Involvement of co-repressor LUH and the adapter proteins SLK1 and SLK2 in the regulation of abiotic stress response genes in Arabidopsis
© Shrestha et al.; licensee BioMed Central Ltd. 2014
Received: 18 November 2013
Accepted: 6 February 2014
Published: 24 February 2014
During abiotic stress many genes that are important for growth and adaptation to stress are expressed at elevated levels. However, the mechanisms that keep the stress responsive genes from expressing under non stress conditions remain elusive. Recent genetic characterization of the co-repressor LEUNIG_HOMOLOG (LUH) and transcriptional adaptor proteins SEUSS-LIKE1 (SLK1) and SLK2 have been proposed to function redundantly in diverse developmental processes; however their function in the abiotic stress response is unknown. Moreover, the molecular functions of LUH, SLK1 and SLK2 remain obscure. Here, we show the molecular function of LUH, SLK1 and SLK2 and the role of this complex in the abiotic stress response.
The luh, slk1 and slk2 mutant plants shows enhanced tolerance to salt and osmotic stress conditions. SLK1 and SLK2 interact physically with the LUFS domain in LUH forming SLK1-LUH and SLK2-LUH co-repressor complexes to inhibit the transcription. LUH has repressor activity, whereas SLK1 and SLK2 function as adaptors to recruit LUH, which in turn recruits histone deacetylase to the target sequences to repress transcription. The stress response genes RD20, MYB2 and NAC019 are expressed at elevated levels in the luh, slk1 and slk2 mutant plants. Furthermore, these stress response genes are associated with decreased nucleosome density and increased acetylation levels at H3K9 and H3K14 in the luh, slk1 and slk2 mutant plants.
Our results indicate that SLK1, SLK2 and LUH form a co-repressor complex. LUH represses by means of an epigenetic process involving histone modification to facilitate the condensation of chromatin thus preventing transcription at the target genes.
Plant’s ability to perceive and respond to various environmental stresses including too little water (drought), too much salt (salinity) and extremes of temperature, depends on appropriate regulation of gene expression. Abiotic stress causes both up and down regulation of gene expression [1–3] . Many of the up regulated genes encode proteins that can be classified into two groups: genes coding for the transcription factors and genes encoding proteins involved directly in response mechanisms . Genes of both classes are controlled at the level of transcription. The molecular mechanisms of specific transcriptional factors that bind to the conserved cis-acting promoter elements in plants are well studied, especially for the abiotic stress-induced up-regulated genes [4–7]. In contrast, mechanisms whereby abiotic stress regulated genes are kept silent in the absence of stress has not been well investigated. To silence gene expression, eukaryotes employ transcriptional repression as a key regulatory mechanism. Transcriptional repression plays a critical role in cell fate specification and body patterning in both animals and plants [8, 9]. Transcriptional activation and repression occur within the context of chromatin organization in eukaryotes. Chromatin structure is a governed process often associated with epigenetic regulation, namely, histone post-translational modifications, histone variants and DNA methylation that alter chromatin compaction resulting in altered accessibility of genes to transcriptional regulation [10, 11].
Transcriptional repression is mediated by an important and extensively studied class of co-repressors, those belonging to the Gro/Tup1 family, including Tup1 in yeast (Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans), Groucho (Gro) in Drosophila, and Transducin-like enhancer of split (TLE) in mammals. These co-repressor proteins, collectively called the Gro/Tup1 family [12, 13], do not possess direct DNA-binding capability. They repress a diverse number of target genes through targeted recruitment to the DNA template via protein-protein interactions with a variety of DNA-bound transcription factors to mediate repression .
The Gro/Tup1 family consists of 13 members in Arabidopsis, and the functions of only a few have been studied [9, 12, 13]. LEUNIG (LUG) was the first Gro/Tup1 family member to be characterized in Arabidopsis. The LUG protein has LisH and LUFS domains at the N-terminus, and resembles Gro/Tup1 in having a Q-rich and seven WD domains. The LisH (lissencephaly homology) domain is a dimerization motif that is present in all plant Gro/Tup1 proteins. The LUFS (named after LUG, LUH, yeast Flo8 and human SSDP) domain is present only in LUG and LUH among the Gro/Tup1 family members present in Arabidopsis. LUFS domain is involved in protein-protein interactions [12, 13]. The LUFS domain in LUG interacts physically with SEUSS (SEU), a Q rich protein with a conserved domain that is similar to the dimerization domain of the LIM-domain binding (Ldb) family of transcriptional co-regulators in mouse and Drosophila[14, 15]. SEU forms a co-repressor complex with LUG and acts as an adapter between LUG and a variety of transcription factors to mediate repression of diverse target genes during floral organ identity, floral patterning and abaxial organ identity in leaves [16–18].
LUH is another member of the Gro/Tup1 family and highly similar to LUG with 44% identity in Arabidopsis[12, 13, 19]. Thus, not surprisingly LUH functions redundantly to some extent with LUG in abaxial organ identity in leaves and identity of floral organs [18, 19]. Furthermore, LUH interacts with SEU in a yeast two hybrid assay suggesting that SEU-LUH complex could functionally substitute for the SEU-LUG complex to mediate repression at target gene which may possibly explain the redundant functions . In addition, LUH also interacts with SLK1 and SLK2 and functions redundantly with LUG in abaxial organ identity . Until recently, LUH function in addition to its minor role in development was not known. Several recent reports indicate that LUH plays an important role in regulating pectin structure and mutants lacking LUH fail to release mucilage from the seed coat. One relevant target of LUH is MUM2, a β-galactosidase involved in the modification of the mucilage [20–22]. At present, there are two plausible mechanisms to account for MUM2 regulation. The first is by LUH acting as a direct positive regulator of MUM2. The other mechanism involves LUH acting as negative regulator of a MUM2 repressor.
Although LUH shows significant sequence similarity with LUG, the molecular function of LUH remains unclear. The only known function of LUH is its major role in mucilage secretion in Arabidopsis. In this study, we present results indicating involvement of LUH in the abiotic stress response. We demonstrate that LUH functions as a transcriptional repressor similar to Gro/Tup1 family proteins. Additionally, we show that the conserved LUFS domain in LUH physically interacts with adaptor proteins SLK1 and SLK2 which do not show repressor activity themselves. The luh, slk1 and slk2 mutant plants shows elevated salt and osmotic stress tolerance and higher expression levels of abiotic stress responsive gene under non-stress conditions. In addition, LUH physically interacts with histone H2B and H3 and either directly or indirectly regulates chromatin structure at the abiotic stress responsive genes. These data provide an insight into the novel roles for LUH, SLK1 and SLK2 in abiotic stress response gene regulation and illuminate LUH function in chromatin remodeling.
luh-4, slk1-1 and slk2-1plants exhibit tolerance to salt and osmotic stress
To verify whether the salt and osmotic stress tolerance in slk1-1, slk2-1 and luh-4 is due to loss of function. We transformed the mutants with native gene promoter containing wild type coding sequence. Transgenic mutants with wild type coding sequence complemented the salt tolerance phenotype and were similar to wild type plants when grown on MS medium supplemented with 125 mM NaCl for salt stress (Additional file 2: Figure S1A). The root length of complemented mutants were comparable to the wild type plants when grown on medium containing 125 mM NaCl and 300 mM mannitol (Additional file 2: Figure S1B, D). In addition, the fresh weight of the complemented mutants were similar to the wild type plants when grown on medium supplemented with 125 mM NaCl and 300 mM mannitol (Additional file 2: Figure S1C, E). Expression level of SLK1, SLK2 and LUH gene in the complemented plants were similar to their expression in the wild type plants (Additional file 3: Figure S2).
To determine whether the double mutants show enhanced tolerance to the salt and osmotic stress compared to the single mutants, we constructed slk1-1 /luh-4 and slk2-1/luh-4 double mutants. The double mutants did not exhibit significant differences in the root length and fresh weight compared to the single mutants when subjected to salt and osmotic stress (Additional file 1: Table S1). Additionally, altered response to the plant hormone abscisic acid (ABA up to 10 μM concentration) and freezing tolerance (-4°C to -10°C) was tested and these responses were unchanged in the single mutants compared to the wild type plants (unpublished data). Collectively these data show that loss of function in LUH, SLK1 and SLK2 results in enhanced tolerance to salt and osmotic stress in the single mutants compared to the wild type plants.
SLK1 and SLK2 interact with the LUFS domain in LUH
To confirm LUH, SLK1 and SLK2 interactions in plants, we performed split luciferase complementation assays in Arabidopsis protoplasts  by fusing luciferase N-terminal fragment translationally to full length LUH and to the LUFS domain alone. The C-terminal fragment was fused to SLK1 or SLK2. In agreement with the yeast two hybrid assays, protoplasts transfected with LUH-SLK1 and LUH-SLK2 plasmids showed elevated levels of luciferase activity compared to vector treated and N-terminal fragment fused with LUH alone (Figure 2C). The SLK2 interaction with LUH was higher compared to SLK1. Moreover, SLK1 and SLK2 interaction with the LUFS domain were as strong as full length LUH supporting the idea that the LUFS domain is sufficient for interaction with SLK1 and SLK2 to form LUH-SLK1 and LUH-SLK2 co-repressor complexes in vivo (Figure 2C).
LUH has repressor activity
Co-repressors in the Gro/Tup1 family mediate repression by recruiting histone deacetylases (HDACs) to the target genes [12, 13]. In order to determine whether a similar mechanism is utilized by LUH, we performed repression assays in protoplasts in presence of TSA, an inhibitor of HDAC activity. Interestingly, LUH failed to repress the reporter gene in the presence of TSA (Figure 3C) indicating that LUH employs a highly conserved process that involves recruitment of HDACs to mediate repression at the target loci .
LUH negatively regulates abiotic stress response genes
MYB2 and NAC019 are transcription factors that are implicated in the regulation of several abiotic stress response genes [26–28]. Furthermore, elevated expression of RD20 confers abiotic stress tolerance . These data indicate that the loss of function in SLK1, SLK2 and LUH results in increased expression of RD20, transcription factors MYB2 and NAC019 and could possibly result in the improved tolerance to the abiotic stress in these mutant plants.
LUH alters the chromatin state at the abiotic stress response genes
We did not determine the acetylation status in H2B due to the lack of plant specific antibodies. In conclusion, LUH interacts with histone H2B and H3 and recruits HDAC to eliminate the acetylation on histone H3 at the positions Lys-9 and Lys-14. Furthermore, the presence of LUH could increase the nucleosome density resulting in the condensation of the chromatin and hindering the active transcription at the target genes.
In Arabidopsis, LUG and TOPLESS (TPL) are the most studied Gro/Tup1 co-repressors that are implicated in developmental processes and hormone signaling [9, 12, 13]. LUH is the homolog of LUG and plays critical role in mucilage excretion [20–22]. Expression profile analysis indicated that the LUH is differentially regulated during abiotic stress compared to LUG and could play a role in the abiotic stress response . Surprisingly, HOS15, belonging to Gro/Tup1 family, was identified in a forward genetic screen involving abiotic stress response, and loss of function in HOS15 results in freezing sensitivity . These studies prompted us to investigate LUH function in abiotic stress response and here we show that LUH is indeed involved in abiotic stress response thus broadening the function of LUH. Loss of function mutation in LUH results in plants that are more tolerant to salt and osmotic stress compared to the wild type plants. LUH interacts with SEU, an adaptor protein that links LUH to the transcription factor, and interestingly, SEU mutants do not show tolerance to salt and osmotic stress. In Arabidopsis, there are three SEU like genes , and we found that loss of function in SLK1 and SLK2 confers salt and osmotic tolerance similar to LUH when mutant plants were subjected to the stress conditions. SEU, SLK1 and SLK2 function redundantly in embryonic development mediated through plant hormone auxin and in the outer integument development in the ovule . Our results provide a novel function for the SLK1 and SLK2 in the abiotic stress response outside their role in the development. Double mutant analysis with slk1-1/luh-4 and slk2-1/luh-4 for salt and osmotic stress indicated that slk1, slk2 and luh functions in the same genetic pathway. We did not observe differential responses in slk1, slk2 and luh compared to wild type plants during freezing and plant hormone ABA treatment.
Genetic analysis indicated that LUH function is dependent on SLK1 and SLK2. Yeast two hybrid and in planta analyses in protoplasts indicated that the LUH interacts with SLK1 and SLK2 confirming an earlier study . Interestingly, LUFS domain in LUH is sufficient for interaction with SLK1 and SLK2 which is similar to the interaction of SEU with the LUFS domain in LUG . SEU, SLK1 and SLK2 are highly similar with a centrally positioned Q-rich region containing a Ldb1/chip conserved domain (LCCD) that is likely to interact with the LUFS domain . Phylogenetic analysis indicated that SEU is more closely related to SLK2 than to SLK1 which could explain the stronger interaction between LUH and SLK2 compared to SLK1 [18, 23].
The molecular functions of SLK1, SLK2 and LUH were unknown. Our results indicate that only LUH has transcriptional repressor activity. Interestingly, co-transfection with either SLK1-BD or SLK2-BD with CaMV 35S::LUH gave repressor activity. These results indicate that SLK1 and SLK2 function as adaptors to recruit LUH, which serves as the repressor within this complex. A recent study showed that LUH functions as an activator in Arabidopsis protoplasts in contrast to the repressor function observed in our study . One possibility for this observed function of LUH could be due to the different reporter systems used in the protoplast assay. In our assay, the reporter has 5X Gal sequence upstream of constitutive CaMV 35S promoter; in contrast, the published study used 4X Gal sequence with the reporter gene without constitutive promoter in between 4X Gal sequence and the reporter gene . In our view, LUH functions as the repressor and this is supported by the observation that in the luh mutant, expression of LUG which has repressor activity [14, 36] restores the mucilage deficiency phenotype in the luh plants . It is possible that in some cellular or developmental contexts LUH may function as an activator, although this mode of regulation has little empirical support.
The repressor activity of the LUH in protoplasts is eliminated by addition of TSA suggesting that the repression is mediated by recruiting HDAC. Arabidopsis encodes 18 HDACs and plays critical role in development, growth and hormone signaling [37–40]. Recent studies indicate that HDA19 genetically and physically interacts with co-repressors LUG and TPL, and has been implicated in flower development [9, 36, 41]. Our preliminary results indicate that LUH does not interact with the HDA19 and the identity of the HDAC that interacts with the LUH remains to be established (unpublished data).
To explain the observed salt and osmotic stress tolerance in slk1, slk2 and luh mutants, we performed quantitative RT-PCR for the abiotic stress response genes that show elevated transcripts level compared to the wild type plants thus contributing to the stress tolerance. Our results indicate that RD20, MYB2 and NAC019 genes are expressed at elevated level in these mutants compared to wild type plants. RD20 gene is a well known abiotic stress inducible marker and participates in stomatal control and transpiration in Arabidopsis thus conferring abiotic stress tolerance . The MYB2 gene encodes a R2R3 MYB domain-containing transcription factor that regulates several salt and drought stress responsive genes [27, 42]. NAC domain-containing transcription factors are prominent plant specific transcription factors and NAC019 is one of the 110 genes that are encoded in the Arabidopsis genome . NAC019 gene is induced by salt and dehydration stress, and over expression in the transgenic plants results in the induction of several stress response genes hence conferring abiotic stress tolerance [38, 43, 44]. Interestingly, NAC019 regulatory region has an MYB binding site, and MYB2 transcription factor binds to the NAC019 regulatory region in a yeast one hybrid assay . However, NAC019 gene activation by MYB2 in planta has not been demonstrated.
The three identified target genes are not adequate to explain the observed salt and osmotic stress phenotype in the luh-4, slk1-1 and slk2-1 mutant plants. Therefore, it appears that several positive factor genes are expressed in these mutants compared to wild type plants that confer abiotic tolerance. Further studies are required to identify additional target genes.
Since SLK1, SLK2 and LUH lack DNA binding domain, the mechanisms of recruitment of SLK1-LUH and SLK2-LUH complexes to the regulatory region of RD20, MYB2 and NAC019 genes are unknown. Among the possible mechanisms, one could be that SLK1 and SLK2 interacts with different sequence specific transcription factor or SLK1 and SLK2 form heterodimeric complexes that bridge the transcription factor and LUH at the target regulatory region. Identification of specific transcription factors that interact with SLK1 and SLK2 and in vivo association at the regulatory region would illustrate the precise mechanism of SLK1-LUH and SLK2-LUH recruitment to the target genes.
Chromatin structure within a gene largely determines its transcriptional state and expression levels and can be changed with modification at the N- terminal tails of histones. One of the key mechanisms in chromatin remodeling is histone acetylation and deacetylation, mediated by the enzymes histone acetyl transferase (HAT) and HDAC respectively [33, 39]. The role of chromatin remodeling is well established in transcriptional gene silencing and in control of flowering response by vernilization in Arabidopsis[10, 45]. Recent studies suggest that abiotic stress response gene expression also depends on chromatin remodeling, yet how this distinctive chromatin state is established is not known . In addition, the function of Gro/Tup1 family of co-repressors in chromatin remodeling in Arabidopsis is not well understood. Our results demonstrate that LUH interacts with histone H3 and H2B. Furthermore, the chromatin state is altered at target genes that are expressed at elevated levels. We observed that nucleosome density at target genes RD20, MYB2 and NAC019 that are expressed in the slk1-1, slk2-1 and luh-4 mutants are reduced compared to the wild type plants. These results are consistent with the observation that higher nucleosome density within a gene inhibits transcription by limiting RNA polymerase processivity . In plants, histone H3 modification at positions Lys-9 and Lys-14 is positively correlated with gene activation, and the deacetylated status with inactive transcription . Our results indicate that histone H3 is acetylated at positions Lys-9 and Lys-14 on the target genes RD20, MYB2 and NAC019 that are highly expressed in the slk1-1, slk2-1 and luh-4 mutants compared to the wild type plants. These data indicate that LUH prevents the expression of target genes by recruiting HDACs that deacetylate histone H3 at positions Lys-9 and Lys-14. Further studies are needed to establish the presence of LUH, SLK1 and SLK2 at the regulatory sequence of the target genes to modify the chromatin status.
LUH is induced during abiotic stress in contrast to LUG suggesting that LUH plays an important role in abiotic stress response. Interestingly SLK1 and SLK2 are induced in response to osmotic stress (unpublished data). There are several possible roles that LUH can participate in regulating abiotic stress response in plants. First, during abiotic stress several genes are induced that confer tolerance to the abiotic stress and increased LUH expression could form complex with SLK1/SLK2 and negatively regulate genes that are detrimental to the abiotic stress tolerance. Second, one of the main mechanisms that plants employ to endure abiotic stress is by reprogramming the developmental pathway so that important growth phases that are sensitive to abiotic stress are delayed . The LUH-SLK1 and LUH-SLK2 complexes could repress the genes that are involved in the transition of growth phase. Third, LUH-SLK1 and LUH-SLK2 complexes could regulate the abiotic stress pathway by controlling the length or level of response by regulating the positive or negative determinant genes by negative feedback loop.
SLK1 and SLK2 function as adapters to form SLK1-LUH and SLK2-LUH complexes with LUH possessing repressor activity. How the SLK1-LUH and SLK2-LUH complexes are recruited to the promoter of the abiotic stress response genes remains to be determined. LUH could exert its repressive effect on the target genes by recruiting histone deacetylase that facilitates deacetylation of histone H3 associated with promoter of target genes. The binding of the LUH to the histone H3 results in the condensation of chromatin and increased nucleosome density, thus preventing gene transcription by RNA polymerase at the target genes. Further studies are needed to determine the transcription factors that interact with SLK1 and SLK2 to recruit LUH to the regulatory sequence of target genes. Microarray analysis in slk1, slk2 and luh mutant plants will provide additional insight into the abiotic stress response genes regulated by SLK1, SLK2 and LUH.
Plant materials and abiotic stress treatment conditions
The Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col0) and Landsberg erecta (Ler) was used as wild type controls. luh-3 (seed stock no. SALK_107245C), luh-4 (seed stock no. SALK_097509), slk1-1 (seed stock no. CS65896), slk2-1 (seed stock no. CS65894) mutant lines were obtained from the Arabidopsis Biological Resource Center (ABRC). All the mutant lines are in the Col0 background except for seu-1 which is in Ler background.
The wild type and mutant seeds were sterilized with 50% bleach and planted on half-strength Murashige and Skoog salt, 1% sucrose, 0.8% agar (MS) media and incubated at 22°C under long-day light conditions in the growth chamber (Percival). For abiotic stress treatment, six day old seedlings were transferred to the MS media with or without 125 mM NaCl or 300 mM mannitol and incubated in the growth chamber under long-day conditions at 22°C. Root length and fresh weight are expressed as a percentage relative to plants grown on MS medium without stress treatment after 15 days for salt and 25 days for mannitol treatment.
Yeast Two hybrid assay
LUH (G12254), SLK1 (G66746) and SLK2 (G10219) cDNA clones were obtained from Arabidopsis Biological Resource Center. The cDNA clones were amplified by PCR with Pfu ultra (Agilent Technologies) and cloned in frame by In-Fusion HD Cloning Plus (Clontech) into vector pGBKT7 (Clontech) at Nde1-Sal1 and pGADT7 (Clontech) at Nde1-BamH1 sites to generate Gal4-BD and Gal4-AD fusions respectively. The LUFS domain (1-88 amino acids) was PCR amplified from LUH cDNA and cloned in frame by In-Fusion HD Cloning Plus into vector pGBKT7 at Nde1-Sal1 site. The histone gene H3 (AT5G65360), H4 (AT5G59690), H2A (AT5G54640) and H2B (AT1G07790) were amplified from total RNA by RT-PCR with respective primers and inserted into vector pGADT7 at Nde1-BamH1site by In-Fusion HD Cloning Plus to generate Gal4-AD fusion. All the sequences were verified by sequencing. The yeast two hybrid interaction assays were performed in Y2H Gold (Clontech) yeast strain according to manufacturer’s protocol and reference 14. The primer sequences are listed in Additional file 6: Table S2.
The protoplast isolation and transfection was performed as described in .
Repression assay in protoplasts
To construct reporter gene for repression assay, 342 bp CaMV 35S promoter was PCR amplified from pMDC32 vector  using primers CaMV_pUASluc2F, CaMV_pUASluc2R and inserted at Hind111-EcoR1 site by In-Fusion HD Cloning Plus in the plasmid pUAS-luc2 (Addgene, plasmid: 24343)  to generate CaMV 35S::LUC vector. The 5XUAS region was PCR amplified from pUAS-luc2 plasmid using primers 5xGal4DBF, 5xGal4DBR and inserted at Bgl11 site in the CaMV 35S::LUC to generate 5XUAS GAL4 CaMV 35S::LUC reporter construct. The 800 bp CaMV 35S promoter was PCR amplified from pMDC32 vector and inserted at EcoR1-Pst1 site by In-Fusion HD Cloning Plus in the plasmid pRL-null (Promega) to generate CaMV 35S::Renilla LUC reporter construct. For the effector constructs, the respective gene from pGBKT7 were PCR amplified using primers pGBTK_GAL4F, pGBTK_GAL4R and cloned at BamH1 site in the vector pXSN  using In-Fusion HD Cloning Plus. To generate CaMV 35S::LUH and CaMV 35S::LUFS, the respective genes were PCR amplified and inserted at the BamH1 site by In-Fusion HD Cloning Plus in the pXSN vector. The protoplast transfection, reporter gene assay and trichostatin-A (TSA) treatment was performed as described in . The primer sequences are listed in Additional file 6: Table S2.
Split luciferase complementation assay
The cDNA was amplified with PCR with respective gene specific primers and inserted at Kpn1-Sal1 site in the CaMV 35S::Nluc or Kpn1-Pst1site in the CaMV 35S::Cluc vector to generate N-luciferase and C- luciferase fusion respectively . The transfection was performed with 5 × 104 protoplasts, 15 μg of each fusion construct and 0.5 μg CaMV 35S::Renilla LUC as an internal control for transfection. The protoplasts were incubated in the dark for 16 h at room temperature and the luciferase assay was performed with dual luciferase reporter assay kit (Promega) and TD-20/20 luminometer (Turner Biosystems). The primer sequences are listed in Additional file 6: Table S2.
Subcellular localization of LUH, SLK1 and SLK2
The cDNAs were amplified with respective gene specific primers and cloned into BamH1 site by In-Fusion HD Cloning Plus in the plasmid pXDG  to generate GFP fusion driven by CaMV 35S promoter. The protoplasts were transfected with 15 μg of each plasmid DNA and incubated in the dark for 16 h at room temperature. The protoplasts were incubated with 1 μg/ml 4, 6-diamidino-2-phenylindole (DAPI), the GFP and DAPI localization was visualized with a Nikon fluorescent microscope (Exclipse E800) equipped with digital camera. The images obtained at different channels were cropped and merged with imageJ program (National Institutes of Health). The primer sequences are listed in Additional file 6: Table S2.
Construction of transgenic plants for complementation assay
The promoter region of LUH (2.6 kb), SLK1 (2.4 kb) and SLK2 (1.6 kb) upstream from start codon were PCR amplified from wild type genomic DNA using promoter specific primers with Sal1 site in the reverse primer. The amplified promoter region of respective genes was cloned in PCR8/GW/TOPO vector (Invitrogen). The coding sequence without stop codon were PCR amplified with gene specific primers using LUH (G12254), SLK1 (G66746) and SLK2 (G10219) cDNA clones and inserted at Sal1 site in the promoter containing TOPO vector by In-Fusion HD Cloning Plus. LUH::LUH, SLK1::SLK1 and SLK2::SLK2 cassettes were transferred into the binary vector pEarleyGate 302, pEarleyGate 301 and pEarleyGate 303  respectively using LR Clonase ll mix (Invitrogen). The binary vector was introduced into Agrobacterium strain GV3101 and transformed into mutant plants using floral dip method . The primary transformants were isolated on MS medium with BASTA selection. The resistant plants were confirmed by PCR and RT-PCR to detect T-DNA insertion and gene expression. The primer sequences are listed in Additional file 6: Table S2.
RNA isolation and quantitative RT-PCR
Total RNA was extracted from 21 day old seedlings using TRIzol reagent (Invitrogen) and purified by RNeasy Plus Mini Kit (Qiagen). For qRT-PCR, 5 μg of DNase treated total RNA was used for cDNA synthesis using oligo (dT) primer and SuperScript III reverse transcriptase (Invitrogen). The target genes were quantified using SYBR Green Supermix reagent (Bio-Rad) with 1:10 dilution of the cDNA and gene specific primers in the Bio-Rad iCycler iQ real time system. ACTIN2 was used as an internal control for normalization in each quantitative PCR experiment. Real time qRT-PCR was repeated with three biological replicates for each sample. The primer sequences are listed in Additional file 6: Table S2.
One gram of 21 day old seedlings was used for ChIP assay. Chromatin preparation and immunoprecipitation were performed as described in . Briefly, the chromatin extracts were prepared from seedlings treated with 1% formaldehyde. The chromatin was sheared to an average length of 500 bp by sonication (ultrasonic processor) and immunoprecipitated with specific antibodies using magnetic protein G beads (Dynabeads protein G, Invitrogen). The antibodies used for ChIP were anti-histone H3 C-terminus (Abcam; 1791), anti-H3K9ac (Abcam; 4441) and anti-H3K14ac (Millipore; 07-353). The immune complexes were washed, eluted from the magnetic protein G beads and reverse cross-linked at 65°C overnight. The DNA was purified using QIAquick PCR purification kit (Qiagen) in a final volume of 50 μL. Three microliter of the DNA was used for each qPCR assay with SYBR Green Supermix reagent (Bio-Rad) in the Bio-Rad iCycler iQ real time system. ACTIN7 was used as an internal control for normalization in each qPCR experiment. The experiment was repeated with three biological replicates for each sample. The amplification region for the target genes are provided in Additional file 5: Figure S4. The primer sequences are listed in Additional file 6: Table S2.
All experiments were performed at least three times. Error bars in each graph indicate mean values ± SE of three repetitions. P values were determined by Student’s t test.
We would like to thank Beatriz Briones for constructing some of the plasmids and performing the luciferase assays. We would like to acknowledge undergraduate research fellowship from College of Sciences Undergraduate Research Program to BS from University of New Orleans. BS and BG are supported by funding from the Louisiana Governor's Biotechnology Initiative. This work was supported by funding from Office of Research and Sponsored Programs at the University of New Orleans and the Louisiana Board of Reagents (LEQSF(2012-15)-RD-A-32).
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