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First published online September 20, 2007; 10.1104/pp.107.102657 Plant Physiology 145:863-874 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
A Nonclassical Arabinogalactan Protein Gene Highly Expressed in Vascular Tissues, AGP31, Is Transcriptionally Repressed by Methyl Jasmonic Acid in Arabidopsis1,[OA]Section of Molecular, Cell, and Developmental Biology, University of Texas, Austin, Texas 78712
In response to wounding and pathogens, jasmonate (JA) serves as a signal molecule for both induction and repression of gene expression. To examine defense-regulated gene repression in Arabidopsis (Arabidopsis thaliana), we have identified a nonclassical arabinogalactan protein (AGP) gene, AGP31, and show that its mRNA decreased to about 30% of its original level within 8 h in response to methyl JA (MeJA) treatment of whole 7-d-old seedlings. Wounding and abscisic acid treatment had similar effects. MeJA suppression primarily depends on the action of the JA-signaling protein, COI1, as shown by much lower MeJA suppression in coi1-1 mutant plants. The main mechanism of mRNA suppression by MeJA is repression of transcription, as shown by nuclear run-on experiments. The AGP31 protein shares features with several known and putative nonclassical AGPs from other species: a putative signal peptide, a histidine-rich region near the N terminus followed by a repetitive proline-rich domain, and a cysteine-rich C-terminal PAC (for proline-rich protein and AGP, containing cysteine) domain. Positive Yariv reagent interaction demonstrated that the protein is an AGP. Monosaccharide analysis of purified AGP31 indicated it is a galactose-rich AGP. Expression of an AGP31-enhanced green fluorescent protein fusion protein in transgenic cells revealed that the AGP31 protein was localized to the cell wall. AGP31 promoter-  -glucuronidase reporter gene analysis showed expression in the vascular bundle throughout the plant, except in the flower. In the flower, -glucuronidase staining occurred throughout the pistil, except in the stigma. The strong preferential expression in vascular tissues suggests that AGP31 may be involved in vascular tissue function during both the defense response and development.
Plants respond to insect attack, mechanical damage, and pathogen infection by regulating specific and overlapping sets of genes to provide wound-healing and protective functions. These responses widely involve both gene activation and repression. Both of these processes appear to be important in coordinating the antagonistic and synergistic interplay of different signaling pathways and downstream effectors to address biotic stress (Lorenzo et al., 2003  ). For example, defense-induced salicylic acid promotes expression of various defense genes, such as pathogenesis-related proteins, while repressing the expression of genes under the control of the jasmonate (JA)-signaling pathway (Li et al., 2004). Recently, a bacterial elicitor has been shown to mediate repression of auxin signaling and enhance disease resistance through induced microRNA accumulation in Arabidopsis (Arabidopsis thaliana; Navarro et al., 2006).
Positive and negative regulation of gene expression, along with diverse nongenomic processes, contribute to the dynamic remodeling of the plant cell wall during responses to herbivory, wounding, and infection. Changes in the wall often play a central role in the defense outcome (Schulze-Lefert, 2004
In a number of cases, changes in HRGP and PRP gene expression have been found to correlate with the presumed function or lack of function in protection during the plant defense response. Accordingly, specific HRGP and PRP mRNAs whose encoded proteins perform postulated or demonstrated adaptive roles are induced by defense-related stimuli (Bradley et al., 1992
In this study, a low-Tyr PRP was identified in Arabidopsis, whose mRNA was down-regulated by various defense-related stimuli: wounding, MeJA, and abscisic acid (ABA) treatments. MeJA repression of the mRNA level was COI1 dependent and occurred at the level of transcription. This PRP is better classified as a nonclassical AGP on the basis of its hallmark reactivity with
AGP31 Encodes a Nonclassical AGP
A BLASTP search was conducted to identify Arabidopsis proteins with features and protein organization similar to the French bean PvPRP1 protein, including low Tyr content (generally <5%). The gene with the greatest similarity throughout the encoded protein length was At1g28290. Using reverse transcription-PCR, we obtained a cDNA clone spanning the complete open reading frame of At1g28290. DNA sequencing confirmed the annotation of At1g28290 (http://www.arabidopsis.org). At1g28290 has one intron and a 208-nucleotide 3'-untranslated region (UTR) deduced from the cDNA sequence. At1g28290 encodes a 359-amino acid Pro-rich protein with four distinct domains: a putative signal peptide followed by a His-rich domain, a middle repetitive Pro-rich domain, and a C-terminal non-Pro-rich domain (Fig. 1A
). In the Pro-rich domain, there are only five types of amino acids, with 45.9% Pro, 18.2% Lys, 15.7% Val, 6.3% Ala, and 3.8% Tyr. These amino acids form different exact repeat units. There are two long repeats with 31 amino acids overlapping with four shorter 20-amino acid repeats (Fig. 1A). These repeats are essentially composed of different variants of the basic repeat unit PP(A/V/T)(K/Y). This degenerate quadripeptide is different from the typical pentapeptide PPVX(K/T) of other PRPs, such as several AtPRPs in Arabidopsis (Fowler et al., 1999
The protein sequence of At1g28290 suggested that it may encode a nonclassical AGP. Preceding the His-rich domain, there is a module APAPAP and there are six single AP duplets scattered in the PRP domain (Fig. 1A). Increasing evidence suggests that these AP modules are sites of carbohydrate attachment in AGPs (Tan et al., 2004 ). In addition to these putative O-glycosylation sites, there are two putative N-glycosylation sites in the PAC region (Fig. 1A). Based on these analyses, we postulated that this protein might be a nonclassical AGP.
Further supporting evidence was provided using the PAC domain sequence to BLAST the GenBank database. This identified several putative homologs among different species. Among these putative homologs, we searched for proteins that have similar structure: predicted signal sequence, N-terminal His-rich stretch, a Pro-rich region with the repeat PPXX, and a PAC domain. After applying these criteria, we identified several proteins, PvPRP1 (French bean), HyPRP1 (cotton [Gossypium hirsutum]), DcAGP1 (carrot [Daucus carota]), NaPRP4, NtTTS1, and NtTTS2 (tobacco), which we consider to be homologs of At1g28290 (Fig. 1B). Among these homologs, DcAGP1, NaPRP4, NtTTS1, and NtTTS2 have been shown to be bona fide nonclassical AGPs (Cheung et al., 1995
To determine whether At1g28290 is a bona fide AGP, we used the 35S promoter to overexpress the At1g28290 protein fused with a 6x-myc tag at the C-terminal end in transgenic plants. A cell wall fraction was isolated from 7-d-old seedlings. Proteins released by high salt extraction of this fraction were incubated with
To examine its carbohydrate composition and likely hydroxylation of Pro residues, we purified native AGP31 protein from transgenic plants overexpressing native AGP31. Transgenic lines were generated by introducing a genomic fragment that contains about 3 kb upstream of translation start codon and about 700 bp downstream of the translation stop codon of the AGP31 gene. Due to the nature of Agrobacterium transformation, multiple copies of fragments are frequently introduced into plants and can exhibit higher mRNA levels. Plants of a transgenic line that showed a higher mRNA level than wild type grown in liquid Murashige and Skoog medium were used to isolate the high-salt eluted cell wall protein. AGP31 was purified by a Ni-NTA metal affinity column from high-salt eluted cell wall protein and an aliquot subjected to trifluoromethanesulfonic (TFMS) acid hydrolysis, a treatment that removes the majority of carbohydrate in glycoproteins. The Coomassie Blue-stained gel showed that acid hydrolysis resulted in a dramatic shift from the broad high-molecular-mass band to a single prominent band of 45 kD in the deglycosylated sample (Fig. 1D). It is noteworthy that the innermost Asn-linked N-acetylglucosamine of an N-linked oligosaccharide is resistant to TFMS treatment (Edge, 2003
Amino acid composition analysis of purified, deglycosylated AGP31 showed approximately equal amounts of Pro and Hyp (Table I
). Another nonclassical AGP, NaPRP4, which is the homolog of AGP31 in Nicotiana alata, also has about equal amounts of Pro and Hyp (Sommer-Knudsen et al., 1996
AGP31 mRNA Is Repressed by Wounding, MeJA, and ABA To investigate AGP31 gene expression during the defense response, the leaves of 4-week-old plants were mechanically wounded and directly wounded leaves were harvested at various times. RNA-blot analysis shows a decrease in AGP31 mRNA level over the 8-h time course (Fig. 2A ). The 1.35-kb size of the AGP31 transcript matches the expected size based on the cDNA sequence. The GLUTATHIONE S-TRANSFERASE1 (GST1) transcript was also hybridized as a known wounding-induced mRNA, whereas the UBIQUITIN10 (UBQ10) transcript was monitored to show equal loading. Because JA and its derivatives are well-known secondary messengers induced by wounding, we also examined the effect of MeJA. MeJA treatment resulted in a decrease in AGP31 mRNA to about 30% of the original level by 8 h (Fig. 2B). After 12 h, the AGP31 mRNA level began to recover but remained below its original level through the 24-h time course. In contrast, control plants treated with the solvent exhibited only a slight decrease in AGP31 mRNA. The marked induction of LIPOXYGENASE2 (LOX2) mRNA, a known MeJA-regulated mRNA, demonstrated effective MeJA exposure. Control plants treated with solvent showed low-level induction of LOX2 mRNA, especially at early time points. However, compared with MeJA-treated plants, the response was much lower and diminished after 4 h.
ABA has been shown to be involved in some plant defense responses and is widely active in the response to water stress and other abiotic stresses. To determine the expression pattern of AGP31 mRNA in response to ABA, we treated seedlings with ABA and harvested after 8 h. Northern-blot analysis showed that AGP31 mRNA was also repressed by ABA treatment (Fig. 2C).
COI1 has been shown to play a pivotal role in JA signaling. Based on microarray data, it has been estimated that about one-half of JA signaling involves the action of this protein, which encodes an F-box protein as part of the E3 ubiquitin ligase complex (Devoto et al., 2005
Repression of AGP31 mRNA Occurs Primarily at the Transcriptional Level
In French bean, repression of PvPRP1 mRNA is due to mRNA stability regulation with no change in the rate of transcription (Zhang et al., 1993
To investigate whether mRNA stability regulation may also play a role, the AGP31 3'-UTR was inserted downstream of a LUC+ reporter coding sequence. Agrobacterium-mediated transformation generally results in the concatenated insertion of several T-DNA copies at a single locus. To avoid possible generation of double-stranded RNAs and RNA interference processes that could variously impact the steady-state mRNA levels, transgenic plants were screened to identify single-copy insertions by Southern blot as described by Forsbach et al. (2003) . Analysis of three lines of transgenic plants containing single-copy insertions showed that the LUC+ reporter mRNA level remained approximately constant in MeJA-treated plants, whereas the endogenous AGP31 and LOX2 mRNAs showed expected regulation (Fig. 5
). The results suggest that MeJA-induced down-regulation of AGP31 mRNA does not involve mRNA destabilization through the 3'-UTR sequence. Whereas mRNA stability regulation commonly operates through the 3'-UTR (Bevilacqua et al., 2003), the evidence does not rule out a role for another part of the transcript or the 3'-UTR plus another sequence in conferring MeJA-induced mRNA stability regulation. Together with the run-on data, our results support predominant regulation by repression of transcription.
AGP31 Is Localized in the Cell Wall
AGP31 protein is predicted to be a cell wall or secreted protein based on its putative signal sequence and past findings of these locations for numerous AGPs. To establish the subcellular localization of AGP31, enhanced GFP (eGFP) was fused to the C-terminal end of AGP31 to form an AGP31::eGFP fusion construct under the control of the 35S promoter. The constructs were stably expressed in Arabidopsis transgenic plants and also bombarded into onion (Allium cepa) epidermal cells to express the fusion protein transiently. Figure 6, A and B
, shows confocal microscope views of the same root section from stable transgenic Arabidopsis plants. The eGFP signal indicated that the fusion protein was localized to the cell periphery (Fig. 6, A and B). In bombarded onion epidermal cells, the eGFP-alone construct showed intracellular localization, including a commonly observed presence in the nuclei (Fig. 6, C and D). To distinguish between cell wall and cell membrane locations, the transiently transformed cells were treated with 0.8 M mannitol to induce plasmolysis. As shown in Figure 6E, mannitol treatment effectively contracted the plasma membrane from the cell periphery in a cell that expressed eGFP alone. The corresponding fluorescence image (Fig. 6F) showed that eGFP fluorescence was also contracted with the cytoplasm. In contrast, in a plasmolyzed transgenic cell expressing the AGP31-eGFP fusion protein, a significant portion of the fusion protein was found at the cell periphery (Fig. 6H), indicating cell wall localization. Some fusion protein was also seen more faintly inside the cell and at the plasma membrane, which would be consistent with intermediate locations of the protein during its biogenesis. The AGP31-myc fusion protein was also highly enriched in the high-salt elution fraction from partially purified cell walls (data not shown). In work from other researchers, AGP31 was also identified in a cell wall proteomics study (Feiz et al., 2006
AGP31 Is Expressed Primarily in the Vascular Bundle and in the Flower Toward understanding the biological function of this protein during defense and normal development, we characterized the AGP31 mRNA expression level in roots, leaves, stems, and inflorescences of Arabidopsis plants. RNA-blot analysis showed that AGP31 mRNA was expressed in all parts of the plant, with the roots and inflorescences having the highest expression levels (Fig. 7A ). To determine the expression pattern of the AGP31 gene more precisely, we made transgenic plants harboring the bacterial uidA gene (GUS) under the control of a 1.4-kb fragment extending upstream from the AGP31 start codon. This construct is termed the AGP31 promoter-GUS reporter construct (PAGP31::GUS). The GUS expression pattern based on histochemical staining for GUS activity revealed that the AGP31 promoter-GUS reporter gene was predominantly expressed in the vascular bundles throughout the plants (Fig. 7, B–F). In flowers, apart from staining vascular bundles in sepals and stamen filaments, GUS was also expressed throughout pistils, except for stigmas (Fig. 7C). In roots, GUS stain was predominant in vascular bundles without appearance in the root tip regions (Fig. 7, D and E). Cross section of the root showed GUS activity was broadly distributed through the vascular bundle, including the phloem and primary xylem (Fig. 7F).
Wounding and MeJA have been shown to induce dozens of genes while repressing an approximately equal number of other genes (Devoto et al., 2005 ). However, the mechanisms of gene repression are poorly understood. Defense-related remodeling of the plant cell wall involves activation of some cell wall protein genes, whereas others are repressed, providing an opportunity to understand molecular regulatory mechanisms that have evolved in the context of altering cell wall properties for improved disease resistance. In this study, we have identified a nonclassical AGP gene, AGP31, whose mRNA is down-regulated by MeJA, wounding, and ABA. We further showed that MeJA suppression is primarily due to transcriptional repression and the signaling pathway involves a central JA regulator, COI1. In addition, AGP31 expression during normal development showed a striking association with the vascular tissue and the pistil exclusive of the stigma.
As part of our interest in understanding gene repression during the defense response, we identified the At1g28290 gene in Arabidopsis as a homolog to the known French bean elicitor and wounding down-regulated PRP gene, PvPRP1 (Sheng et al., 1991
Although the functions of these domains remain to be clarified, there is some evidence for roles in diverse aspects of plant development and defense. The His-rich domain usually functions in metal binding in other proteins. For example, the His-rich domain of citrus dehydrin binds Cu2+ (Hara et al., 2005
It is worth noting that, although members of the nonclassical AGP subclass discussed in this article have similar overall structures, differences in their spatial expression and regulation suggest there may be different and common functions. For example, carrot DcAGP1 mRNA is unaffected by wounding (Baldwin et al., 2001
The physiological functions of nonclassical AGPs have remained more elusive than individual classical AGPs whose functions have begun to be uncovered by genetic analyses (Yang et al., 2007
A common feature among all AGPs is that they are highly glycosylated cell wall or secreted proteins in which the carbohydrate moiety usually accounts for about 90% of AGP mass. The carbohydrate exists predominantly as type II arabinogalactans and shows distinctive binding to a class of synthetic phenylazo dyes. Binding one member of this class of dyes,
Using the promoter-GUS fusion technique, we showed that expression of AGP31 was localized to vascular bundles throughout the plant, including phloem and primary xylem cells. AGP31 mRNA repression by MeJA and ABA correlates spatially with the vascular tissue being a major site for biosynthesis of both phytohormones (Stenzel et al., 2003
We conclude that down-regulation of AGP31 mRNA by MeJA is achieved by the combination of repression of transcription and a relatively short mRNA half-life (less than 4 h is suggested by the kinetics of mRNA loss). The mRNA half-life may be constitutively short or regulated from a longer half-life found in untreated cells. However, the finding that the AGP31 3'-UTR did not impart MeJA-induced instability to a LUC+ reporter transcript makes it more likely that mRNA stability regulation is not a major factor because a common region controlling mRNA stability is the 3'-UTR (Bevilacqua et al., 2003
Transcription factors mediating AGP31 transcriptional repression by MeJA are unknown. A number of Arabidopsis transcription factors have been identified that repress several JA-inducible genes, including ERF4 (McGrath et al., 2005
Plant Materials and Growth Conditions Arabidopsis (Arabidopsis thaliana) plants were grown on agar plates with 0.5x Murashige and Skoog salts, 1% Suc, and 0.8% phytoagar (Bio World) under continuous light at 20°C to 23°C. The wild-type plant is ecotype Columbia. The coi1-1 mutant was a gift from Dr. J.G. Turner (University of East Anglia). Arabidopsis cell culture was obtained from Dr. W.F. Thompson (North Carolina State University).
The cell wall fractions were isolated from 8-g 11-d seedlings according to Feiz et al. (2006)
AGPs were isolated from high-salt eluted cell wall proteins according to Schultz et al. (2000)
To isolate native AGP31, a transgenic plant overexpressing native AGP31 was generated by transforming plants with Agrobacterium carrying an EcoRI and HindIII genomic fragment from bacterial artificial chromosome F3H6 (obtained from The Arabidopsis Information Resource). This fragment contains 3,130 bp upstream of AGP31 translation start codon and 701 bp downstream of AGP31 translation stop codon, respectively. A transgenic plant overexpressing AGP31 was used to isolate native AGP31. Plants were grown in Murashige and Skoog liquid medium supplemented with 1% Suc and full-strength Murashige and Skoog vitamins (Czako et al., 1993
Isolated AGP31 was deglycosylated by the TFMS acid method using the Glyco-Profile IV kit (Sigma). Deglycosylated AGP31 and native AGP31 were separated by SDS-PAGE on 15% Tris-Gly gel.
Protein bands were excised from Coomassie Blue-stained gel and digested by trypsin. MALDI-TOF MS was carried out by the core facility service of the University of Texas at Austin to identify the protein bands.
Amino acid analysis was carried out by AAA Service Laboratory as described (Roach and Gehrke, 1970
Monosaccharide composition analysis was carried out by the Complex Carbohydrate Research Center as described (Merkle and Poppe, 1994
AGP (7.5 µg) from wild-type and transgenic plants was loaded on 4% to 15% gradient gel and blotted to nitrocellulose membrane (Bio-Rad) by a semidry transfer unit (Bio-Rad). Membranes were blocked in 5% bovine serum albumin in phosphate-buffered saline + 0.5% Triton 100. The primary antibody, anti-myc polyclonal antibody (Sigma), was diluted 1:1,000, and the secondary antibody was diluted 1:25,000. The signal was captured using the enhanced chemiluminescent method following the manufacturer's instructions (Pierce).
Four-week-old plants grown in soil were used for wounding treatment. About one-third of the leaves were pressed with pliers and only wounded leaves were used for RNA extraction. For MeJA treatment, 11-d-old plants grown vertically in standard agar plates (0.5x Murashige and Skoog salts, 1% Suc, and 0.8% phyto agar) were sprayed with 500 µM MeJA or transferred to 50 µM MeJA plates (0.5x Murashige and Skoog salts, 1% Suc, and 0.8% phyto agar, 50 µM MeJA). For ABA treatment, 11-d-old plants were transferred to 10 µM ABA plates (0.5x Murashige and Skoog salts, 1% Suc, and 0.8% phyto agar, 10 µM ABA). After treatment for the indicated times, whole plants were harvested and frozen in liquid nitrogen.
Total RNA was isolated from plant and cell culture samples according the protocol of Ahn (2000)
Nuclei were isolated from 10-g Arabidopsis cell culture according to the previously described protocol (Yu et al., 1998
35S::GFP was generated by cloning the eGFP coding region into the KpnI and BamHI sites of pCHF3. 35S::AGP31::eGFP was generated by cloning the eGFP coding region into the BamHI site of 35S::AGP31::myc to replace the 6x-myc fragment. Constructs were bombarded into onion (Allium cepa) epidermal cell using a PDS-1,000/He particle delivery system (Bio-Rad) as described (Arnim, 2002
About 1.4 kb upstream of the translation start site of the AGP31 promoter were amplified by PCR from genomic DNA using primers 5'-CCAGAATTCGCAATTACGCTCAAAGTCTCC-3' and 5'-CGCGAGCTCTTTGTTTTGTTTTTGGGTTA-3' and cloned into EcoRI and SacI sites of binary vector pCHF3 (Borevitz et al., 2000
We would like to thank Dr. Maria Person and Ms. Michelle Gadush at the Core Facilities of the University of Texas at Austin for their excellent technical assistance in MALDI-MS. This project is supported in part by the Department of Energy-funded (DE–FG09–93ER–20097) Center for Plant and Microbial Complex Carbohydrates. Received May 22, 2007; accepted September 10, 2007; published September 20, 2007.
1 This work was supported by the University of Texas at Austin Office of Vice President for Research (grant to M.C.M.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Mona C. Mehdy (mmehdy{at}mail.utexas.edu).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.102657 * Corresponding author; e-mail mmehdy{at}mail.utexas.edu.
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ABSTRACT
RESULTS
-probe (Bio-Rad) membranes. Blots were hybridized following the manufacturer's instructions. Probes were amplified by PCR from genomic DNA or reverse-transcribed cDNA and labeled by the DECAprime II kit (Ambion). Signals were quantitated by the National Institutes of Health ImageJ program. 
-32P-UTP (3,000 Ci/mmol, 10 µCi/µL; Perkin-Elmer), and 80 units RNAsin (Promega). The mixture was incubated at 30°C for 30 min. The reaction was stopped by extracting the reaction using the RNA extraction buffer used in RNA isolation. Subsequently, labeled RNA was extracted as total RNA isolation. Five micrograms of linearized plasmid DNAs were blotted onto a