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. 2018 Feb 26;176(4):3103–3119. doi: 10.1104/pp.17.01530

Modulation of Plant Salicylic Acid-Associated Immune Responses via Glycosylation of Dihydroxybenzoic Acids1

Xu-xu Huang 1, Guo-qing Zhu 1, Qian Liu 1, Lu Chen 1, Yan-jie Li 1, Bing-kai Hou 1,2
PMCID: PMC5884596  PMID: 29483147

The glycosyltransferase UGT76D1 catalyzes the glycosylation of dihydroxybenzoic acids and modulates plant salicylic acid homeostasis and immune responses.

Abstract

Salicylic acid (SA) plays a crucial role in plant innate immunity. The deployment of SA-associated immune responses is primarily affected by SA concentration, which is determined by a balance between SA biosynthesis and catabolism. However, the mechanisms regulating SA homeostasis are poorly understood. In this study, we characterized a unique UDP-glycosyltransferase, UGT76D1, which plays an important role in SA homeostasis and associated immune responses in Arabidopsis (Arabidopsis thaliana). Expression of UGT76D1 was induced by treatment with both the pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 and SA. Overexpression of UGT76D1 resulted in high SA accumulation, significant up-regulation of pathogen-related genes, and a hypersensitive response (HR)-like lesion mimic phenotype. This HR-like phenotype was not observed following UGT76D1 overexpression in SA-deficient NahG transgenic or sid2 plants, suggesting that the phenotype is SA dependent. Biochemical assays showed that UGT76D1 glycosylated 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), the major catabolic forms of SA, to their Glc and Xyl conjugates in vitro and in vivo. Moreover, in a mutant background blocked in the formation of 2,3-DHBA and 2,5-DHBA, UGT76D1 overexpression did not cause a HR-like lesion mimic phenotype. Following infection with Pst DC3000, UGT76D1 knockout mutants displayed a delayed immune response, with reduced levels of DHBA glycosides and SA, and down-regulated SA synthase expression. By contrast, UGT76D1 overexpression lines showed an enhanced immune response and increased SA biosynthesis before and after pathogen infection. Thus, we propose that UGT76D1 plays an important role in SA homeostasis and plant immune responses by facilitating glycosylation of dihydroxybenzoic acids.

Plants can initiate the specific hypersensitive response (HR), a localized programmed cell death (PCD) response, at infection sites following challenge by biotrophic pathogens (Heath, 2000; Van Doorn, 2011). This response is likely to be a consequence of the progress of pathogen growth restriction (Coll et al., 2010,Coll et al., 2011). The expression and regulation of HR relies on many components, such as salicylic acid (SA), reactive oxygen species (ROS), jasmonic acid (JA), and ethylene (Coll et al., 2011). However, we currently have only a fragmented understanding of the roles of the components leading to HR.

The identification of a number of lesion mimic mutants (LMMs), which display spontaneous HR-like cell death, has increased our understanding of plant HR (Walbot et al., 1983; Lorrain et al., 2003; Bruggeman et al., 2015). For instance, the LMM ssi4 from Arabidopsis (Arabidopsis thaliana), with a gain-of-function mutation of R-protein SSI4, constitutively activates the ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent SA signaling pathways and displays a conditional spontaneous lesion formation, demonstrating the function of the R protein in triggering HR (Shirano et al., 2002; Zhou et al., 2004). One of the constitutive expresser of pathogenesis-related proteins (cpr) mutants, cpr22, accumulates high levels of SA and displays a spontaneous lesion phenotype (Yoshioka et al., 2001). Reducing the SA level in the accelerated cell death (acd6) mutant or blocking SA signaling in the acd5 mutant attenuates the cell death phenotype (Rate et al., 1999; Greenberg et al., 2000). Transgenic plants expressing NahG (bacterial salicylate hydroxylase), which confers low SA concentration, are also capable of inhibiting lesion formation, providing evidence that SA participates in the regulation of cell death (Rate et al., 1999; Shah et al., 2001; Yoshioka et al., 2001). SA can induce the expression of some pathogenesis-related (PR) genes and promotes the production of ROS, and together they regulate cell death (Jabs, 1999; Alvarez, 2000; Straus et al., 2010). In addition to disease resistance, SA also participates in multiple processes in plants including senescence, which is a further kind of PCD. It has been reported that leaf senescence and disease resistance use the same SA signaling pathway (Vlot et al., 2009).

The biosynthesis of SA in plants has been proposed to occur through two alternative pathways, namely, the Phe ammonia-lyase (PAL) and the isochorismatesynthase (ICS) pathways. In the PAL pathway, benzoic acid and ortho-coumaric acid are likely to be the two intermediates for SA biosynthesis (Yalpani et al., 1993; Ryals et al., 1994; Chong et al., 2001). It is believed that the ICS pathway produces most of the SA generated in plants (Wildermuth et al., 2001; Dempsey et al., 2011). A past study showed that the SA level in sid2 (an ICS mutant) was reduced to only 5 to 10% of the wild-type level after pathogen infection (Wildermuth et al., 2001).

SA biosynthesis is regulated by multiple and complex factors. For instance, EDS1 and NON-RACE-SPECIFIC DISEASE RESISTANCE1 have been identified as upstream regulators of SA biosynthesis in the effector-triggered immune response (Aarts et al., 1998). PHYTOALEXIN DEFICIENT4 (PAD4) and SENESCENCE-ASSOCIATED CARBOXYLESTERASE101 (SAG101) are two components of EDS1-mediated immunity (Feys et al., 2001,Feys et al., 2005). In addition, SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1 and its homolog CALMODULIN-BINDING PROTEIN 60g have been shown to be transcription activators of ICS1 expression (Zhang et al., 2010).

In addition to upstream regulation, active SA level is also modulated by downstream metabolic modifications, including glycosylation, methylation, amino acid conjugation, and hydroxylation. Different modifications of SA are likely to have different functions in plant defense. Two main glycosyltransferases, UDP-dependent glycosyltransferase 74F1 (UGT74F1) and UGT74F2, can transform SA into its glucoside (SAG) or its Glc ester (SGE) (Lim et al., 2002; Dean and Delaney, 2008). SAG and SGE are the inactive forms of SA and can be transformed back into active SA when plants are challenged by pathogens (Dean and Delaney, 2008). Amino acid-conjugated SA has been reported to be a potential activator of plant immunity (Chen et al., 2013). Hydroxylated SA, including 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), is the major metabolic form of SA. Recently, the enzymes catalyzing SA to 2,3-DHBA (S3H) and to 2,5-DHBA (S5H) have been identified, and SA was found to accumulate in s3h or s5h mutants (Zhang et al., 2013,Zhang et al., 2017).

The roles of 2,3-DHBA and 2,5-DHBA in Arabidopsis are not known; however, 2,5-DHBA in tomato (Solanum lycopersicum) is thought to play a signaling role in the activation of inducible defenses. The signaling role of 2,5-DHBA is thought to be complementary to SA because 2,5-DHBA induces the formation of a different set of PR proteins that are not induced by SA (Bellés et al., 1999). Because of the cytotoxicity of DHBA, 2,3-DHBA and 2,5-DHBA always exist as sugar conjugates in plants (Bartsch et al., 2010). However, it appears that sugar conjugation of DHBA has other physiological significance besides detoxification. 2,5-DHBA 5-O-β-d-xylopyranoside has been found to accumulate to high levels in cucumber (Cucumis sativus) and tomato after inoculation with different pathogens (Fayos et al., 2006). It was also reported that the sugar conjugates of 2,3-DHBA and 2,5-DHBA increased in Arabidopsis during expression of pathogen resistance (Bartsch et al., 2010). Research suggested that glycosylation of DHBA may be involved in plant immunity. Some glycosyltransferases have been identified to be capable of catalyzing the transformation of 2,3-DHBA and 2,5-DHBA in vitro to form their glucosyl or xylosyl conjugates, although very little has been carried out toward understanding the physiological relevance of these conjugates (Lim et al., 2001,Lim et al., 2002; Chen and Li, 2017). As a consequence, it is still unclear whether DHBA glycosylation functions in plant defense response and, if so, what its molecular mechanism entails.

Here, we identified and characterized the pathogen-induced glycosyltransferase UGT76D1. We found that UGT76D1 was involved in the lesion mimic HR, including the formation of natural necrotic spots, ROS accumulation, SA homeostasis, and PR gene expression in rosette leaves of Arabidopsis. Furthermore, our experiments revealed that UGT76D1 can catalyze the formation of 2,3-DHBA and 2,5-DHBA glycosides in vitro and in vivo. We found that blocking DHBA glycosylation abolished the UGT76D1-induced HR. Together, our data suggest that the glycosylation of DHBA plays a previously unrecognized role in the plant innate immune response through modulating SA homeostasis.

RESULTS

UGT76D1 Expression Is Induced by the Plant Pathogen Pst DC3000

Through searching the publicly available microarray data for UDP-dependent glycosyltransferase (UGT) genes associated with plant secondary metabolism, we found that several Arabidopsis UGT genes were induced by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). UGT76D1 is one of the most highly pathogen-responsive UGT genes. Since the UGT76D subfamily contains only this unique member, UGT76D1 may have an important and specific role in plant immune responses. To examine the reliability of microarray data, RT-qPCR was used to analyze the expression of UGT76D1 at different time points after treatment with Pst DC3000 or SA. The results showed that expression of UGT76D1 was strongly induced by either Pst DC3000 or SA (Fig. 1, A and B). UGT76D1pro:GUS transgenic plants were constructed and GUS staining also demonstrated the high induction of UGT76D1 expression by pathogen infection (Fig. 1C), but not by MgCl2 treatment used as a negative control (Fig. 1D). Thus, UGT76D1 is a pathogen-responsive gene and may be involved in plant defense responses.

Figure 1.

Figure 1.

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Expression of UGT76D1 is induced by SA and Pst DC3000. A and B, RT-qPCR analysis of UGT76D1 expression induction by SA and Pst DC3000 treatment. Mock treatment in A is DMSO solvent. Mock treatment in B is 10 mm MgCl2 suspension buffer. The value of the wild type with mock treatment for 0 h was set at 1. Data are means ± sd of three biological replicates (**P < 0.01, *P < 0.05, Student’s t test). C and D, GUS staining of UGT76D1Pro:GUS expression following Pst DC3000 (C) and MgCl2 (mock; D) inoculation. Bars = 1 mm (upper panels) and 200 μm (lower panels).

UGT76D1 Is Involved in the Spontaneous HR-Like Lesion Mimic Response

To study the function of UGT76D1 in plants, we constructed UGT76D1 knockout mutants and transgenic lines overexpressing UGT76D1. Through the CRISPR/Cas9 system, two independent mutant lines of UGT76D1 (ko-33 and ko-36) were isolated, each with a nucleotide deletion in the target site of the 5′ coding region (Supplemental Fig. S1, A–D), which causes a frameshift mutation. Nineteen independent UGT76D1 overexpression lines were obtained, and two lines (OE-17 and OE-32) with a high expression of UGT76D1 were used in this study (Supplemental Fig. S1E). Under long-day conditions, mutant lines exhibited a phenotype comparable to that of wild-type plants. On the other hand, UGT76D1 overexpression lines showed obvious spontaneous necrotic lesions, a phenotype that mimics hypersensitive cell death (Fig. 2A). In the T2 generation of OE-17 and OE-32 lines, the lesion phenotype cosegregated with the antibiotic resistance marker (Supplemental Table S1). The lesions became more severe under short-day conditions (Supplemental Fig. S2). In addition to the spontaneous necrotic lesions, leaf senescence in UGT76D1 overexpression lines also occurred earlier than in wild-type plants (Supplemental Fig. S3).

Figure 2.

Figure 2.

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Analysis of UGT76D1 mutant and overexpression lines. A, Phenotype of 4-week-old wild type (WT), UGT76D1 overexpression lines (OE-17 and OE-32), and UGT76D1 mutant lines (ko-33 and ko-36) grown under 22°C and long-day conditions (16 h light/8 h dark). Right panel is a magnified photograph showing the spontaneous lesion mimic phenotype in rosette leaves of overexpression lines. Bar = 1 cm for both panels. B, Trypan blue staining showing cell death in UGT76D1 overexpression and mutant lines. Four-week-old plants grown under long-day condition were used for the staining assay. The bottom panels are the magnified photographs of top panels. Bars = 1 cm (top panels) and 100 μm (bottom panels). C, DAB staining showing the H2O2 content of UGT76D1 overexpression and mutant lines. Four-week-old plants grown under long-day conditions were used for the staining assay. The bottom panels are magnified photographs of top panels. Bars = 1 cm (top panels) and 100 μm (bottom panels). D and E, RT-qPCR analysis of the expression of defense response genes PR1 and PR2 in 4-week-old UGT76D1 overexpression lines and mutant lines. The value of wild-type plants was set at 1.0. Data are means ± sd of three biological replicates (**P < 0.01, *P < 0.05, Student’s t test). F and G, Quantification of free SA and total SA levels in UGT76D1 overexpression and mutant lines. Leaves of 4-week-old plants grown under long-day conditions at 22°C were collected and used for the measurements. Data are means ± sd of three biological replicates (**P < 0.01, *P < 0.05, Student’s t test).

LMMs are mutants with spontaneous HR-like cell death without pathogen infection (Lorrain et al., 2003; Bruggeman et al., 2015). The phenotype of UGT76D1 overexpression lines was similar to that of LMM mutants, suggesting a constitutively activated hypersensitive response and localized cell death in the UGT76D1 overexpression lines. Trypan blue staining further confirmed cell death in UGT76D1 overexpression lines (Fig. 2B). ROS overproduction can lead to cell death and is a family of crucial signal molecules in the activation of PCD during HR (Malik et al., 2014). The 3,3′-diaminobenzidine (DAB) staining showed that UGT76D1 overexpression lines accumulated considerable amounts of hydrogen peroxide, whereas UGT76D1 knockout mutant lines exhibited hydrogen peroxide levels comparable to those in the wild-type plants (Fig. 2C), a phenomenon which correlated with the HR-like phenotypes of these lines. We next examined the expression of the SA-regulated PR1 and PR2 defense genes. PR1 and PR2 were drastically up-regulated in UGT76D1 overexpression lines, whereas they were downregulated in the UGT76D1 knockout mutant lines (Fig. 2, D and E), suggesting the involvement of UGT76D1 in the SA-associated innate immune response.

The difference in changes of PR1 and PR2 expression between UGT76D1 overexpression and knockout mutant lines, as well as the spontaneous HR-like lesion mimic phenotype occurring in the UGT76D1 overexpression lines, but not in the mutant lines, prompted us to investigate SA levels in these lines. Our analysis showed that free and total SA levels were considerably higher in UGT76D1 overexpression lines than that in wild-type plants, but were moderately reduced in the mutant lines compared to the wild type (Fig. 2, F and G). These data suggest that UGT76D1 may be involved in the plant innate immune response through modulating SA homeostasis. To further explore whether UGT76D1 was involved in pathogen-induced HR, we tested the ability of UGT76D1 mutants to trigger the HR against Pst DC3000 carrying avrRpt2. We found that UGT76D1 knockout mutants suppressed the HR induced by Pst DC3000 carrying avrRpt2 and the disease symptoms occurring at 48 h after inoculation (Fig. 3A). Cell death in each line was visualized with Trypan blue staining (Fig. 3B). These results suggested that UGT76D1 may be a positive activator in at least the Pst DC3000 (avrRpt2)-induced HR.

Figure 3.

Figure 3.

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Hypersensitive response phenotype of UGT76D1 mutant lines following avirulent pathogen induction. A and B, HR phenotypes (A) and Trypan blue staining showing cell death (B) of wild-type (WT) and UGT76D1 mutant (ko-33 and ko-36) leaves following inoculation with Pst DC3000 (avrRpt2) (OD600 = 0.02). MgCl2 treatment was used as mock control. At least 36 leaves were assessed for each genotype during each experiment, with a similar phenotype displayed in most. Representative leaves are shown at 24 and 48 h after inoculation. Bars = 1 cm.

SA Is Required for the UGT76D1-Activated Lesion Mimic Phenotype

To determine whether the UGT76D1-activated lesion mimic phenotype is SA dependent, UGT76D1 overexpression lines were crossed transgenic plants expressing NahG, a SA hydroxylase that degrades SA (Delaney et al., 1994). Hybrids with high expression of both UGT76D1 and NahG were selected and named OE-17 NahG and OE-32 NahG. It was shown that NahG can restore a wild-type phenotype (Fig. 4A) and prevents a spontaneous lesion mimic phenotype despite UGT76D1 overexpression in hybrid lines (Fig. 4B). Corresponding with the rescued leaf phenotype, cell death (indicated by Trypan blue staining), the H2O2 content (indicated by DAB staining), and the expression of PR1 and PR2 were restored to the levels of wild-type plants following NahG expression in UGT76D1 overexpression lines (Fig. 4, C–E). Most importantly, the contents of free and total SA were reduced to undetectable levels in the UGT76D1 overexpression NahG hybrids (Fig. 4, F and G). Thus, we propose that SA accumulation is the main factor triggering the UGT76D1-activated lesion mimic phenotype.

Figure 4.

Figure 4.

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SA is required for UGT76D1-activated lesion mimic phenotype. A, Phenotypes of 4-week-old wild-type (WT), UGT76D1 overexpression (OE-17 and OE-32), NahG-expressing (NahG), and UGT76D1 overexpression/NahG hybrid (OE-17 NahG and OE-32 NahG) plants. Bar = 1 cm. B, RT-PCR analysis of the expression of UGT76D1 in UGT76D1 overexpression/NahG hybrid lines alongside control lines described in A. ACTIN2 was used as the internal control. C, Trypan blue staining (top panel) showing the cell death level and DAB staining (bottom panel) showing the H2O2 contents in leaves from 4-week-old plants of UGT76D1 overexpression/NahG hybrid lines alongside control lines described in A. Bars = 1 cm. D and E, RT-qPCR analysis of PR1 and PR2 expression in UGT76D1 overexpression/NahG hybrid lines alongside control lines described in A. The value of wild-type plants was set at 1. Data represent mean ± sd of three biological replicates (**P < 0.01, *P < 0.05, Student’s t test). F and G, Free and total SA contents in UGT76D1 overexpression/NahG hybrid lines alongside control lines described in A. Data are means ± sd of three biological replicates (*P < 0.05, Student’s t test). ND, Not detectable.

UGT76D1 Possesses Biochemical Activity toward DHBA Glycosylation in Vitro

To investigate the mechanism by which UGT76D1 was involved in maintaining SA homeostasis, we next carried out in vitro screening of substrates catalyzed by this glycosyltransferase. Fusion proteins of UGT76D1 with the GST tag were heterologously expressed in Escherichia coli and then purified (Fig. 5A). When SA was tested as a candidate substrate for UGT76D1, UGT76D1 exhibited no detectable enzymatic activity toward SA, suggesting that SA was not the natural substrate (Fig. 5B). We further extended the candidate substrates to SA metabolites, phenolic compounds, plant hormones, and flavonoids (Supplemental Table S2). UGT76D1 had high enzymatic activities toward only the hydroxylated products of SA, namely, 2,3-DHBA and 2,5-DHBA (Fig. 5, C and D). No activity or only trace activity was observed toward other compounds in our analyses (Supplemental Table S2). It was previously reported that 2,3-DHBA and 2,5-DHBA mainly form both glucosides and xylosides in planta, although the physiological roles of these glycosides were unknown (Dempsey et al., 2011). Thus, both UDP-Glc and UDP-Xyl were used as the sugar donors in our biochemical assays. We found that UGT76D1 can catalyze 2,3-DHBA and 2,5-DHBA to form their corresponding glucosides and xylosides (Fig. 5, C and D). Liquid chromatography-mass spectrometry (LC-MS) analysis confirmed the identity of the glucosides and xylosides produced from 2,3-DHBA and 2, 5-DHBA (Supplemental Fig. S4). A previous study had found that 2,3-DHBA and 2,5-DHBA can be conjugated by UGT89A2 to form 2,3-DHBA or 2,5-DHBA xylosides (Li et al., 2014). To determine the DHBA and sugar donor preferences of UGT76D1 and UGT89A2, specific enzyme activity was tested in vitro. UGT89A2 exhibited a strong preference toward UDP-Xyl over UDP-Glc. In contrast, UGT76D1 preferred UDP-Glc to UDP-Xyl (Supplemental Table S3).

Figure 5.

Figure 5.

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UGT76D1 catalyzes the in vitro glycosylation of 2,3-DHBA and 2,5-DHBA, but not SA. A, SDS-PAGE detection of the purified recombinant UGT76D1-GST fusion protein. Proteins were visualized by Coomassie blue staining. GST, Glutathione S-transferase; UGT76D1, UGT76D1 fusion protein. B, HPLC analysis of SA glycosylation by UGT76D1. C and D, HPLC analysis of 2,3-DHBA and 2,5DHBA glycosylation by UGT76D1 G, UDP-Glc; X, UDP-Xyl. GST protein was used as the negative control.

UGT76D1 Functions in DHBA Glycosylation in Vivo

To further investigate the biochemical function of UGT76D1 in vivo, glycosides of 2,3-DHBA and 2,5-DHBA were extracted and measured from UGT76D1 overexpression and knockout mutant lines. The HPLC profiling of the DHBA sugar conjugates showed that UGT76D1 overexpression lines accumulated much more 2,3-DHBA and 2,5-DHBA glycosides than that in the wild type under nonchallenged conditions. However, in nonchallenged UGT76D1 mutant lines, only 2,3-DHBA xyloside was significantly affected, accumulating at levels lower than that in the wild type (Fig. 6, A and B). The relative level of DHBA glucosides in the UGT76D1 knockout mutants was very low and comparable to that in the wild type.

Figure 6.

Figure 6.

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The relative levels of sugar conjugates of 2,3-DHBA and 2,5-DHBA in UGT76D1 overexpression and mutant lines. A and B, HPLC profiling (A) and the quantitation (B) of DHBA sugar conjugates in the wild type (WT), UGT76D1 overexpression lines (OE-17 and OE-32), and UGT76D1 mutant lines (ko-33 and ko-36) in the absence of pathogen exposure. Data were normalized to the corresponding DHBA glycoside in the wild type to account for the changes in UGT76D1 overexpression and mutant lines. C and D, HPLC profiling and the quantitation of DHBA sugar conjugates in the wild type and UGT76D1 mutant lines following Pst DC3000 (DC) or 10 mm MgCl2 (Mock) treatment for 48 h. Data were normalized to the corresponding DHBA glycoside in the wild type treated with Pst DC3000 to account for the changes in UGT76D1 mutant lines. The DHBA sugar conjugates formed by UGT76D1 catalysis in vitro were used as the glycoside standards. Peak 1, 2,5-DHBA glucosides; Peak 2, 2,3-DHBA glucosides; Peak 3, 2,5-DHBA xlyoside; Peak 4, 2,3-DHBA xylosides; Peak 5, internal reference (caffeic acid). 2,3DHBA-G, 2,3-DHBA glucosides; 2,3DHBA-X, 2,3DHBA xyloside; 2,5DHBA-G, 2,5-DHBA glucosides; 2,5DHBA-X, 2,5-DHBA xlyoside. Data are means ± sd of three biological replicates (**P < 0.01, *P < 0.05, Student’s t test).

The very low base levels of DHBA glucosides made them difficult to measure accurately. To overcome this difficulty, we applied 100 μm 2,3-DHBA and 2,5-DHBA to plants and remeasured the DHBA glucoside concentrations. Although the level of 2,5-DHBA glucosides was comparable between UGT76D1 knockout mutants and the wild type, 2,3-DHBA glucoside level in the mutants was much lower than that in the wild type (Supplemental Fig. S5, A and B). Meanwhile, it was observed that UGT76D1 overexpression lines accumulated much higher concentrations of both 2,3-DHBA and 2,5-DHBA glucosides than that in the wild type upon application of DHBA; a similar result occurred with UGT76D1 overexpression lines without DHBA application. Because expression of UGT76D1 is Pst DC3000 inducible, the concentrations of 2,3-DHBA glycoside and 2,5-DHBA glycoside in UGT76D1 mutants and the wild type were remeasured 48 h after infiltration with Pst DC3000 or with MgCl2 as the negative control. Our analysis revealed that the accumulation of both 2,3-DHBA and 2,5-DHBA glucosides was significantly reduced in Pst DC3000-inoculated UGT76D1 mutant plants compared to that in the wild type (Fig. 6, C and D). The concentrations of both 2,3-DHBA and 2,5-DHBA xylosides were only slightly reduced in Pst DC3000-inoculated UGT76D1 mutants. These data suggested that UGT76D1 functions in DHBA glycosylation in vivo and may be the main enzyme responsible for the glucosylation of 2,3-DHBA and 2,5-DHBA.

We also attempted to determine the concentrations of the free forms of 2,3-DHBA and 2,5-DHBA in the UGT76D1 overexpression and knockout mutant lines. We found that the 2,3-DHBA level was too low to be detectable with or without pathogen inoculation, a result that is consistent with those from previous studies (Bartsch et al., 2010; Zhang et al., 2013). For free 2,5-DHBA, however, an interesting and unexpected change was observed. After Pst DC3000 inoculation, UGT76D1 overexpression lines accumulated much more of the free form of 2,5-DHBA than was determined in the wild type, whereas mutant lines had greatly reduced levels of the free form of 2,5-DHBA compared to that in the wild type (Supplemental Fig. S6). There are several hypotheses to explain this unexpected change. First, overexpression or mutation of UGT76D1 could alter metabolic flux toward the conjugated DHBA form, resulting in feedback activation or inhibition of upstream enzymes of DHBA biosynthesis. Similar situations have been found in previous studies (Chong et al., 2002; Tognetti et al., 2010). Second, one part of the DHBA pool may arise from cleavage of the glucosylated form by glucosidases. Third, because of the cytotoxicity of DHBAs, an overcompensated degradation of DHBAs may occur in UGT76D1 mutants.

Immune Response Activated by UGT76D1 Depends on the Formation of 2,3-DHBA and 2,5-DHBA Glycosides

Previous research indicated that S3H can convert SA to 2,3-DHBA, whereas the formation of 2,3-DHBA is blocked in the mutant s3h (Zhang et al., 2013). To investigate the effects of UGT76D1 on the immune response in the absence of 2,3-DHBA, the preferred substrate of UGT76D1, we produced hybrids between UGT76D1 overexpression lines and s3h mutants. It was found that the s3h background inhibited the spontaneous lesion mimic phenotype caused by UGT76D1 overexpression (Fig. 7A). DAB and Trypan blue staining also showed reduced levels of H2O2 and cell death in the UGT76D1 overexpression s3h hybrid lines (Fig. 7B). RT-qPCR analysis revealed that, although the expression of UGT76D1 remained at high levels in the UGT76D1 overexpression s3h hybrids (Figs. 7C), the expression of both PR1 and PR2 was significantly reduced (Fig. 7D). We also measured the SA levels in UGT76D1 overexpression s3h hybrid plants and found that the concentrations of both free SA and total SA were markedly reduced (Fig. 7E). These data indicated that the formation of 2,3-DHBA glycosides under the presence of substrate 2,3-DHBA would be important for UGT76D1-activated immune response.

Figure 7.

Figure 7.

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UGT76D1-activated immune response depends on DHBA glycosylation. A, Phenotypes of 4-week-old wild-type (WT), UGT76D1 overexpression (OE-17 and OE-32), s3h mutant (s3h), and UGT76D1 overexpression/s3h hybrid (OE-17 s3h and OE-32 s3h) plants grown under long-day conditions at 22°C. Bar = 1 cm. B, DAB (top row panels) and Trypan blue (bottom row panels) staining showing levels of H2O2 and cell death, respectively, in leaves from 4-week-old plants of UGT76D1 overexpression/s3h hybrid lines alongside control lines described in A. Bars = 100 μm. C, RT-PCR analysis of UGT76D1 expression in UGT76D1 overexpression/s3h hybrid lines alongside control lines described in A. ACTIN2 was used as the internal control. D, RT-qPCR analysis of relative PR1 and PR2 expression in UGT76D1 overexpression/s3h hybrid lines alongside control lines described in A. The value of wild-type plants was set at 1. Data are means ± sd of three biological replicates (**P < 0.01, *P < 0.05, Student’s t test). E, The free and total SA levels in UGT76D1 overexpression/s3h hybrid lines alongside control lines described in A. Data are means ± sd of three biological replicates (**P < 0.01, *P < 0.05, Student’s t test). F, Phenotypes of transgenic plants overexpressing UGT76D1 in a wild-type or s3h s5h double mutant background. The right panel shows the enlarged leaf phenotype. Leaf lesions are indicated with red arrowheads. Bars = 1 cm (left panel) and 0.5 cm (right panel).

Recently, S5H has also been described (Zhang et al., 2017). It was reported that the concentrations of 2,3-DHBA and 2,5-DHBA were reduced in the s3h s5h double mutant, which exhibited a dwarf and early senescence phenotype (Zhang et al., 2017). Here, we further examined the effect of increased UGT76D1 expression on the defense response in the s3h s5h double mutant background. We found that UGT76D1 overexpression in s3h s5h double mutants did not cause the HR-like lesion mimic phenotype seen in the wild-type background (Fig. 7F), further demonstrating that the function of UGT76D1 in activating the immune response is dependent on DHBA glycosylation.

DHBA Glycosylation Catalyzed by UGT76D1 Modulates SA Biosynthesis during Pathogen Infection

SA is believed to be the crucial signaling molecule in SA-dependent plant defense responses, where PR1 and PR2 are known to be important marker genes, the expression of which is regulated by SA. It appears that the levels of SA and the expression of PR1/PR2 always correlate with the lesion mimic phenotype of the UGT76D1 overexpression and mutant lines, suggesting an important relationship between UGT76D1-catalyzed DHBA glycosylation and SA synthesis. To further investigate the physiological effects of UGT76D1 on SA synthesis in the plant defense response, we analyzed the changes in SA concentration and PR expression in UGT76D1 overexpression and mutant lines challenged by Pst DC3000 or the MgCl2 control. The expression of PR1 and PR2 was detected at different time points after plants were inoculated with Pst DC3000 or the control buffer. It was found that PR1 and PR2 had high expression levels in the overexpression lines even at the beginning of the treatment. As the time after pathogen challenge increased, the expression of PR1 and PR2 increased rapidly in UGT76D1 overexpression lines. However, the expression of PR1 and PR2 increased only slowly in the wild type. In the UGT76D1 mutant lines, the expression levels of PR1 and PR2 were even lower than that in wild-type at every time point of pathogen treatment (Fig. 8, A and B), suggesting that the defense response was retarded by the loss of function of UGT76D1. We analyzed the free and total SA concentrations in the UGT76D1 overexpression and mutant lines after exposure to Pst DC3000 or MgCl2 for 48 h. Although SA concentrations in UGT76D1 overexpression lines were little more than that in the wild type 48 h after inoculation, the SA concentrations in the knockout mutant lines were much lower than that in the wild type (Fig. 8, C and D). These data suggested that the physiological role of UGT76D1 in the SA-dependent defense response was a consequence of modulating SA levels.

Figure 8.

Figure 8.

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The immune response and SA biosynthesis in UGT76D1 overexpression and mutant lines following treatment with Pst DC3000. A and B, Relative PR1 and PR2 expression in 3-week-old wild-type (wild-type) and UGT76D1 overexpression (OE) and mutant (ko) plants following Pst DC3000 (DC) or MgCl2 (Mock) treatment. For either treatment, expression levels in the wild type at 0 h were set at 1. C and D, Free and total SA levels in 3-week-old UGT76D1 overexpression (OE-17 and OE-32) and mutant (ko-33 and ko-36) lines following treatment with Pst DC3000 or MgCl2 (Mock) for 48 h. E, RT-qPCR analysis of EDS1, PAD4, and ICS1 expression in UGT76D1 overexpression and mutant lines without any experimental treatment. Gene expression levels in the wild type were set at 1.0. F, RT-qPCR analysis of EDS1, PAD4, and ICS1 expression in UGT76D1 mutant lines following treatment with Pst DC3000 for 24 and 48 h. Gene expression levels in the wild type following 24 h Pst DC3000 treatment were set at 1.0. A to F, Data are means ± sd of three biological replicates (**P < 0.01, *P < 0.05, Student’s t test).

The mechanism by which SA concentration is modulated by UGT76D1 was also investigated. We analyzed the expression of SA synthesis-related genes in UGT76D1 overexpression and mutant lines with or without challenge by Pst DC3000. Without Pst DC3000 treatment, the expression of several key SA-related genes, namely, the SA biosynthesis gene ICS1 and the SA biosynthesis regulatory genes EDS1 and PAD4, were substantially increased in the UGT76D1 overexpression lines, whereas they were only slightly decreased in the mutant lines (Fig. 8E). After challenge by Pst DC3000 for 24 or 48 h, the expression of the key SA biosynthetic gene ICS1 was dramatically decreased in the UGT76D1 mutant lines (Fig. 8F), although the expression levels of EDS1 and PAD4 were still only slightly decreased. It is likely that UGT76D1 was involved in the positive feedback regulation of SA biosynthesis, particularly via the regulation of the ICS1 gene.

To verify the role of ICS1 in the UGT76D1-mediated innate immune response, we generated hybrids between UGT76D1 overexpression lines with sid2, an ICS1 mutant. Although expression of UGT76D1 remained at a high level in the hybrid lines, the UGT76D1-activated lesion mimic phenotype was totally suppressed (Fig. 9, A and B). We also measured the SA concentrations in the hybrid lines. It was found that both the free SA and total SA were reduced to levels lower than that in wild-type plants (Fig. 9C). These data indicated that the pathogen-induced glycosyltransferase UGT76D1 functions in the defense response through modulating salicylic acid biosynthesis. The glycosylation of DHBA by UGT76D1 may be involved in a positive feedback loop of SA biosynthesis, accelerating SA accumulation in the process of plant defense response.

Figure 9.

Figure 9.

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UGT76D1-activated immune response depends on SA biosynthesis. A, Phenotypes of 4-week-old wild-type (WT), UGT76D1 overexpression (OE-17 and OE-32), sid2 mutant (sid2-1), and UGT76D1 overexpression/sid2 hybrid (OE-17 sid2-1 and OE-32 sid2-1) plants. Bar = 1 cm. B, RT-PCR analysis of UGT76D1 expression in UGT76D1 overexpression/sid2 hybrid lines alongside control lines described in A. ACTIN2 was used as the internal control. C, Quantification of free SA and total SA levels in UGT76D1 overexpression/sid2 hybrid lines alongside control lines described in A. Leaves of 4-week-old plants grown under long-day conditions at 22°C were collected and used for the measurements. Data are means ± sd of three biological replicates (Student’s t test, *P < 0.05, * * P < 0.01).

DISCUSSION

UGT76D1 Is a Novel DHBA Glycosyltransferase

Previous experiments had shown that SA is the main precursor of 2,3-DHBA and 2,5-DHBA (Ibrahim and Towers, 1959). The two DHBAs usually exist in a glycosylated form (glucoside or xyloside) and were considered to be among the major inactive forms of SA (Bartsch et al., 2010). However, the enzyme responsible for the formation of those DHBA glycosides had previously been unknown. Recent studies indicated that UGT89A2 is a 2,5-DHBA xylosyltransferase in Arabidopsis (Li et al., 2014). However, overexpression of the UGT89A2 gene did not result in any overt phenotype (Chen and Li, 2017). In this study, UGT76D1 was identified as a novel DHBA glycosyltransferase in Arabidopsis. UGT76D1 can glycosylate both 2,3-DHBA and 2,5-DHBA, using either UDP-Glc or UDP-Xyl as sugar donors. Our analyses indicated that lines overexpressing UGT76D1 accumulated much higher concentrations of DHBA glycosides, particularly the DHBA glucosides, than did the wild type. However, knockout mutant lines of UGT76D1 contained much lower concentrations of DHBA glucosides following pathogen challenge. These data suggest that the role of UGT76D1 in the defense response is mainly with respect to the glucosylation of DHBAs. This hypothesis received support from our enzyme activity analyses. Our results indicated that UGT76D1 prefers UDP-Glc to UDP-Xyl as the sugar donor for DHBA glycosylation. By contrast, although UGT89A2 exhibited enzyme activity toward the DHBAs, it exhibited a strong preference toward UDP-Xyl over UDP-Glc. UGT76D1 and UGT89A2 seem to be too distant on the phylogenetic tree, whereas they can act on the same substrate, DHBA. Actually, this is a common phenomenon. For example, UGT73C6 and UGT78D1, two UGTs belonging to different groups in the GT1 family, can act on the same flavonol molecules (Jones et al., 2003). Another example involves auxin glycosylation. Both UGT74E2 and UGT75D1 can catalyze the glycosylation of the auxin indole-3-butyric acid (Tognetti et al., 2010; Zhang et al., 2016), whereas UGT84B1 is reported to act on the auxin indole-3-acetic acid (Jackson et al., 2002). What is the biological significance for plants to have several different UGTs acting on the same substrate? It is speculated that plant evolution has involved the formation of functionally redundant multiple glycosyltransferases active toward the same type of substrates. A synergistic or coordinated interaction between different glycosyltransferase members may be meaningful for the fine tuning of metabolic homeostasis. On the other hand, the activities of these enzymes will depend on cell specificity of enzyme expression, relative availability of substrates and sugar donors, and relative compartmentalization of the enzyme and substrates, which may have the potential to enhance the plant’s flexibility in development or in adaptation to diverse environments.

Pathogen-Induced UGT76D1 May Act as a Positive Component to Promote Hypersensitive Response

In plants, the innate immune system has evolved to protect them from the pathogens which attack them, but the mechanisms involved in mediating this defense are poorly understood. Plant cells are usually capable of perceiving signals from pathogens and inducing resistance to the challenge, which is achieved sometimes by launching a cell suicide mechanism to produce a localized PCD, known as HR, at the infection site (Heath, 2000). The fates of cells at the infection site and in adjacent positions are different due to the SA concentration gradient. In the infection site, high levels of SA induced the degradation of NPR1 (an inhibitor of PCD in effector-triggered immunity) and then cell death. By contrast, in cells adjacent to the infection site, the SA level is not high enough to lead to cell death, so the cells survive and enhance resistance (Fu and Dong, 2013). UGT76D1 is a glycosyltranferase gene induced by pathogens. Transgenic plants with high levels of expression of UGT76D1 displayed spontaneous local cell death, a lesion mimic and HR-like phenotype. This finding suggested that UG76D1 may be a positive component in the regulation of the HR-related immune response. When the SA hydroxylase gene NahG was transferred into UGT76D1 overexpression lines, the lesion mimic phenotype, the high levels of H2O2 and the upregulated PR expression disappeared. Thus, high levels of SA are required for lesion formation in UGT76D1 overexpression lines. Under normal conditions, the basal expression of UGT76D1 is very low. When plants were challenged by pathogens such as Pst DC3000, UGT76D1 expression was induced. Based on these findings, we proposed that UGT76D1 acts as a positive component, playing an important role in the SA-activated hypersensitive response.

The Glycosylation of DHBAs May Be an Important Process Responsible for the UGT76D1-Mediated Innate Immune Response

The 2,5-DHBA glucosides accumulated in tomato and cucumber plants after infection by different pathogens (Bellés et al., 1999; Fayos et al., 2006), while 2,3-DHBA xylosides increased in Arabidopsis after induction by Pst DC3000 (avrRpm1; Bartsch et al., 2010). It is likely that DHBA glycosides have important roles in the plant defense response. In this study, UGT76D1 overexpression lines can accumulate much higher concentrations of 2,3- and 2,5-DHBA glycosides (glucosides and xylosides) than that in the wild type. By contrast, the UGT76D1 mutants produced less 2,3- and 2,5-DHBA glucosides than that in the wild type after inoculation with Pst DC3000, suggesting the greater role of 2,3-DHBA and 2,5-DHBA glucosylation in the UGT76D1-activated immune response. S3H and S5H are enzymes responsible for the conversion of SA to 2,3-DHBA and 2,5-DHBA, and the s3h s5h double mutants abolished the production of 2,3-DHBA and 2,5-DHBA (Zhang et al., 2013,Zhang et al., 2017). In our experiments, when UGT76D1 was overexpressed in s3h mutant or s3h s5h double mutant backgrounds, we found that the HR-like lesion mimic phenotype caused by UGT76D1 overexpression was totally suppressed. These results suggested that the lack of glycosylation of 2,3-DHBA on its own or of both 2,3-DHBA and 2,5-DHBA abolished the UGT76D1-activated immune response. How does the s3h single mutant suppress the lesion mimic phenotype caused by UGT76D1? We proposed several possible reasons. First, it was reported that S3H can convert SA to both 2,3-DHBA and 2,5-DHBA in vitro (Zhang et al., 2013); therefore, s3h mutation may be efficient for blocking the formation of 2,3-DHBA and 2,5-DHBA. In addition, s3h mutant nearly abolished the formation of 2,3-DHBA, while s5h mutant still maintain the formation of 2,5-DHBA to a greater extent (Zhang et al., 2017), which may be due to the enzyme activity of S3H in s5h mutant and also suggest a more crucial role for S3H in the metabolic flux from SA to DHBAs. Consistent with this hypothesis, our results indicated that SA level was restored to the s3h level in the UGT76D1OE s3h hybrid plants. Second, because of the difference in molecular structures, 2,3-DHBA glucoside may have different roles from 2,5-DHBA glucoside in the defense responses, thus resulting in the elimination of the lesion phenotype when UGT76D1 was overexpressed in a s3h background. Third, UGT76D1-activated immune response is likely to depend on a threshold of 2,3-DHBA and 2,5-DHBA concentrations.

It was reported that SA accumulated in the s3h s5h double mutant or the s3h single mutant. However, the s3h and s3h s5h mutants did not show the HR-like lesion phenotype as was exhibited in the UGT76D1 overexpression lines, but only showed dwarf or earlier leaf senescence traits when compared to the wild type (Zhang et al., 2013,Zhang et al., 2017). Previous studies had indicated that leaf senescence and HR are both examples of PCD, triggered by different stimuli (Love et al., 2008; Rivas-San Vicente and Plasencia, 2011). Here, UGT76D1 was demonstrated to be involved in the HR-like immune response, which was different from the involvement of S3H or S5H in senescence, suggesting that UGT76D1 may respond to the different stimuli from S3H and S5H. In the past, DHBA glycosides were viewed simply as the storage forms of DHBA. In this study, however, we revealed that the sugar conjugates of DHBA, or the process toward their formation, are likely to be an important trigger to initiate PCD in the plant HR response.

In this study, we also found that UGT76D1 has trace activity toward coumarins and some other compounds in vitro. Previous studies indicated that scopoletin, a coumarin compound, may be a reactive oxygen intermediate scavenger and may play a role in redox homeostasis during the HR of tobacco (Nicotiana tabacum) to Tobacco mosaic virus (Chong et al., 2002). Here, because UGT76D1 has only a trace activity toward coumarins in vitro, we propose that the glycosylation of coumarins may not be the major stimulus to HR phenotype mediated by UGT76D1.

The Glycosylation of DHBA May Be Involved in the Positive Feedback Regulation of SA Synthesis

SA is a crucial signal for plant defense responses. Its levels and dynamic changes in tissues will directly influence the SA-dependent defense responses of plants. Thus, SA concentrations in plant defense responses must be precisely controlled (Shah, 2003; Fu and Dong, 2013). When challenged by a pathogen, plants need to rapidly synthesize more SA at the challenge sites to promote cell death. An amplification loop of immune response may exist. For example, SA itself can activate the expression of R genes and further promote downstream signals to synthesize more SA (Yang and Hua, 2004). Previous studies have usually focused their attention on the upstream regulation of SA, but with very little attention being paid to downstream regulation of SA. Whether the downstream events of SA biosynthesis pathway have an important role in modulating SA concentration is largely unknown. Recently, it was reported that the concentrations of DHBA glycosides increased after plants were inoculated with Pst DC3000 (Bartsch et al., 2010). It was also noticed that expression of both S3H and S5H were induced by plant pathogens, affecting the SA level (Zhang et al., 2013,Zhang et al., 2017). These findings suggested the importance in SA signaling of downstream metabolic processes of SA. In a previous study, glycosyltransferase UGT76B1 was found to be involved in the modulation of SA levels and defense responses (von Saint Paul et al., 2011). Isoleucic acid (2-hydroxy-3-methyl-pentanoic acid) was identified to be a substrate for UGT76B1, and UGT76B1 is known to function as a novel player in SA-JA signaling crosstalk (von Saint Paul et al., 2011). However, the step at which isoleucic acid influences SA level or its action mechanism is currently not clear. In another report, UGT76B1 was found to have SA glycosyltransferase activity, and some immune-priming compounds can repress the activities of SA glycosyltransferases including UGT74F1and UGT76B1 (Noutoshi et al., 2012). These examples suggest the complexity of regulation mechanisms in SA levels and defense response.

In this study, our focus was on the glycosylation of 2,3- and 2,5-DHBA, the two main forms of hydroxylated SA. UGT76D1 was found to encode DHBA glycosyltransferase, which can convert both 2,3-DHBA and 2,5-DHBA to their glycosides, particularly the glucosides. More importantly, we found that the levels of SA can be changed after disturbing the expression of UGT76D1 in the pathogen infection process. Our analyses indicated that the key SA synthase gene, ICS1, was significantly upregulated in UGT76D1 overexpression lines but downregulated in UGT76D1 knockout mutants. The findings presented here suggested that the glucosylation of DHBA catalyzed by UGT76D1 may be involved in the positive feedback regulation of SA synthesis. Therefore, this study revealed that the glucosylation of DHBA plays a previously unrecognized role in modulating SA homeostasis and plant immune response.

In conclusion, we propose a putative working model for UGT76D1 (Fig. 10). When plants are challenged by pathogens, expression of UGT76D1 is induced to a high level, and the formation of DHBA glycosides is accelerated. At the same time, DHBA glycosylation may activate SA synthase and then increase SA synthesis through an unknown positive feedback loop, possibly through increasing the metabolic flux toward SA and the DHBAs. High levels of SA accumulation lead to oxidative burst and PR gene expression and ultimately lead to PCD and increased plant resistance to pathogens. Considering the xylosyltransferase activity of UGT89A2 toward DHBAs, we cannot exclude the role of UGT89A2 in this immune-priming process. However, the detailed mechanism by which DHBA glycosylation regulates SA biosynthesis requires further investigation.

Figure 10.

Figure 10.

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Proposed scheme for the regulation of SA homeostasis and plant defense responses by UGT76D1. When plants are challenged by pathogens, expression of UGT76D1 is induced to a high level, and the formation of DHBA glycosides is accelerated. At the same time, DHBA glycosylation may activate SA synthase and then increase SA biosynthesis through an unknown positive feedback loop, possibly by increasing the metabolic flux toward SA and the DHBAs. High levels of SA accumulation lead to an oxidative burst and PR gene expression, and ultimately lead to PCD and increased plant resistance to pathogens. Considering the xylosyltransferase activity of UGT89A2 toward DHBAs, we cannot exclude the role of UGT89A2 in this immune-priming process.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 (Col-0) was used in this study. For selection of transgenic plants, SA treatment, and GUS staining, seeds were sterilized and imbibed for 3 d at 4°C in the dark and sown on plates with Murashige and Skoog medium and 1% (w/v) Suc, solidified with 0.8% (w/v) phytoagar. The plates were transferred to a growth room with a temperature at 22°C in a long day (16 h light/8 h dark). For all other analyses, plants were grown for 1 week as described above and transferred to soil under a long-day condition (16 h light/8 h dark, ∼100 µmol photons m−2 s−1) for the indicated times. A short-day condition (8 h light/16 h dark) was used to determine whether the lesion phenotype of UGT76D1 overexpression lines is influenced by light cycle.

GUS Staining

Sequences about 1500 bp upstream of the UGT76D1 transcription start were amplified from the Arabidopsis genome by PCR using UGT76D1 promoter-specific primers (Supplemental Table S4). The amplified sequences were inserted into the pBI121 plasmid to replace the CaMV35S promoter. Arabidopsis was transformed with the UGT76D1pro:GUS vector to generate UGT76D1pro:GUS transgenic plants through the floral dip method (Clough and Bent, 1998).

For GUS staining assays, leaves of 2-week-old transgenic seedlings of UGT76D1pro:GUS were soaked in 10 mm MgCl2 (control) or Pst DC3000 (OD600 = 0.01). Seedlings were then collected in different time points for GUS staining. At each time point, GUS activity was measured in at least 12 individual plants, most of them with similar results were used to take photos.

GUS staining was performed as described by Wang et al. (2012). Briefly, two independent lines of the UGT76D1pro:GUS transgenic plants were first fixed with 90% acetone on ice for 20 min and then washed with staining buffer [50 mm sodium phosphate, pH 7.2, 0.2 mM K3Fe(CN)6, 0.2 mM K4Fe(CN)6, and 0.2% Triton X-100] without X-Gluc (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) twice before adding staining buffer with X-Gluc (final concentration of 2 mm) and incubation overnight at 37°C. Two independent transgenic lines showing similar results were used as sources for the photos.

Plasmid Construction and Creation of Transgenic Arabidopsis Lines

Full-length cDNA of UGT76D1 was obtained from total RNA of Arabidopsis Col-0 by RT-PCR using specific primers (Supplemental Table S4). The cDNA was inserted into plasmid pBluescript SK and then transferred to the plant expression vector pBI121 to replace the GUS gene. The constructed plant expression plasmids were used to transform Arabidopsis, using the floral dip method (Clough and Bent, 1998). The transgenic plants were selected for kanamycin resistance. After selection for three generations, the homozygous UGT76D1 overexpression lines were obtained. Two independent overexpression lines (OE-17 and OE-32), with high expression levels of UGT76D1 and similar phenotypes, were selected for further study. UGT76D1 mutant lines were obtained, using the CRISPR/Cas9 system (Feng et al., 2013; Li et al., 2013). The 19-bp sequence (5′-GCGTCCTACCTTTCTTCCC-3′), with high specificity to the UGT76D1 gene, was selected as the targeting sequence for UGT76D1. Two knockout mutant lines (ko-33 and ko-36) with different deletions in the 5′ coding region of UGT76D1 were selected for further study.

DAB and Trypan Blue Staining

Approximately 4-week-old plants were used in these staining experiments. For detecting H2O2, DAB staining was performed as described by Thordal-Christensen et al. (1997). Whole rosette leaves were immersed in a 1% solution of DAB in Tris-HCl buffer (pH 6.5) for 16 h at 25°C under dark conditions and then cleared by boiling destaining solution (ethanol:acetic acid:glycerol = 3:1:1) for 10 min. H2O2 accumulation was visualized by the yellow-brown color.

For detecting cell death, Trypan blue staining was carried out according to the method described previously (Yang et al., 2015). Plant leaves were boiled 1 min in Trypan Blue solution (10 mL of lactic acid, 10 mL of glycerol, 10 g of phenol, and 10 mg of Trypan Blue, dissolved in 10 mL of distilled water) diluted in 1 volume of 100% ethanol. Leaves were then bleached in choral hydrate (2.5 g mL−1) overnight and conserved in 50% glycerol.

Protein Purification and Enzyme Assays

Full-length cDNA of UGT76D1 was inserted into plasmid pBluescript SK and then transferred to the prokaryotic expression vector pGEX-2T. The UGT76D1-GST fusion protein was expressed in Escherichia coli XL1-Blue and then purified using the method described previously (Hou et al., 2004). The enzyme assays were performed at 30°C using methods described previously (Lim et al., 2001) with modifications. About 2 μg of UGT76D1 fusion protein was added to 200 μL enzyme reaction mixture. The reaction mixture contained 100 mm Tris-HCl (pH 7.6), 14 mm 2-mercaptoethanol, 5 mm UDP-Glc or UDP-Xyl, 2.5 mm MgSO4, 10 mm KCl, and 1 mm substrate. The tested substrates in this study are listed in Supplemental Table S2. The reaction products were analyzed by HPLC and confirmed by LC-MS.

Measurements of SA, DHBA, and DHBA Glycosides

Four-week-old plants were harvested for the analysis of SA and DHBA glycoside concentrations. Free SA and total SA were extracted and measured by the methods described previously (Nawrath and Metraux, 1999). The internal reference 2,6-dichloroisonicotinic acid was used to estimate the loss rates during extraction. SA was detected with a 295-nm excitation wavelength and a 405-nm emission wavelength, using a fluorescence detector. DHBA glycosides were extracted and measured by the protocol described previously (Zhang et al., 2012,Zhang et al., 2013), with modifications. Briefly, DHBA glycosides were extracted from leaves homogenized in 80% (v/v) methanol overnight at 4°C. Caffeic acid and ferulic acid were used as the internal references to estimate the loss rates during extraction. Target compounds were analyzed by HPLC (Shimadzu) on a C18 column at a flow rate of 0.7 mL min−1. The mobile phases were composed of sodium acetate (50 mm, pH 5.5) and methanol. DHBA glycosides were detected using a UV detector at 310-nm wavelength. The identity of these compounds following HPLC profiling by retention time were confirmed by LC-MS (Thermo Scientific) and UV absorbance. 2,3-DHBA and 2,5-DHBA were extracted and measured as previously described (Zhang et al., 2017).

To quantify SA concentration, a SA standard curve was constructed. The peak area of SA showed a close linear relationship with concentration in the standard curve. Because the DHBA glycosides are not commercially available, the relative concentrations of DHBA glycosides were analyzed in this study. The peak areas of DHBA glycosides from the overexpression lines and knockout mutant lines were determined and compared to the corresponding values from the wild type.

RNA Extraction and Gene Expression Analysis

For analysis of gene expression, the rosette leaves from 4-week-old plants (six plants grown in different pots at the same time for each technical replicates) were collected for RNA extraction. Total RNA was extracted using the Trizol reagent (TaKaRa). Reverse transcription was performed using Prime Script RT reagent kit (TaKaRa). RT-qPCR was performed with a Bio-Rad thermal-cycling system (CFX Connect), using a SYBR Green PCR Master Mix kit (TaKaRa). ACTIN2 was used as the internal control. Results were normalized to the reference gene ACTIN2 using the ΔΔCt method. Primers used in RT-qPCR are listed in Supplemental Table S4.

Pst DC3000 Cultivation and Challenge Assays

Pst DC3000 was used for most of the pathogen assays, while Pst DC3000 (avrRpt2) was used for HR assays. Both bacterial strains were cultured in King’s B liquid medium (King et al., 1954) at 30°C, washed twice in 10 mm MgCl2, and resuspended at OD600 = 0.001 (for Pst DC3000) or 0.02 [for Pst DC3000 (avrRpt2)]. The resulting suspension solution was injected into the leaves of 4-week-old plants; MgCl2 (10 mm) was used as the negative control. Inoculated leaves were collected at different time points to determine the expression of SA-related genes in response to pathogen challenge. SA levels were determined at 48 h after injection.

Statistical Analysis

All experiments were carried out with at least three independent biological replicates, with each measurement also being carried out in triplicate. Data are presented as the mean ± sd. Data were statistically analyzed using Student’s t test. Asterisks indicate significant differences relative to the wild type or control (*P < 0.05 and **P < 0.01).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers UGT76D1 (At2g26480), PR1 (At2g14610), PR2 (At3g57260), EDS1 (At3g48090), PAD4 (At3g52430), and ICS1 (At1g74710).

Supplemental Data

The following supplemental materials are available.

  • Supplemental Figure S1. Generation and identification of UGT76D1 mutants and overexpression lines.

  • Supplemental Figure S2. Exaggerated necrotic lesion phenotype of UGT76D1 overexpression lines under short-day conditions (8 h light/16 h dark).

  • Supplemental Figure S3. The leaf senescence phenotype of UGT76D1 overexpression and mutant lines.

  • Supplemental Figure S4. LC-MS analyses confirmed the glycoside formation from UGT76D1 catalyzed 2,3-DHBA and 2,5-DHBA under positive ion mode.

  • Supplemental Figure S5. Analyses of DHBA glucoside in UGT76D1 overexpression and mutant lines after application of DHBA.

  • Supplemental Figure S6. LC-MS analyses of free forms of 2,3-DHBA and 2,5-DHBA in UGT76D1 overexpression and mutant lines with or without inoculation of Pst DC3000.

  • Supplemental Table S1. Cosegregation data of LMM phenotype and kanamycin resistance marker in T2 generation.

  • Supplemental Table S2. Candidate substrates detected for UGT76D1 enzymatic activity.

  • Supplemental Table S3. Glucosyltransferase activity of UGT76D1 and UGT89A2 toward 2,3-DHBA and 2,5-DHBA with different sugar donors.

  • Supplemental Table S4. Primers used in this study.

Acknowledgments

We thank Dr. Shuhua Yang (China Agricultural University) for providing the bacteria strains Pst DC3000, Pst DC3000 (avrRpt2), and the sid2 mutant seeds. We also thank Dr. Kewei Zhang (Zhejiang Normal University, China) for providing the s3h mutant and s3h s5h double mutant seeds and Dr. Jia Li (Lanzhou University, China) for providing the NahG transgenic seeds.

Footnotes

1

This research was supported by the National Natural Science Foundation of China (91217301, 31570299, and 31770313 to B.H.).

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