SUMO modification of LBD30 by SIZ1 regulates secondary cell wall formation in Arabidopsis thaliana

A wide range of biological processes are regulated by sumoylation, a post-translational modification involving the conjugation of SUMO (Small Ubiquitin-Like Modifier) to protein. In Arabidopsis thaliana, AtSIZ1 encodes a SUMO E3 ligase for SUMO modification. siz1 mutants displayed defective secondary cell walls (SCWs) in inflorescence fiber cells. Such defects were caused by repression of SND1/NST1-mediated transcriptional networks. Yeast two-hybrid assay indicated that SIZ1 interacts with the LBD30 C-terminal domain, which was further confirmed using bimolecular fluorescence complementation and immunoprecipitation. Mass spectrometry and co-immunoprecipitation indicated that SIZ1 mediates SUMO conjugation to LBD30 at the K226 residue. Genes controlling SCW formation were activated by the overexpression of LBD30, but not in the LBD30(K226R) mutant. LBD30 enhancement of SCW formation resulted from upregulation of SND1/NST1-mediated transcriptional networks. This study presents a mechanism by which sumoylation of LBD30, mediated by SIZ1, regulates SCW formation in A. thaliana.


Introduction
Plant cells are surrounded by walls that provide structural support and regulate growth. All plant cells form primary cell walls, which are synthesized during cell expansion and differentiation, while specialized cell types can also deposit a secondary wall on the inside of the primary wall once cell elongation has finished. Examples of the SCW are found in vascular tissues, such as in fiber cells and tracheary elements, as well as in other mechanically important tissues, for example, collenchyma cells. The major constituents of the SCW are cellulose, non-cellulosic polysaccharides and lignin. These polymers are cross-linked, providing cell walls with both mechanical strength and hydrophobic properties. Such characteristics are needed for upright growth, long-distance transport of solutes [1], selectivity of nutrient and water transport in root endodermis [2], defense against pathogens [3], and phenomena such as pod shattering [4], anther dehiscence [5] and flower abscission [6].
In the cells undergoing SCW biosynthesis, SCW cellulose synthase complexes in the plasma membrane produce β-(1-4) glucan chains that assemble into microfibrils in the orientation guided by cortical microtubules [7]. The microfibrils are extruded into the cell wall matrix and interact with Golgi-synthesized hemicellulose, generally xylan and mannan, to form a stable network [8]. Lignin monomers are transported to the space within the polysaccharide network where they are oxidized and polymerized to make matured SCW [9]. Genes responsible for the SCW biosynthesis process are regulated by a group of transcriptional activators and repressors, which constitute a hierarchical regulatory network controlling SCW formation in various locations [10]. For example, SND1 and NST1 control SCW deposition in fiber cells [11][12][13] while VND6 and VND7 are responsible for vessel cells SCW formation in A.thaliana [14,15]. Increasingly, post translational regulation of SCW formation is also being studied. For example, N-glycosylation regulates the enzyme activity of PtrMAN6 in suppression of SCW formation in Populus [16]. The phosphorylation of cellulose synthase AtCesA7 affected SCW cellulose biosynthesis in A.thaliana [17].
Sumoylation, conjugation of SUMO to substrate proteins, is a reversible and dynamic protein modification that regulates a range of biological processes [18]. SUMO conjugation forms a covalent bond between the C-terminal glycine carboxyl group of SUMO and the ε-amino group of a lysine residue, mostly occurring at the consensus motif CKXD/E (C, hydrophobic amino acid; K, lysine for conjugation; X, any amino acid; D/E, acidic amino acids) of target proteins [19]. Completion of sumoylation requires an enzymatic cascade of SUMO E1 activating enzyme, SUMO E2 conjugating enzyme and SUMO E3 ligase [18]. This process can be reversed through desumoylating proteases [20]. Generally sumoylation results in either stabilization of the target protein by protecting it against ubiquitylation [21,22] or destabilization by promoting the sumoylated protein for proteoasomal degradation [23]. Sumoylation can also alter protein cellular localization and modulate protein function or enzymatic activity [24]. In plants sumoylation plays a variety of roles in stress responses, growth, flowering, photomorphogenesis, nutrient homeostasis, and other biological processes [25,26].
In this study, we observed SCW defects in the A. thaliana siz1 mutants. Genetic and biochemical analyses indicate that the SCW defects were caused by failure of the LBD30 sumoylation which was mediated by SIZ1. The study reveals a mechanism that sumoylation functions as a regulatory expedient in SCW formation in A. thaliana.

siz1 mutants display SCW defects in inflorescence fiber cells
We screened an A. thaliana T-DNA insertion pool (Col-0 background) for the phenotypic abnormality of SCW formation in the inflorescence stem through microscopy observation. Two T-DNA insertion alleles, siz1-2 and siz1-3, which impair AtSIZ1 SUMO E3 ligase function [35] (Fig 1A), displayed morphological The siz1 mutant plants were smaller with shorter inflorescence stems compared to WT ( Fig  1A). To determine whether SIZ1 directly affects SCW formation, we employed an RNAi strategy to inhibit AtSIZ1 expression specifically in the cells forming secondary walls. The promoter of the fiber cell-specific SND1 was used to drive SIZ1-RNAi in P SND1 AtSIZ1-i transgenic  (Fig 1F and 1G) and siz1-3 (Fig 1K and 1L) showed a significant reduction in wall thickness compared to the wild-type (WT) (Fig 1B and 1C). Transmission electron microscopy analysis confirmed that siz1 mutants form much thinner cell walls in the fiber cells (Fig 1D, 1E, 1H, 1I, 1M and 1N), while the wall thickness of vessel cells showed little difference between the siz1 mutants and WT (Fig 1J).
https://doi.org/10.1371/journal.pgen.1007928.g001 plants (S1A Fig). In transgenic lines, expression of AtSIZ1 was suppressed by about 50% (S1E Fig). The wall thickness of the fiber cells in inflorescence stem was reduced compared to WT (S1B, S1C and S1F Fig). These suggest that SIZ1 plays a role in SCW formation in inflorescence fiber cells.
To investigate how SCW formation is changed in the siz1 mutants, we analyzed the chemical composition of their cell walls and examined expression of the SCW-related genes. In inflorescence stem crystalline cellulose and lignin were reduced by more than 20% in siz1 plants compared to WT (Fig 2A and 2B).

SIZ1 interacts with LBD30
AtSIZ1 promoter was active in cortex cells and interfascicular fibers of the inflorescence stem undergoing SCW formation (S2 Fig). SIZ1 is a nuclear-localized protein [27] and functions in facilitating SUMO conjugation to target proteins [47]. Using AtSIZ1 as the bait against a cDNA library made from A. thaliana inflorescence stem undergoing SCW formation, we conducted yeast two-hybrid (Y2H) screening to identify its target proteins for sumoylation. Among 191 identified candidates, four were found to be different parts from the ASYMMET-RIC LEAVES2/LATERAL ORGAN BOUNDARIES DOMAIN (AS2/LBD) protein, LBD30, encoded by At4g00220 locus [48,49]. We examined LBD30 expression in public databases and found that it is highly expressed in the inflorescence stem (S3 Fig). LBD/AS2 family proteins have a characteristic LOB domain at N terminus that possess DNA-binding ability [49,50]. We re-examined the interaction between AtSIZ1 and LBD30 in an Y2H system and found that AtSIZ1 interacted with LBD30 through its C-terminus (LBD30-C, amino acids 121-228) ( Fig  3A). This interaction was verified through

AtSIZ1 mediates SUMO1 modification of LBD30
SIZ1 interacted with LBD30, but expression of LBD30 was not altered in siz1 mutants (S4 Fig). LBD30 is predicted to contain a sumoylation motif (CKXE) with K226 as a potential SUMO conjugation residue (S1 Table) and showed a high possibility to be sumoylated among a list of SCW formation-related proteins [1] (S1 Table). Then we examined whether LBD30 could be SUMO conjugated at the CKXE motif. Using tandem mass spectrometry analysis, a mutant AtSUMO1 (T91R) protein, which allows production of a signature peptide containing a diglycine remnant at the sumoylation site [51], was identified at K226 in LBD30 ( Fig 4A). To verify this sumoylation, recombinant LBD30 was generated and the sumoylated LBD30 was detected in sumoylation assay ( Fig 4B). When LBD30 was mutated to generate a K226R variant (LBD30 K226R ), the substitution of K226 to R resulted in failure of SUMO1 conjugation to LBD30 ( Fig 4C) without affecting its nuclear localization (S5 Fig). Furthermore, we examined if the mutant LBD30 can be sumoylated by AtSIZ1 in planta. By combinational expression of LBD30 or LBD30 K226R , AtSIZ1 and AtSUMO1 in tobacco leaves, immunoblotting indicated only LBD30 is SUMO-conjugated (S6 Fig). Next, AtSUMO1 and LBD30 or LBD30 K226R were co-expressed in siz1-2 and WT A. thaliana. AtSUMO1 conjugation to LBD30 was only detected in the transgenic plants with WT background expressing LBD30 and AtSUMO1 ( Fig  4D). These demonstrated that SIZ1 mediates LBD30 sumoylation at the K226 residue.

AtSIZ1-mediated sumoylation of LBD30 affects SCW formation and development
We investigated the effect of LBD30 sumoylation in the transgenics overexpressing LBD30 and LBD30 (K226R) . Overexpression of LBD30 caused drastic phenotypic changes, severe dwarfism, short petioles and downward curled leaves. Ectopic lignin deposition was detected in cotyledons in 24 out of 28 T1 transgenic plants (Fig 5A-5C). In contrast, overexpression of LBD30 K226R showed little phenotypic changes (Fig 5A and 5D). Similarly, expression of LBD30 in siz1-2 caused no phenotypic change in 28 transgenic plants out of 36 T1 plants and minor changes in remaining 8 plants compared to the siz1-2 plants (Fig 5A, 5E and 5F). LBD30 sumoylation played a role in development and secondary cell wall biosynthesis.
Then, we investigated whether the SCW defects in siz1 plants is caused by failure of LBD30 SUMO modification. We examined the transcripts of SND1 and NST1 in the transgenics overexpressing LBD30 (S7A Fig). The transgenic plants were unable to develop normal inflorescence stem ( Fig 5A) but expression of SND1 and NST1 was drastically up-regulated in the 2 weeks-old seedlings ( Fig 5G). This upregulation of SND1 and NST1 expression was insignificant in the transgenics carrying LBD30 K226R or in the transgenics overexpressing LBD30 in siz1 mutant background (Fig 5G). When LBD30 was overexpressed in nst1/snd1 double Myc-tagged AtSIZ1 and HA-tagged LBD30 were expressed or coexpressed in tobacco leaves. Proteins were detected by immunoblotting with an anti-Myc antibody and an anti-HA antibody in crude lysates and in protein extracts after immunoprecipitation with an anti-Myc antibody and an anti-HA antibody, respectively. co-precipitation ( Fig 3B) and bimolecular fluorescence complementation (BiFC) assays ( Fig 3C). In addition, by co-expression of LBD30 and AtSIZ1 in tobacco (N. benthamiana) leaves, the two proteins interacted with each other (Fig 3D). Together, these results demonstrated that AtSIZ1 interacts with LBD30 at C-terminal. On the other hand, we evaluated the effect of LBD30 sumoylation on SND1 and NST1 expression using a dual luciferase assay in A. thaliana protoplasts. The effecter was constructed by using a 35S promoter to drive expression of LBD30 and LBD30 (K226R) . A firefly luciferase driven by SND1 or NST1 promoter was used as a reporter (Fig 5H and 5I). LBD30 showed a significantly higher activity in activation of SND1 or NST1 promoter than LBD30 (K226R) (Fig 5H and 5I), suggesting that LBD30 SUMO conjugation affected SND1 and NST1 expression. Thus, the SUMO modification of LBD30 played a role in regulating SCW formation through the SND1/NST1-directed transcriptional network.

Discussion
In higher plants all cells form primary cell wall. In some type cells, additional SCWs are formed inside the primary wall, providing plants with mechanical support for erect growth and channels for long-distance transportation of water, nutrients, and photosynthetic products. Formation of the SCWs in various type cells need to be precisely regulated in a spatio-temporal manner during growth and development [52]. To ensure a precise deposition of SCWs in some type cells, multiple levels of regulation have to be developed in plants. Disturbance of the regulatory networks causes abnormal growth and development [1]. At the transcriptional level, complex regulatory networks are involved in SCW formation [8,53]. SCW formation in different cell types is initiated through cell type-specific transcription regulators [11][12][13][14][15]. Many signaling molecules regulating SCW formation have yet-to-be characterized [54].
At the protein level, post-translation modifications, such as protein phosphorylation and N-glycosylation [16,17], are also being studied for their roles in regulating SCW formation. While a large number of proteins are modified with SUMO-conjugation and such modification affects a variety of biological processes [18], this study presents a detailed picture of how sumolyation can lead to the upregulation of SCW formation. Specifically, we found LBD30 sumoylation is required for activation of the SND1/NST1-mediated transcriptional networks in SCW formation.
AtSIZ1-mediated sumoylation is involved in a variety of growth and development processes such as flowering, response to light, immunity and nutrient element metabolisms in A. thaliana [31-34, 36, 55]. In this study, we observed that siz1 mutants displayed defective SCWs in LBD30 is a transcription factor belonging to the Lateral Organ Boundaries Domain (LBD) family [56,57]. LBD30 and its homolog LBD18 in A. thaliana were preferentially expressed in vascular tissues and LBD18 played a role in regulating tracheary element differentiation [57].
Defective SIZ1 or mutated LBD30 at K226 position led to loss of LBD30 function during the formation of SCW in interfascicular fiber cells. The evidence indicated that LBD30, when it was sumoylated by SIZ1, played a role in activating the SND1/NST1-mediated transcriptional networks (Fig 6) which regulate SCW formation in the fiber cells of inflorescence stem.
Generally, stress conditions cause activation of SCW formation [58,59]. Several transcription factors sumoylated by AtSIZ1 are related to stress responses, including ICE1 in freezing stress [39], HsfA2 in heat stress [60], PHR1 in phosphate (Pi) deficiency [35], MYB30 and ABI5 in the abscisic acid-dependent drought stress [42,43]. It is worthy of further study whether LBD30 sumoylation acts as a linking device between stress responses and SCW formation.
Generally LBD family proteins regulate plant development through interaction with other transcription factors [50]. A number of transcription factors have been identified to bind to SND1 and NST1 promoters to activate their expression [1,61]. In this study, we found that transcription factor LBD30 was sumoylated by SIZ1 and such protein modification affected activation of the SND1/NST1-mediated transcriptional networks for SCW formation in fiber cells. Though it remains to be investigated how LBD30 sumoylation performs its function in activation of the transcriptional networks, one possibility is that LBD30 sumoylation may affect the transcription factor interactions that are necessary for activation of SND1/NST1 expression. This possibility might justify the observation that LBD30 sumoylation showed different strength of effect on SND1 and NST1 expression between transgenics and protoplast system. Interaction of LBD30 with other factors in planta affected the SND1 and NST1 promoter activity.
The finding that LBD30 sumoylation acts as another layer of regulation to aid in the precise control of SCW formation provides additional insight into a key process that is essential for upright growth and the long-distance transport of water and solutes in plants and has implications in cell wall modification via regulation of LBD30 sumoylation in crop improvement.

Plant materials and culture conditions
The A. thaliana Col-0 ecotype (WT) and the T-DNA insertion mutant lines, siz1-2 (SALK_065397) [35], siz1-3 (SALK_034008) [35] and snd1/nst1 double mutant (CS67921) [11][12][13], were grown in a phytotron at 22˚C with a photoperiod of 16 h of light and 8 h of darkness. Transformation of A. thaliana was performed using the Agrobacterium tumefaciensmediated floral dip method [62]. Transgenic plants were selected on MS medium containing 50 μg/ml hygromycin. Positive T2 transgenic plants were used for further analysis, with the exception of LBD30 overexpressing plants in the Col-0 background, where T1 plants were used because the T1 transgenic displayed severe growth defects and hardly produced seeds.

Gene cloning and plasmid construction
cDNAs for AtSIZ1 (At5g60410), LBD30 (At4g00220), AtSUMO1 (At4g26840) and the promoter regions of AtSIZ1(3535bp), SND1(2858bp) and NST1(2913bp) were PCR-amplified from a cDNA pool of A. thaliana as well as from genomic DNA with specific primers listed in S2 Table. For the Y2H assay, the coding region of LBD30 and AtSIZ1 were inserted respectively into the pGBKT7 and pGADT7 plasmids (Clontech) and introduced into AH109 yeast cells (Clontech) following the manual. For BiFC analysis, LBD30-YC and YN-AtSIZ1 were constructed as previously described [63] and mobilized into A. tumefaciens strain GV3101 and transformed into Nicotiana benthamiana tobacco leaf cells [63]. For purification of recombinant proteins, the LBD30, a mutated LBD30 (K226R) and AtSIZ1 coding regions were cloned into the pET-28b (Novagen) and pGEX-4T-1 (GE Healthcare) plasmids to produce the His 6 -LBD30, His 6  For transcriptional activation analysis, the coding regions of LBD30 and LBD30 (K226R) and the promoter regions of the SND1 and NST1 genes were cloned into the effector (35S-transcription factor) and reporter (firefly luciferase) vectors (pGreenII vector, Promega) and then coexpressed in A. thaliana protoplasts [64]. For analysis of AtSIZ1 expression, an AtSIZ1 promoter fragment was cloned and fused to a β-glucuronidase (GUS) reporter gene in the pCambia1301 vector for A. thaliana transformation. To investigate the function of AtSIZ1 in inflorescence stems, two different genomic DNA fragments specific to AtSIZ1 were amplified separately to form hairpin structures under the control of the SND1 gene promoter. These constructs were designed to cause RNAi suppression (SND1promoter-AtSIZ1RNAi1 and SND1promoter-AtSIZ1RNAi2) specifically in A. thaliana inflorescence stems.

Microscopy analyses
The basal internodes of inflorescence stems of 8-week-old plants with the same flowering date were collected as described before. Briefly, the internodes were fixed in FAA overnight and embedded in paraffin (Sigma-Aldrich 18635) after dehydration through a graded ethanol series. Ten-micrometer-thick sections were cut and stained with toluidine blue for light microscopy. Free-hand cross sections of A. thaliana inflorescence stems were stained with 0.5% phloroglucinol (Sigma-Aldrich P3502) (w/v) in 12% HCl for 3 min, and immediately observed under a bright-field microscope (OLYMPUS BX53). For transmission electron microscopy, ultrathin sections were cut and observed as described [65]. To visualize lignin auto-fluorescence under UV light and the sub-cellular localization of GFP-fusion proteins, A. thaliana cotyledons were grown on MS plates and A. thaliana leaf protoplasts were observed using a fluorescent microscope (OLYMPUS BX53). For the BiFC analysis, tobacco leaf cells were stained with DAPI [66]and visualized using a confocal microscope (LSM 510 META; Zeiss).

Analysis of cell wall components
Fluorescence stems from at least three independent 8-week-old A. thaliana WT or mutant plants were collected and ground in liquid nitrogen to a fine powder to prepare alcohol insoluble residue (AIR) as previously described [67]. After the de-starched procedure [67], the crystalline cellulose content and monosaccharide composition were analyzed according to a previously published protocol [68]. The lignin content was determined following the methods in [69].

Gene expression analysis
Total RNA isolated from the lower center part of the inflorescence stem of 4-week-old A. thaliana plants and whole seedlings of 2-week-old WT, mutants and transgenic plants were extracted using the E.Z.N.A. Total RNA Kit (Omega) according to the manufacturer's instructions. cDNA was synthesized by treatment with reverse transcriptase and oligo (dT) primer (TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix, Transgene Biotech) and quantitative PCR assays were conducted with a MyiQ real-time PCR detection system (Bio-Rad) using SYBR Green (TransStart Top Green qPCR MIX) following the user manual. The A. thaliana ACT2 gene (AT3G18780) was used as an internal control to normalize the data. The mathematical analysis for qPCR quantification was delta-delta Ct method [70]. The quantitative PCR (qPCR) experiment was performed in biological triplicates.

GUS staining assay
Free-hand cross-sections of the lower internodes of the inflorescence stems from 4 week old AtSIZ promoter-GUS transgenic A. thaliana were examined for GUS activity as previously described [71].

Protein-protein interaction assay
To identify AtSIZ1 interacting proteins, a Y2H library was generated using cDNA derived from 4-week-old A. thaliana inflorescence stems and used to screen for target proteins, using the Make Your Own Mate & Plate Library System (Clontech), according to the manufacturer's directions. For the BiFC analysis, the constructs were transformed into Agrobacterium strain GV3101, and the resulting strains were used to transform N. benthamiana leaf cells, either individually or in combination. The leaves were examined after 48 h of incubation. To investigate the physical interaction between AtSIZ1 and LBD30 in vitro, recombinant His 6 -LBD30 and GST-AtSIZ1 proteins were expressed in Escherichia coli and purified with Ni-NTA Agarose (Qiagen) and Pierce GST Agarose (Thermo Scientific), according to the manufacturer's instructions. GST and GST-tagged AtSIZ1 proteins from the cell lysates were first immobilized on the GST Agarose (Thermo Scientific). After washing away unbound proteins with 1×PBS, the immobilized GST and GST-AtSIZ1 proteins were incubated with the cell lysate of Escherichia coli expressing His 6 -LBD30. After several washing steps with 1×PBS, the complexes were eluted with 2×SDS loading buffer and boiled at 100˚C for 5 min. The eluted proteins were separated by SDS-PAGE, transferred to a PVDF membrane and the protein was immunoblotted with an anti-His antibody (1:5000 dilution, Abmart).

Sumoylation assay
The in vitro sumoylation was performed using the SUMOlink TM SUMO-1 Kit (Active Motif). Briefly, recombinant His 6 -LBD30 and His 6 -LBD30 (K226R) proteins were expressed in E. coli and purified. A total of 3 μg of target protein was added to 20 μl reaction buffer and incubated at 30˚C for 3 h. The reaction was stopped by adding 10 μl of 2×SDS-PAGE loading buffer. Sumoylated of His 6 -LBD30 was detected by immunoblot analysis using an anti-His mouse monoclonal antibody (1:5000 dilution, Abmart) and a SUMO-1 rabbit antibody (1:2000 dilution, Active Motif).
The reaction mixture was also separated by SDS-PAGE. After staining with Coomassie Blue R-250, the sumoylated His 6 -LBD30 protein band was cut into 1 mm wide pieces for digestion and liquid chromatography-tandem mass spectrometry (LC-MS ⁄MS) analysis of the LBD30 sumoylation site. Protein digestion for LC-MS/MS analysis was performed by the Beijing Protein Institute. Briefly, the protein bands were destained with 50% v/v acetonitrile (ACN) [72] and 25 mM ammonium bicarbonate and dried in 100% ACN and the gel slices were incubated with a 10 ng μl -1 trypsin solution in 25 mM ammonium bicarbonate at 37˚C for 12h. The extracts were then dried in a stream of N 2 and resuspended in 5% ACN in 0.1% v/v formic acid FA. LC-MS ⁄MS analysis was performed using an Ultimate3000 liquid chromatography system (Dionex) connected to a Q Exactive mass spectrometer (Thermo Scientific) as decribed previously [72] with modifications. The extracts were separated by a C18 reverse-phase column with a 1 hour gradient of mobile phase (phase A, 5% ACN in 0.1% FA; phase B, 95% CAN in 0.1% FA) at a flow rate of 300 nL / min. The separated sample was then injected into the mass spectrometer and a method of full scans were acquired with AGC target value of 1E6, resolution of 70,000 FWHM at 200 m/z, and maximum ion injection time (IT) of 100 ms. The mass spectura were extracted by BioWork version 3.3.1 sp1 (Thermo Fisher). All MS/MS samples were analyzed using Mascot software (Marix Science).
For the in vivo sumoylation assay, the Myc tagged AtSIZ1, the FLAG-tagged AtSUMO1 and the HA-tagged LBD30 or LBD30 (K226R) were expressed in tobacco leaves. The FLAG-At-SUMO1 transgenic A. thaliana plants (Col-0 background) were crossed with LBD30-HA or LBD30 (K226R) -HA transgenic A. thaliana plants (siz1-2 background). F2 progeny of transgenic plants with WT and siz1-2 background overexpressing FLAG-AtSUMO1 and LBD30-HA or LBD30 (K226R)-HA were obtained. Total proteins were extracted and immunoprecipitated with an anti-FLAG mouse monoclonal M2 affinity gel (Sigma-Aldrich). The sumoylated LBD30 was detected by immunoblotting with an anti-HA Rat monoclonal high-affinity antibody (1:2000 dilution, Roche) after IP.

Dual luciferase assay
Protoplasts used in the transient effector-reporter analysis were isolated from 2-week-old A. thaliana seedlings as previously described [64]. The coding sequences of LBD30 and LBD30 (K226R) were cloned into the effector plasmid. The promoters of SND1 and NST1 were cloned into the firefly luciferase reporter vector (pGreenII, Promega). The Renilla luciferase gene driven by the CaMV 35S promoter served as a control to normalize for transformation efficiency. Luciferase activities were measured with a dual-luciferase reporter assay system (Promega).