Transcriptional regulation of the cinnamyl alcohol dehydrogenase gene from sweetpotato in response to plant developmental stage and environmental stress
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Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in the biosynthesis of lignin. We have isolated full length of a cDNA encoding CAD (IbCAD1) that was previously identified as the most abundant gene in an EST library of sweetpotato suspension cells. Phylogenetic analysis revealed that IbCAD1 belongs to the family of defense-related CADs. High levels of IbCAD1 mRNA were found in the roots of sweetpotato, but not in the leaves and petioles. The IbCAD1 gene transcripts were highly induced by cold, wounding, and reactive oxygen species. Analyses of transcriptional regulation of the IbCAD1 gene in transgenic tobacco plants carrying the IbCAD1 promoter–GUS revealed that IbCAD1 promoter expression was strong in the roots, but barely detectable in the cotyledons. IbCAD1 promoter activity increased with increasing root age, and strong promoter expression was observed in the lateral root emergence sites and in root tips. Weak GUS expression was observed in lignified tissues of vascular system of mature leaves and stems. IbCAD1 promoter activity was strongly induced in response to the biotic and abiotic stresses, with the strongest inducer being wounding, and was also induced by salicylic acid (SA) and jasmonic acid (JA) as well as by abscisic acid (ABA) and 6-benzylaminopurine. Taken together, our data suggest that IbCAD1 can be involved in JA- and SA-mediated wounding response and ABA-mediated cold response, respectively. The IbCAD1 gene may play a role in the resistance mechanism to biotic and abiotic stresses as well as in tissue-specific developmental lignification.
KeywordsCinnamyl alcohol dehydrogenase Promoter activity Biotic and abiotic stresses Hormone response Lignification
Lignin is the second most abundant biopolymers in nature after cellulose. It forms an integral part of the secondary cell walls of specialized conducting and supporting tissues of plants, facilitating water transport and providing mechanical strength, respectively (Boudet 2000). The lignification of tissues is also thought to play a role as a defense barrier to limit pathogen invasion (Boerjan et al. 2003; Goujon et al. 2003). The biosynthesis of lignin proceeds via the general phenylpropanoid pathway, which generates a pool of hydroxycinnamyl-CoA esters. These hydroxycinnamyl-CoA esters are then channeled into the lignin-specific pathway to produce monolignols through the action of two enzymes, cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD). CCR catalyzes the conversion of hydroxycinnamyl-CoA esters to their corresponding cinnamaldehydes, and these are subsequently converted to their corresponding alcohols (p-coumaryl, coniferyl, and sinapyl alcohol) by CAD (Walter et al. 1988; Goujon et al. 2003).
In angiosperm species, CAD is encoded by a multigene family. Nine CAD genes (AtCAD1 to AtCAD9) have been identified in Arabidopsis and classified into three groups based on both homology and biochemical properties in vitro (Sibout et al. 2003; Kim et al. 2004, 2007). The first group consists of AtCAD4 (AY302081) and AtCAD5 (AY302082). These genes are associated with the highest CAD enzyme activity and homology (74–83% similarity) with respect to the bona fide CADs, such as Eucalyptus EgCAD2 and rice OsCAD2, and act as primary genes in lignin biosynthesis in Arabidopsis. EgCAD2 is the best characterized CAD involved in developmental lignin synthesis (Goffner et al. 1992). The second group of Arabidopsis CADs consists of AtCAD2 (AY302077), AtCAD3 (AY302078), AtCAD7 (AY302079) and AtCAD8 (AY302080). These all have a relatively lower CAD catalytic activity. AtCAD7 (AY302079) and AtCAD8 (AY302080) show the highest homology (~78%) to poplar sinapyl alcohol dehydrogenase (PtSAD) (Li et al. 2001). PtSAD has been shown to be highly specific for sinapaldehyde substrates and is possibly responsible for syringyl unit deposition in poplar. AtCAD7 and AtCAD8 are induced in response to pathogen infection (Kiedrowski et al. 1992). The members of the last group, AtCAD1 (AY288079), AtCAD6 (AY302075) and AtCAD9 (AY302076), lack detectable CAD catalytic activities in vitro, but they are expressed predominantly in vascular (lignin deposition) tissues. The expression of AtCAD1 was found to be induced by bacterial pathogen (Tronchet et al. 2010). The true biochemical roles of these CAD enzymes still need to be elucidated, particularly so as differences in the lignin composition of tissues in different plant species result from the different activities of several CAD isoenzymes, each with a different specificity.
Detailed knowledge of the role of CADs in relation to the biosynthesis of developmental lignins would contribute extensively to our understanding of the in vivo enzyme activity of CADs and subsequent lignin composition in tissues. In comparison to wild-type Arabidopsis plants, the content of lignin in the stem of double mutant Atcad4/Atcad5 plants is reduced by 60% and the level of coniferyl and sinapyl alcohol is reduced by 94% (Sibout et al. 2003, 2005). The rice gold hull and internode2 (gh2) mutant, which results from a mutation in the OsCAD2 gene, has reduced levels of p-hydroxyphenol, guaiacyl and syringyl monomers—in almost the same ratio—compared to the wild type (Zhang et al. 2006). Transgenic plants having reduced levels of lignins are more readily attacked by fungi and bacteria, as shown by double mutant Atcad4/Atcad5 plants, whose disease symptoms are distinctly more severe than those of the wild type (Tronchet et al. 2010). The relationship between CAD and biotic stress has been well-established. The mRNA level of alfafa MsaCADs and ryegrass LpCADs was observed to increase following wounding treatments (Brill et al. 1999; Lynch et al. 2002), and the addition of fungal elicitor to bean cell cultures leads to the rapid accumulation of CAD transcripts (Walter et al. 1988). In addition, syringyl lignin was found to be accumulated during the hypersensitive resistance response in wheat (Menden et al. 2007). All of these results indicate that the amount and unit composition of lignin is influenced by both developmental and environmental conditions. However, little information is yet available on the role of CAD in relation to abiotic stress.
We have isolated a cDNA (IbCAD1) encoding CAD and the upstream region of the IbCAD1 gene from sweetpotato and have investigated this IbCAD1 gene transcript and the activity of its promoter in response to various development stages. To gain an understanding of the regulation mechanism of IbCAD1 gene by biotic and abiotic stresses, we subjected wild type and transgenic plants to various stresses and then monitored promoter activity and tissue-specific expression. The effect of various plant hormones on IbCAD1 promoter activity was also examined. We report here the results of these analyses and suggest that the IbCAD1 gene may be involved in lignification induced by both abiotic and biotic stresses and in tissue-specific developmental lignification.
Materials and methods
Sweetpotato plants (Ipomoea batatas cv. Yulmi) and transgenic tobacco plants were maintained in pots in a greenhouse at 25°C under a 16/8-h light/dark photoperiod. They were propagated from cuttings taken from the top growing tip and grown for 3 weeks for the stress treatments. For the Agrobacterium-mediated transformation procedure, tobacco (Nicotiana tabacum cv. Xanthi) seeds were surface sterilized and sown on MS medium (Murashige and Skoog 1962) containing 3% sucrose and 0.8% agar.
Southern blot analysis
Whole cells of sweetpotato cultured in cell suspension were frozen and ground to a fine powder under liquid nitrogen, and genomic DNA was isolated according to the manufacturer’s instructions (QIAGEN, Germany). The genomic DNA (10 μg) was then digested with EcoRI and HindIII independently, followed by electrophoretic separation in a 0.8% agarose gel. These restriction enzymes do not have any recognition sequences within the IbCAD1 cDNA sequence. After a complete denaturation and subsequent renaturation, the gel was blotted onto a positively charged nylon membrane (Hybond-N+, Amersham Biosciences, UK). Biotin (biotin-14-dCTP, Invitrogen, USA) was used as a probe and labeled by PCR amplification using IbCAD1 cDNA as a template. The PCR analysis was performed in a 20 μl volume containing 1.25 U ExTaq DNA polymerase (Takara, Japan), 2 μl of 10× Taq buffer, 4 μl of 5× dNTPs mix (0.25 mM biotin-14-dCTP, 0.25 mM dCTP, 0.5 mM dATP, 0.5 mM dGTP and 0.5 mM dTTP), and 10 pmol of T3 and T7 primers. The PCR cycling conditions consisted of 94°C for 5 min, 30 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min at 72°C, with a final extension step of 7 min at 72°C. The labeled probe was purified using a PCR Purification kit (QIAGEN). The membrane was hybridized at 65°C for 16 h and then washed twice with 2× SSC/1% SDS at room temperature for 10 min, twice with 0.1× SSC/1% SDS at 65°C for 20 min, and twice with 1× SSC at room temperature for 10 min. Hybridized signals were detected using the Southern-Star™ System (Tropix, USA).
Reverse transcription-PCR analysis
Semi-quantitative reverse transcription (RT)-PCR was performed to analyze the expression pattern of IbCAD1. Total RNA (2 μg) was utilized for the synthesis of first strand cDNA, and the RT was performed using oligo (dT)20 (Invitrogen, USA) as a primer and Superscript™ II reverse transcriptase (Invitrogen). Aliquots consisting of one-tenth of the RT product were used as a template for each of the subsequent PCR amplifications. To amplify the IbCAD1, we designed PCR primers for gene-specific amplification in the 5′- and 3′-untranslated region (UTR) of the IbCAD1 cDNA. Sequences of the forward (F) and reverse (R) primers used to amplify cDNA were: F, 5′-ATCTTGATTGTCTCAATCTA3′, R, 5′-GGACATTATTACATTACAC-3′. Tubulin was used as the internal control (F, 5′-CAACTACCAGCCACCAACTGT-3′; R, 5′-CAAGATCCTCACGAGCTTCAC-3′). PCR amplification was carried out in a 20 μl reaction mixture containing 0.2 mM of each dNTPs, 1 pmol of each forward and reverse primers, and 0.125 U of Taq polymerase (NEB, USA), including cDNA products as a template. After an initial denaturation at 94°C for 5 min, amplification was carried out at 94°C for 30 s, 60°C for 1 min, 72°C for 1 min, with a final incubation for 7 min at 72°C. There were 30 and 20 PCR cycles for IbCAD1 and tubulin, respectively. The amplified PCR products were separated in a 1% agarose gel.
Construction of genomic library and isolation of the IbCAD1 promoter
The 5′-flanking region of IbCAD1 was isolated from the genomic DNA of sweetpotato cells grown in suspension culture following the instructions of the Universal GenomeWalker kit (Clontech, USA), which is a simple method for cloning unknown genomic sequences adjacent to a DNA fragment of known sequence. The first step consisted of constructing adaptor-ligated sweetpotato genomic DNA libraries. This was accomplished by completely digesting separated aliquots of sweetpotato genomic DNA with different restriction enzymes, namely, NaeI, NruI, HpaI, and ScaI. Each batch of digested product was then ligated separately to the GenomeWalker Adaptor. In the primary PCR, the four adaptor-ligated genomic libraries were used as a template. The PCR was performed in 50 μl aliquots, each containing 1 μl of each template, 5 μl of 10× PCR reaction buffer, 4 μl of dNTPs (2.5 mM each), 1 μl of outer adaptor primer (AP1, 5′-GTAATACGACTCACTATAGGGC-3′), 1 μl of gene-specific primer (GSP1, 5′-AAAGGAGAAAGAACTCCAGAGGTGTCC-3′), and 0.5 μl of ExTaq polymerase (Takara, Japan). The PCR cycling conditions consisted of seven cycles at 94°C for 25 s and 72°C for 3 min; 32 cycles of 94°C for 25 s and 67°C for 3 min; one final cycle of 67°C for 7 min. The primary PCR products were then diluted and used as a template in a second PCR using a nested adaptor primer (AP2, 5′-ACTATAGGGCACGCGTGGT-3′) and a nested gene-specific primer (GSP2, 5′-CCACAGGCCTTCACTGGGTGCTCGTTT-3′). After the amplification step, the major PCR product was isolated and cloned into pGEM-T Easy vector (Promega, USA) for sequencing.
Construction of the IbCAD1 promoter–GUS fusion and plant transformation
To generate a binary vector for the transformation of tobacco plants, the IbCAD1 promoter region was amplified with gene-specific primers (F, 5′-ACGCGTCGACCTGGTAACAAAACTATTGGAT-3′; R, 5′-CGGGATCCTTTCCTTGTTGCAGGGGGAT-3′). The reverse primers were designed in front of the ATG start codon of the IbCAD1 gene. The underlined sequences, which are SalI and BamHI sites, were introduced at the end of primers in order to facilitate subcloning. The PCR product was then inserted into the SalI/BamHI sites of pBI101 carrying the GUS reporter gene, and the resulting construct was utilized to transform tobacco by Agrobacterium tumefaciens (GV3101) infection (Martinez-Trujillo et al. 2004). The transformants were identified by genomic DNA PCR. Primary transformants were grown to maturity in soil in a greenhouse to produce seeds. The seeds were germinated on MS medium after sterilization in distilled water:chlorox:0.5% Tween 20 (3:2:1). The IbCAD1 promoter activity in transgenic tobacco was analyzed using T2 plants.
Stress and hormone treatments
Sweetpotato plants were grown in a greenhouse at 25°C for 3 weeks and subjected to several stresses. As cold stress, sweetpotato plants in a pot were exposed to 15°C for 24, 48, and 72 h, respectively. For the methyl viologen (MV) treatment, 50 μM of a solution containing 0.125% Triton X-100 was sprayed onto whole plants and the plants incubated for 6, 12, and 24 h, respectively. For the H2O2 treatment, the second and third leaves from the top were removed from plants and incubated in 440 mM hydrogen peroxide (H2O2) solution at 25°C for 12 and 24 h, respectively; sterile water was used as a control for the H2O2 treatment. Wounding stress was performed according to Sasaki et al. (2002). Fully expanded leaves were detached from the plant and immediately cut into pieces with a razor blade. After the midrib had been removed, six pieces from each leaf, from six different leaves (n = 36 leaf pieces), were placed on filter paper moistened with distilled water and incubated for 1, 4, 8, 12, and 24 h, respectively, at 25°C under continuous illumination. Untreated and stress-treated plant samples were collected, frozen in liquid nitrogen, and stored at −70°C for RNA purification.
Four-day-old seedlings were used for the stress treatments of transgenic tobacco plants containing the IbCAD1 promoter. For the cold stress, 4-day-old seedlings in MS medium were exposed to 4°C for 48 h. For the H2O2 stress, transgenic seedlings were transferred to MS medium containing 0.1 M H2O2 at 25°C for 24 h. For the wounding treatment, detached leaves and stems of mature plants were cut into slices and incubated for 24 h at 25°C. For the hormone treatments, 4-day-old tobacco seedlings were transferred to MS medium containing 100 μM abscisic acid (ABA), 1 mM salicylic acid (SA), or 50 μM jasmonic acid (JA) for 24 h or to MS medium containing 10 μM 6-benzylaminopurine (BA) for 72 h. Untreated and hormone-treated plant samples were collected, and crude extracts were prepared for the analysis of GUS activity.
Fluorometric and histochemical GUS assay
The crude plant extract was prepared and used for the quantitative fluorometric assays of GUS activity according to Jefferson et al. (1987) using 4-methylumbelliferyl glucuronide (MUG; Sigma, USA). GUS values were expressed as nmoles of MUG per minute per milligram protein. Protein concentrations were determined by the method of Bradford (1976). The histochemical analysis of GUS activity was performed by incubating plant tissues with 1 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-Gluc, Gold Biotechnology, USA) for 16–24 h at 37°C (Jefferson et al. 1987). Chlorophyll was removed from the tissues by immersion in 70% ethanol. The samples were observed and photographed under a SZ61 zoom stereo microscope (Olympus, Japan) equipped with digital camera system (Canon, USA).
Isolation and sequence analysis of IbCAD1 cDNA
In an earlier study, we constructed an expressed sequence tag (EST) library from sweetpotato cells grown in suspension culture and identified highly abundant and cell culture-specific genes, of which the most abundant and unique gene was CAD (Kim et al. 2006). Here, we have isolated and characterized the full length of the cDNA clone (IbCAD1, GU380306). The IbCAD1 cDNA consisted of 1,355 nucleotides, and the largest open reading frame (ORF) encoded a polypeptide of 357 amino acids. It encoded a 38.5-kDa protein with an isoelectric point of 6.26. The putative polyadenylation signal AATAAT was located 94 bases upstream from the start of the poly(A)+ tail. We found no clear targeting domains within the sequence, suggesting a cytoplasmic location of the protein (WoLF PSORT Prediction; probability cytoplasm 12.0, chloroplast 1.0).
Effects of environmental stresses on the transcription levels of the IbCAD1 gene
Nucleotide sequence analysis of the IbCAD1 promoter
To isolate a 5′ flanking region of the IbCAD1 gene, we designed specific primers in the ORF region of the IbCAD1 cDNA and performed PCR-based genome walking. A 1,260-bp product was obtained, and the nucleotide sequence of the product was compared with that of the IbCAD1 cDNA. This PCR product encoded 16 amino acids, including an ATG start codon, and corresponded exactly with the 5′-UTR and N-terminal region of the IbCAD1 cDNA. The nucleotide site that corresponded in sequence to the end of 5′-UTR of the IbCAD1 cDNA was designated as +1. A putative TATA box (−65 to −58) and CAAT box (−1043 to −1040, −831 to −828, −138 to −135, and −27 to −24), which act as general signals for eukaryotic gene expression, were observed in IbCAD1 promoter.
Spatial and temporal regulation of the IbCAD1 gene during development
IbCAD1 promoter activity was investigated in the leaves, petioles, stems, and roots of mature plants grown in soil. It was relatively low in 4-week-old mature plants, except for the roots. GUS activity increased only slightly in the leaves, petioles, and stems with increasing plant age. In contrast, there was a dramatic increase in GUS activity in the roots as the plant matured (Fig. 5A), with GUS activity in the roots of 8-week-old plant roots being 2.9- to 6.8-fold higher than that of 4-week-old plant roots. The lateral root initiation site was strongly stained in 4-week-old plants (Fig. 5Bf–g), while much stronger staining was observed in lateral roots and primary roots of 8-week-old plants (Fig. 5Bh). Weak GUS staining was observed in the fibers, phloem, and xylem of the stem and in the midrib and veins of leaves (Fig. 5Bi–k). In the reproductive tissues, the anther, trichomes in the filament, and pollen grains all showed strong GUS staining, while the stigma showed weak GUS staining (Fig. 5Bl–o). Based on these results, the IbCAD1 promoter directs strong expression in the meristematic tissue of the root, but weak expression in the lignified tissue of the leaves and stem.
Up-regulation of IbCAD1 gene expression by both biotic and abiotic stresses
To provoke a wounding response, we wounded detached leaves and stems from 6-week-old mature plants grown in soil by punching or cutting and then incubated the leaf pieces on moistened filter paper. Relative to intact tissues, the GUS activity directed by the IbCAD1 promoter was dramatically increased (15.2- to 174.1-fold) in wounded tissues (Fig. 6C). As shown in Fig. 6Ca, the wounding response was highly localized. Strong GUS staining was also observed at wounded site in the stem, and a histochemical study of cross-sections of the stem revealed that the IbCAD1 promoter was strongly expressed in the xylem, phloem, cortex, and epidermis as well as in the trichomes (Fig. 6Cb, c). Taken together, these results show that the IbCAD1 promoter was highly activated by both biotic and abiotic stresses and that the stress response was tissue-specific.
Effects of hormones on the IbCAD1 promoter activity
Cinnamyl alcohol dehydrogenase (EC 188.8.131.52) is the last enzyme in the lignin biosynthetic pathway, and it mediates the reduction of coniferaldehyde, p-coumaraldehyde, and sinapaldehyde into guaiacyl, p-coumaryl, and syringyl monolignols, respectively. Lignification is a tightly regulated and dynamic process subject to modulation at different levels during normal development and in response to different stresses (Boudet 2000). In this study, we isolated the CAD cDNA (IbCAD1) from sweetpotato, which can be phylogenetically categorized as belonging to the family of defense-related CADs. The Gly 299 residue in IbCAD1 is a key determinant of substrate specificity and is a sinapaldehyde-specific residue according to Bomati and Noel (2005). These researchers reported that the Gly residue at the base of the active site of PtSAD and FxaCAD is responsible for the high specificity of these proteins for the substrate sinapaldehyde. PtSAD is required for the biosynthesis of syringyl lignin (Li et al. 2001). Classical CAD enzymes involved in developmental lignification, such as AtCAD4, AtCAD5, and EgCAD2, have Phe instead of Gly in the active site for substrate specificity. AtCAD5 is able to use all substrates effectively, while AtCAD4 shows a very poor affinity for sinapaldehyde (Kim et al. 2004). Thus, IbCAD1 may have high affinity for the substrate sinapaldehyde based on its structure and, consequently, be involved in the defense-related process.
Angiosperm CAD is encoded by a multigenic family consisting of members thought to have distinct roles, while gymnosperm CAD is encoded by a single gene (MacKay et al. 1995; Zinser et al. 1998). Different CAD genes in angiosperms may be expressed in different tissues or at different stages during growth and development (Kim et al. 2007; Barakat et al. 2009) in response to the type of lignin required. Several authors have suggested that the chemical composition of defense lignin is different to that of developmental lignin. For example, syringyl lignin is accumulated in the plant cell wall during the hypersensitive resistance response in wheat (Menden et al. 2007) and sinapaldehyde-specific CAD activity is activated in F. oxysporum-treated flax cells (Hano et al. 2006). While much is known on how CAD is induced by biotic stress, such as fungal elicitor (Dixon and Paiva 1995; Brill et al. 1999), relatively little information is available on the role of CAD in relation to lignification under conditions of abiotic stress. We have investigated the transcriptional regulation of the IbCAD1 gene in response to various stresses. RT-PCR analysis shows that IbCAD1 gene expression in sweetpotato was strongly induced by both abiotic stress (cold) and biotic stress (wounding), including ROS (MV and H2O2). ROS is a common signal that acts as a trigger of the downstream stress response. Fujita et al. (2006) reported that ROS may mediate crosstalk between biotic and abiotic stress-responsive gene-expression networks. Our RT-PCR results are consistent with those of our promoter analysis of IbCAD1. The activity of the IbCAD1 promoter was induced by the cold and H2O2 treatments in this study, but the stress response was quite different in different tissues; for example, at the seedling stage, the cotyledons were more sensitive than the roots to IbCAD1 promoter induction. Among the stressors tested, wounding was found to be the strongest inducer of IbCAD1, and the wounding response was highly localized. These data suggest that the expression of the IbCAD1 gene is regulated in a tissue-specific manner under conditions of biotic and abiotic stresses.
Hormone signaling pathways regulated by ABA, SA, JA, and ethylene as well as the ROS signaling pathway all play key roles in the crosstalk between biotic and abiotic stress responses (Fujita et al. 2006). ABA is extensively involved in the plant’s response to abiotic stresses, such as drought, low temperature and osmotic stress, and also regulates a variety of growth and developmental processes. In contrast, SA and JA play central roles in biotic stress signaling following pathogen infection and wounding. We identified several hormone-responsive cis-regulatory elements in the IbCAD1 promoter region, such as ABRE (ABA) (Yamaguchi-Shinozaki and Shinozaki 2005), ARR binding site (cytokinin) (Yokoyama et al. 2007), as-1 element (auxin, SA) (Niggeweg et al. 2000), MYB responsive element, MYC responsive element (Yamaguchi-Shinozaki and Shinozaki 2005), and GARE (gibberellin) (Sutoh and Yamaguchi 2003). AtMYC2, which was identified as an activator involved in the ABA-mediated drought stress signaling pathway, upregulates the expression of genes that are involved in the JA-mediated wounding response stress signaling pathway (Abe et al. 2003; Boter et al. 2004; Lorenzo et al. 2004). Transgenic plants that overexpress both AtMYC2 and AtMYB2 show a greater sensitivity to ABA and enhanced osmotic stress tolerance (Abe et al. 2003). When we tested the GUS activity driven by the IbCAD1 promoter, we found that IbCAD1 promoter activity was enhanced when the plants were treated with various plant hormones, being most strongly induced by JA and SA, respectively. The IbCAD1 promoter was also induced by ABA and cytokinin (BA), but to a lesser degree. Taken together, these results indicate that IbCAD1 may function through a JA- and SA-mediated wounding response and an ABA-mediated cold response.
The expression of the IbCAD1 gene was regulated during growth and development in a tissue-specific manner. Our histochemical analysis showed that weak GUS expression was observed in the xylem, phloem, and fiber of stems, and in the midrib and veins of mature leaves, which are lignified tissues of the plant vascular system. However, the IbCAD1 gene was strongly expressed in the area of the initiation sites for lateral roots developing from primary roots as well as in root tips—but not in the vascular cylinder of lignified root tissue. It has been reported that there is no lignification in root meristem cells, which is primarily involved in the constant progression of cell division (de Obeso et al. 2003). GUS expression was also strong in anther and pollen. We have previously shown that IbCAD1 is the most abundant gene in the EST library of cells grown in suspension culture and that it is highly expressed at the actively dividing-log phase. In light of these results, the expression of the IbCAD1 promoter indicates a new role of CAD protein in lateral root formation, cell-wall synthesis, and the promotion of cell division that is unrelated to constitutive lignin formation. We suggest that IbCAD1 may also be involved in lignifications induced by both abiotic and abiotic stresses and in tissue-specific developmental lignification. Further analysis of the IbCAD1 gene will provide an insight into the signaling pathway involved in the regulation of biotic and abiotic stresses in lignin biosynthesis as well as into the role of CAD protein during cell division.
This work was supported by grants (No. 20080401034022 and 20100301061032) from Biogreen 21 Program funded by the Rural Development Administration, Republic of Korea, and a grant from the 2006 Inje University research grant.
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