SAMS activity. Semi-quantitative RT-PCR analysis also showed that induction of TmSAMS1 was specific to the epidermis while TmSAMS2 was specific to mesophyll after Bgt infection. Comparison of the TmSAMS1 and TmSAMS2 sequences shows that the coding regions are highly conserved; 88.2% of nucleotide sequences and 96.8% of the deduced amino acid sequences are identical, which is similar to the SAMS gene in Arabidopsis, sam1 and sam2; (88.7 and 96.7%, respectively, Peleman et al. 1989). Peleman et al. (1989), using 3¢ UTR region specific probes, showed that Arabidopsis had a similar expression pattern in different organs, which might be explained by the three highly conserved sequences in the promoter regions of the two genes. Neither TmSAMS1 nor TmSAMS2 contained extended promoter sequences. Therefore, we do not know whether or not the similar expression patterns were attributable to the same conserved sequences in the promoter region in wheat and Arabidopsis. Kawalleck et al. (1992) reported that in a parsley cell culture, transcription of the SAHH gene was highly activated after application of fungal elicitor, a result that is very similar to the present finding where TmSAHH was induced highly after Bgt infection. SAM acts as a methyl donor in many transmethylation reactions and also acts as an intermediate compound for polyamine, ethylene, nicotianamine and biotin biosynthesis. Thus, high levels of SAM synthesis and of activated methyl turnover are required for these reactions to proceed at elevated rates. The basal level of TmSAMS and TmSAHH gene expression may not be sufficient to achieve these higher rates in response to pathogen infection or other stresses. We found concomitant expression of TmSAMDC, TmCOMT and TmACCO with genes involved in the pathways of generation of methyl units, suggesting that the pathways of generation of methyl units is linked to the polyamine, lignin and ethylene pathways and plays an important role in host defense response against Bgt infection. In contrast, constitutive expression of TmPEAMT and down regulation of TmNAS suggests that glycine betaine and nicotianamine, respectively, are not, as might be expected, linked to Bgt infection in wheat. Similar expression patterns of SAMS and COMT were previously reported in alfalfa cell cultures where fungal

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elicitor was used (Gowri et al. 1991). Cowley and Walters (2002) showed that infection of barley with Blumeria graminis f. sp. hordei led to increased concentrations of putrescine, spermidine and spermine in infected leaves and that these changes were accompanied by increased activities of the biosynthetic enzymes, ornithine decarboxylase (ODC), arginine decarboxylase (ADC), and SAMDC. An increased production of ethylene and upregulation of ACCO by fungal pathogen infection was described in tobacco (Chen et al. 2003). Similar upregulation of TmACCO was also found by us. Although there are no data available on the flux of SAM or lignin or polyamine or ethylene and the levels of corresponding enzymes, it may be that highly expressed genes involved in the pathways of generation of methyl units supply enough SAM to achieve enhanced lignin, polyamine and ethylene levels by the up-regulated TmCOMT, TmSAMDC and TmACCO, respectively, after Bgt infection. How much SAM is distributed to the lignin, polyamine and ethylene pathways, respectively, after Bgt attack also remains to be determined. In this study, TmMTHFR, TmMetSyn, TmSAMS1, TmSAMS2, and TmSAHH were also up regulated by various abiotic stresses. In addition to pathogen infection, salinity, drought, low temperature, and mechanical damage have been reported to induce gene expression in specific or cross-talk patterns (Cheong et al. 2002). The key gene of the methyl cycle, TmSAMS1, is highly expressed in response to all abiotic stresses applied in this study. Some plants often synthesize glycine betaine (Waditee et al. 2005), or polyamine (Cona et al. 2006) as ‘‘stress metabolites’’ to potentially mitigate unfavorable conditions. Similar expression pattern of AnSAMS1 and AnPEAMT were reported in Atriplex under salinity stress (Tabuchi et al. 2005). Enhanced production of PEAMT is known to increase glycine betaine production (McNeil et al. 2001). In our study, TmPEAMT was highly induced in a similar expression pattern to TmSAMS1 in response to NaCl, drought and cold stresses, suggesting that up-regulated transcription of genes involved in the pathways of generation of methyl units may supply methyl units to the glycine betaine pathway to mitigate environmental stresses. Transcripts of TmSAMS2 and TmSAMDC were highly accumulated in response to drought stress, indicating a possible link between these two genes for the supply of methyl units towards polyamine synthesis under this specific stress. Upregulation of both TmSAMS and TmCOMT occurred in response to four types of abiotic stresses, suggesting the involvement of an activated methyl cycle in the regulation of methyl units for lignin synthesis under abiotic stresses. Also in this study, transcripts of TmTHFD/C and TmSHMT showed either constitutive or down-regulated expression in response to biotic and abiotic stresses. Moreno et al. (2004)

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reported that the level of SHMT did not show any variation in a dicot plant, Arabidopsis, under different pathogens challenges. Therefore, we suggest that transcriptional regulations of TmTHFD/C and TmSHMT are not directly involved in response to these stresses. In this study, the elevated expressions of pathogen-induced genes involved in the pathways of generation and supply of methyl units by biotic and abiotic stresses suggest that cross-talk might exist between responses to Bgt attack and reactions to abiotic stresses. In conclusion, upregulation of the transcription of genes involved in the pathways of biosynthesis and supply of methyl units by biotic and abiotic stresses reveals that generation of sufficient methyl units is crucial and prerequisite to challenge by multiple stresses. However, further investigation is required using approaches such as reverse genetics to verify this hypothesis; these investigations are underway in our laboratory. In addition, present findings give rise to the following question: which upstream pathway of C-1 metabolism is activated to supply adequate C-1 for the pathway of biosynthesis of methyl units under various stresses? An elevated level of THF or serine by the activation of their respective biosynthetic pathways might provide the required C-1 for the methyl cycle to form enough methyl units. Serine synthesis through GDC/SHMT is the most likely route as it is the major source for C-1 units in plants (Prabhu et al. 1996; Li et al. 2003). Although transcript of TmSHMT was not accumulated in this investigation, the protein level may be activated under stresses. It is well known that changes in transcript levels do not necessarily reflect changes in protein concentration or activity within the cell. Further elucidation of this question is also now underway in our laboratory. Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada in the form of a Discovery Grant to JK. We thank R. Hirji for technical assistance, and the DNA technology unit of NRC-PBI Saskatoon for sequencing and oligonucleotide synthesis.

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