Protoplasma DOI 10.1007/s00709-016-0983-x
ORIGINAL ARTICLE
Different functions of the histone acetyltransferase HAC1 gene traced in the model species Medicago truncatula, Lotus japonicus and Arabidopsis thaliana Irina Boycheva 1 & Valya Vassileva 2 & Miglena Revalska 1 & Grigor Zehirov 2 & Anelia Iantcheva 1
Received: 3 December 2015 / Accepted: 6 May 2016 # Springer-Verlag Wien 2016
Abstract In eukaryotes, histone acetyltransferases regulate the acetylation of histones and transcription factors, affecting chromatin structural organization, transcriptional regulation, and gene activation. To assess the role of HAC1, a gene encoding for a histone acetyltransferase in Medicago truncatula, stable transgenic lines with modified HAC1 expression in the model plants M. truncatula, Lotus japonicus, and Arabidopsis thaliana were generated by Agrobacteriummediated transformation and used for functional analyses. Histochemical, transcriptional, flow cytometric, and morphological analyses demonstrated the involvement of HAC1 in plant growth and development, responses to internal stimuli, and cell cycle progression. Expression patterns of a reporter gene encoding beta-glucuronidase (GUS) fused to the HAC1 promoter sequence were associated with young tissues comprised of actively dividing cells in different plant organs. The green fluorescent protein (GFP) signal, driven by the HAC1 promoter, was detected in the nuclei and cytoplasm of root cells. Transgenic lines with HAC1 overexpression and knockdown showed a wide range of phenotypic deviations and developmental abnormalities, which provided lines of evidence for the role of HAC1 in plant development. Synchronization of A. thaliana root tips in a line with HAC1 knockdown Handling Editor: Heiti Paves Electronic supplementary material The online version of this article (doi:10.1007/s00709-016-0983-x) contains supplementary material, which is available to authorized users. * Anelia Iantcheva
[email protected] 1
AgroBioInstitute, Blvd. Dragan Tzankov 8, 1164 Sofia, Bulgaria
2
Institute of Plant Physiology and Genetics, Acad. Georgi Bonchev Str., Bl. 21, 1113 Sofia, Bulgaria
showed the involvement of this gene in the acetylation of two core histones during S phase of the plant cell cycle. Keywords Histone acetyltransferase . Model legumes . Plant growth and development . Transcriptional regulation . Replication . Gene activation
Introduction Posttranslational modifications of histones, such as acetylation, methylation, phosphorylation, ubiquitination, and glycosylation, are critical components in biological processes that affect almost all aspects of plant development. They can alter chromatin structure, thereby having a pivotal role for gene transcription or silencing. Posttranslational modifications are believed to act sequentially or in a combinatorial pattern termed a Bhistone code^ that directs the chromatin to conformational changes, thus regulating the availability of genes to transcriptional machinery. Histone acetylation and deacetylation play important roles in regulating gene expression. Transcriptionally active genes are generally highly acetylated, whereas inactive genes are associated with hypoacetylated histones (Hebbes et al. 1988; Sterner and Berger 2000; Boycheva et al. 2014). Fundamental participants in histone acetylation are specialized enzymes, named histone acetyltransferases (HATs), which transfer acetyl groups (CH3COO-) to the NH3+ groups of lysine residues, thereby ensuring access of transcription factors to DNA (Brownell and Allis 1996; Kuo and Allis 1998; Roth et al. 2001). The basic structural unit of chromatin, the nucleosome, consists of double-stranded DNA, two copies each of the four core histone proteins H2A, H2B, H3, and H4, and one copy of the H1 linker protein (Luger et al. 1997; Kornberg and Lorch 1999; Richmond and Davey 2003). Nucleosome positioning defines
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the accessibility of DNA to chromatin modifying enzymes and multiple protein factors, such as general transcription factors, sequence-specific DNA-binding activators, and nonDNA-binding transcription co-activators/adaptors. Chromatin posttranslational modifications, in particular histone acetylation, contribute to the relief of nucleosomal repression. The access of transcription factors to DNA sequence is facilitated by the reduction of binding affinity between histones and DNA through the addition of negatively charged acetyl groups to the positively charged aminoterminal tails of histones that are rich in lysine (lys+) and arginine (arg+) amino acids (Richards and Elgin 2002). HATs are classified into two major classes: HAT-A and HAT-B (Brownell and Allis 1996; Roth et al. 2001). The HAT-A class has been divided into families: GCN5-related N-terminal acetyltransferases (GNATs), the MYST (MOZ, Ybf2/Sas3, Sas2, and Tip60) family, p300/CREB binding protein (CBP) HATs, and transcription initiation factor TAFII250 [for TATA-binding protein (TBP)-associated factor] (Boycheva et al. 2014). The p300/CREB family contains five genes in the Arabidopsis genome (AtHAC1/PCAT2, AtHAC2/ PCAT1, AtHAC4/PCAT3, AtHAC5/PCAT4, and AtHAC12) (Bertrand et al. 2005). These basic proteins are transcriptional regulators that mediate interactions of HAT, bromodomains and three cysteine-histidine-rich domains (cys/his-1, cys/his2, and cys/his-3) (Mizzen et al. 1996), ZZ-type zinc finger domains, TAZ-type zinc finger domains, and KIX (Vo and Goodman 2000). The protein-protein interactions are established by zinc finger domains (ZZ and TAZ) (Ponting et al. 1996), whereas Cys-rich HAT domain at the Cterminal characterizes the HAT activity in vitro (Bordoli et al. 2001). The HAC1 gene, analyzed in this study, belongs to HAT-A class acetyltransferases p300/CREB family and is located in the cell nucleus and acetylate nucleosomal core histones. Little is known about the role of HAC1 in the development of Arabidopsis thaliana and other plant species, including legumes, while its animal homologue p300/CREB is well characterized (Deng et al. 2007). In our study, HAC1 was originally identified by reverse genetic approach in a population of Tnt1 retrotransposontagged mutants of the model legume Medicago truncatula (Revalska et al. 2011). Tnt1 is known to target preferentially gene-rich regions of the genome (d’Erfurth et al. 2003; Tadege et al. 2008). To identify disrupted genes in the created mutant collections, Flanking Sequence Tag (FST) information has been generated and deposited for public use at the Samuel Roberts Noble Foundation (http://bioinfo4.noble.org/ mutant/). After sequencing of some mutant lines, an insertion of a Tnt1 transposon in exon in the mutant line So5945A has been found (Revalska et al. 2011). This insertion is located in the coding region of the histone acetyltransferase gene (HAC1; insert 5; So5945). Sequencing of the M. truncatula genome provides an excellent opportunity to
develop marker systems, identify new genes, and positionally clone genes of interest in legumes, thus enabling an extraction of valuable information on relevant gene families (Young and Udvardi 2009). In the past decade, two legume species, M. truncatula and Lotus japonicus, have been established as model plants for studying the biology and genetics of legumes (Young and Udvardi 2009). Both plants possess small genome and short life cycle and share considerable genetic synteny with forage crops alfalfa (Medicago sativa) and trefoil (Lotus corniculatus) (Young et al. 2005; Young and Udvardi 2009). This favors understanding the relationships between genotype and phenotype by discovering novel and unknown genes. The accumulated knowledge could be transferred from models to economically important crops and could assist in designing new strategies for crop improvement. The candidate gene approach allowed the identification of possible targets of HAC1 that are involved in plant flower development. In dicotyledonous plants, flowers possess four basic parts: sepals, petals, stamens, and pistil, arranged in concentric circles. Sepals are the outermost whorl of a flower, petals form the second, stamens are the third, and the fourth innermost whorl consists of one or more pistils. The identity of these four flower organs is determined by the combined action of all three classes of homeotic genes, known as А, В, and С. In A. thaliana, the A class gene APETALA1 (AP1) affects the formation of the first and second concentric circles, while the class B gene APETALA3 gene (AP3) is required for the second and third circles (Bertrand et al. 2003). The early events during flower morphogenesis, including flower meristem identity and patterning, are regulated by the helix-turnhelix transcription factor LEAFY (LFY) and the MADS box transcription factor AP1, while petal and stamen identities are controlled by AP3 (Sundstrom et al. 2006; Winter et al. 2015). Genetic and molecular lines of evidence have shown that LFY and AP1 regulate transcriptional programs to switch the transition from vegetative growth to reproductive development, orchestrating together floral development and growth (Winter et al. 2015). The main flowering pathways in A. thaliana, defined as the autonomous, vernalization, and photoperiodic, are influenced by HAT genes (Komeda 2004; Amasino 2005). Another MADS box gene, encoding for the transcription factor Flowering Locus C (FLC), acts as a strong repressor of flowering, blocking the transition from vegetative to reproductive development. Suppression of its expression induces flowering (Michaels and Amasino 1999; He and Amasino 2005). It has been shown that the FLC expression level is regulated via histone acetylation (Ausin et al. 2004; Tian et al. 2005), while histone deacetylation is associated with downregulation of FLC, mediated by two main genes: Flowering Locus D (FLD) and Flowering Locus VE. Histone acetylation is usually associated with the activation of gene expression. The expression level of FLC is
Different functions of the HAC1 gene in three model plants
upregulated in hac1 mutants, whereas the transcript levels of all known autonomous pathway genes are unchanged, suggesting that HAC1 affects flowering time by epigenetic modification of factors upstream of FLC (Deng et al. 2007). The current study is aimed to examine different functions of a gene encoding for the p300/CBP histone acetyltransferase HAC1 in the model legume M. truncatula. The HAC1 promoter activity was estimated by GUS and GFP reporter systems in the model legumes species M. truncatula and L. japonicus and compared to A. thaliana as a reference model. Morphological and transcriptional analyses of transgenic plants with HAC1 overexpression and knockdown allowed clarifying the role of HAC1 in plant growth and development. Comparative analysis of HAC1 in the three model species will help in understanding the functional similarities/differences shared by these plants and address the important question of whether we can use A. thaliana as a reference to understand legume genomes.
was targeted at nucleotide positions 138–264. The ORF of the Lotus ortholog Lj0g253430 was silenced at positions 90– 216. The generated constructs were introduced into Agrobacterium tumefaciens strain C58C1, which was maintained on YEB nutrient medium solidified with agar (1.5 %) and supplemented with 100 mg/L rifampicin (Rif), 100 mg/L spectinomycin (Sp), and 50 mg/L gentamycin (Gm). Plant material, growth conditions, and genetic transformation Stable transgenic lines with HAC1 overexpression (OE), RNAi-mediated knockdown, and transcriptional reporters were generated for the three model species by A. tumefaciensmediated transformation. Medicago truncatula
Material and methods Gene cloning and vector construction for plant genetic transformation To generate different HAC1 constructs, the Gateway cloning system was used (Invitrogen Life Technologies, Inc., www. lifetechnologies.com). The Entry clones were designed through recombining the promoter sequence of HAC1 (Mt0g02950, Plaza 2.5, www.medicago.org) with the pDONRP4P1R donor vector, and the open reading frame (ORF) of the gene with the pDONR221 donor vector. Expression clones were generated by recombining the promoter Entry clone with the pEX-K7SNFm14GW (promoterNLS-GUS/GFP) destination vector possessing neomycin phosphotransferase (nptII) gene as a selection marker for transgenic plants. The HAC1 gene entry clone was transferred into the destination vectors for overexpression (pK7WG2 and pK7FWG2) under the control of CaMV 35S promoter and nptII gene for plant selection (Karimi et al. 2007). To specifically knockdown HAC1 in M. truncatula, RNA interference (RNAi) strategy (Limpens et al. 2004) was used. For in silico prediction of the gene region with high silencing capacity that is optimal for the synthesis of double-stranded RNA (dsRNAs), Xwin Razor software was used. To identify the unique gene-specific tag sequence, potential target mRNAs were analyzed, and the specificity was evaluated by a single BLASTn search in the database of M. truncatula (Medicago truncatula Genome Project, http://jcvi.org/ medicago). To create the expression clone, the binary Gateway vector pK7GWIWG2D(II) for hairpin RNA expression was used. A fragment of 130 bp, corresponding to nucleotide positions 132–262 of the ORF of MtHAC1, was silenced. The ORF of the Arabidopsis ortholog At1g79000
To initiate in vitro plant material, seeds of the highly regenerable genotype M. truncatula cv. Jemalong 2HA (Nolan et al. 1989) were used. They were surface sterilized with 6 % (v/v) solution of sodium hypochlorite for 15 min, rinsed at least five times with sterile distilled water, and germinated on MS basal medium (Murashige and Skoog 1962). Then, the germinated seedlings were propagated by cuttings. In vitro plant materials were maintained in Magenta boxes (60 × 60 × 96 mm, Sigma) in a growth chamber at 24 °C, 16-h photoperiod, and light intensity of 30 μmol m−2 s−1. Medium composition for plant regeneration and transformation Leaf and petiole explants were collected from 35-dayold in vitro plants and subjected to genetic transformation. Transgenic plants of M. truncatula were obtained using a combined protocol, described by d’Erfurth et al. (2003), Chabaud et al. (1996), and Iantcheva et al. (2009). Leaf and petiole explants were wounded with a sterile scalpel blade and precultivated for a 2-day period in darkness on the solid Shab callus induction medium, consisting of SH macro- and micronutrients, vitamins, 5 mg/L auxin 2,4-D (2,4dichlorophenoxyacetic acid), and 1 mg/L BAP (6benzylaminopurine). Then, the explants were inoculated with bacterial suspension (OD600 = 0.5) under shaking (100 rpm) for 1 h. The transformed plant material was co-cultivated for an additional 48 h, then transferred for 2 weeks to Shab medium with 50 mg/L kanamycin (Km) and 400 mg/L carbenicillin (Cb) to enable selection of the transformed tissue and remove Agrobacterium. In order to complete the process of callus initiation, after callus appearance along the edge of the wound, the explants were transferred to callus induction medium (CIM) (MS macro- and micronutrients, vitamins, 2 mg/L zeatin, 1 mg/L 2,4-D), containing the same concentrations of Km and Cb for two more weeks. To form green
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embryo zones, after development of a clearly visible callus tissue, explants were transferred for 2 weeks to the 09-03 medium [MS macro- and micronutrients, 0.9 mg/L BAP, and 0.3 mg/L NAA (α-naphthaleneacetic acid)], containing 200 mg/L Cb and without the selective agent Km. Embryo formation and development continued on selective MS1 medium (0.05 mg/L BAP and 250 mg/L casein hydrolysate) with 50 mg/L Km. After production of cotyledonary stage embryos, plant material was transferred for rooting to selective MS medium containing 50 mg/L Km. The rooted plantlets were cultivated on selective MS basal medium (50 mg/L Km) and, after sampling for analyses, transferred to soil and grown in a greenhouse for seed production.
bacterial infection. The selective medium was refreshed every 20 days. After 60 days, embryogenic calli were transferred to EIM for 25 days under no selective pressure. The first green zones on calli were seen after 10–15 days, subsequently growing and forming clusters of closely packed thick globular embryos. Further embryo development was undertaken on EDM for two to three passages (20 days each). The developed dark green globular embryos formed cotyledonary leaves after at least two passages (40 days). Kanamycin selection was employed during embryo development, conversion, and rooting. Selective Cb concentration was half reduced in EIM and EDM and fully removed from ECR medium. Arabidopsis thaliana
Lotus japonicus Seeds of L. japonicus ecotype B-129 Gifu, obtained from Dr. Hiroshi Kouchi, were gently scarified with sandpaper and surface sterilized with 70 % (v/v) ethanol for 30 s and 0.1 % (v/v) mercury chloride (HgCl2) for 8 min, washed in sterile distilled water at least five times, and sowed on MS basal medium (Murashige and Skoog 1962). The germinated seedlings were then propagated by cuttings. Leaf explants were collected from 30- to 40-day-old in vitro plants and used for Agrobacterium-mediated transformation. In vitro plant material was grown in Magenta boxes (60 × 60 × 96 mm, Sigma) in a growth chamber with a photoperiod of 16 h, 30 μmol m−2 s−1 light intensity, and a temperature of 24 °C. Medium composition for plant regeneration and transformation CIM was based on B5 (Gamborg et al. 1968) solid medium containing 4 mg/L 2,4-D, 0.8 mg/L kinetin, 1 mg/L adenine (6-aminopurine, Sigma-Aldrich), 500 mg/L casein hydrolysate, 500 mg/L myo-inositol, 3 % sucrose (w/v), and 0.7 % (w/v) phyto agar (P 1003, http://www.duchefa.com). Embryo induction medium (EIM) was based on MS medium supplemented with 0.9 mg/L BAP and 0.3 mg/L NAA, 3 % sucrose (w/v), and 0.7 % (w/v) phyto agar, as described by Iantcheva et al. (2009). The embryo development medium (EDM) for L. japonicus was based on B5 medium supplemented with 0.2 mg/L BAP, 3 % sucrose (w/v), and 0.7 % (w/v) phyto agar. For embryo conversion and rooting (ECR), basal MS medium was used. Transformation procedure Wounded leaf and petiole explants from in vitro wild-type plants were precultured before inoculation with bacterial suspension on CIM for 48 h. Explants were inoculated with a bacterial suspension at an optical density of 0.3 at 600 nm (OD600) for 1 h and cocultivated for 2 days on solid CIM without the selective antibiotic Km. Explants were then transferred for callus induction to solid selective medium, containing 50 mg/L Km for selection of the transformed tissue, and 400 mg/L Cb to remove
To generate transgenic plants, Agrobacterium-mediated transformation of A. thaliana ecotype Columbia-0 (Col-0) via the floral-dip method (Clough and Bent 1998) was used. T3 and T4 homozygous lines were analyzed: transformants carrying the reporter genes GUS and GFP fused to the endogenous HAC1 promoter (pHAC1:GUS/GFP), transformants with HAC1 overexpression (HAC1-OE), and RNAi-mediated downregulation (HAC1-RNAi). Plants were grown in soil in a greenhouse at 21 °C under long-day conditions (16 h light/ 8 h dark). In vitro plant material was obtained after seed sterilization for 2 min in 70 % ethanol and 12 min in commercial bleach. Then, the seeds were rinsed with sterile distilled water and plated on square plates containing MS medium solidified with 0.8 % agar (Murashige and Skoog 1962), supplemented with 20 g/L sucrose and 0.43 g/L 2-(N-morpholino)ethanesulfonic acid (MES). Seedlings were grown in a growth chamber at 24 °C, with a 16-h photoperiod under light intensity of 30 μmol m−2 s−1. Synchronization and inhibitor treatments The root meristem from transgenic and wild-type seedlings from A. thaliana was synchronized as described by Cools et al. (2010). At first, seeds were plated on nylon meshes and grown on MS medium for 5 days (Prosep), then transferred to MS medium containing 1 mM hydroxyurea (HU) (SigmaAldrich) for 8 h. Root tips (1 mm) were harvested for transcriptional analyses. Seedlings from wild-type L. japonicus and T1-HAC1-OE 2 line were treated on MS medium supplemented with 80 and 100 μM curcumin (diferuloylmethane) for 24 and 48 h. Root tips (1 mm) were harvested for transcriptional analyses. Hydroponic cultivation The 35-day-old in vitro wild-type and T1 transgenic plants from the two model legume species were transferred into hydroponic containers, containing 1.2 L Fahraeus nutrient solution (1 g/L−1 CaCl2⋅2H2O, 1.2 g/L−1 MgSO4⋅7H2O, 1 g/L−1 K2HPO4, 1.5 g/L−1
Different functions of the HAC1 gene in three model plants
Na2HPO4⋅12H2O, 21 mg/L−1 Ca(NO3)2⋅4H2O as a starting nitrogen dose, Gibson micronutrients, and 0.005 g/L−1 iron citrate, pH 6.5–6.8). Plants were cultivated at a temperature of 25 °С/20 °С day/night, 16-h photoperiod under light intensity of 300 μmol m−2 s−1. During the transfer into the hydroponic containers, the bacterial suspension (approximately 2.108 cells of Sinorhizobium meliloti 1021 or Mesorhizobium loti MAFF 303099) was added to each container. Depending on plant age, the nutrient solution was changed once or twice a week. Light microscopy Leaves from mature 3-week-old plants were cleared in absolute ethanol until chlorophyll was removed and then mounted for 24 h in lactic acid on glass slides. The samples were observed under Carl Zeiss Axio Scope upright microscope. Leaf area was measured in scanned images and calculated as the mean sum of 12 individual leaf blades from three independent experiments. To determine mean epidermal cell area, epidermal cells were measured at two different zones of each leaf. Epidermal cell number was estimated as means of 12 leaves. Leaf area, cell size, and cell number were analyzed using ImageJ 1.41 software. Fluorescence and confocal microscopy Images of plant segments expressing GFP were collected using a SZX7 fluorescence stereomicroscope (460–490 nm excitation and 510– 550 nm emission) with a DP73 digital camera (Olympus). Fluorescence imaging of roots was acquired with an Axiovert100M confocal laser scanning microscope and LSM510 software package (Zeiss). GFP was excited at 488 nm with an argon laser. Flow cytometry DNA ploidy levels were assessed in 21-dayold first mature leaves of seedlings of A. thaliana OE 2 and RNAi 10 lines and compared to the wild-type. Leaves were chopped with a razor blade in 200 μL CyStain UV Precise nuclei extraction buffer, then DNA was stained by 800 μL staining buffer (Partec), and samples filtered using a 30-μm mesh. Measurements were carried out with a CyFlow flow cytometer (Partec) and data analysis was performed using CXP Analysis software (Partec). Six leaves were analyzed and three technical repeats were assayed. Real-time quantitative PCR (qRT-PCR) analysis Total RNA was extracted from 20-day-old T1 seedlings and leaves, stems, and nodules from mature 90-day-old hydroponic plants of the wild-type, OE, and RNAi lines. The transcript level of AP1 and AP3 was determined in flowers from the wild-type Arabidopsis plants, T4-HAC1-OE 2 line, and T4 RNAi 10 lines. Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen), according to the manufacturer’s protocol. Firststrand cDNA synthesis was carried out using the Fermentas First-Strand cDNA Synthesis Kit. Gene expression levels
were analyzed in triplicate on a 7300 Real-time PCR System (Applied Biosystems) with primers listed in ESM 2. Actin (ACT) and ubiquitin (UBQ10) were used as reference genes for data normalization. Histochemical assay for GUS activity Plant tissue from independent in vitro- and greenhouse-grown transgenic T1 lines from the two model legumes and T3 lines from A. thaliana were stained for GUS activity. Plant material was incubated in 90 % acetone for 30 min at 4 °C, then washed in phosphate buffer at room temperature, and incubated in staining solution (for 10 mL: 5 mg 5-bromo-4-chloro-3-indolyl-β-d-glucuronide dissolved in 50 μL formamide; 5 mL 100 mM sodium phosphate buffer (pH 7.0); 200 μL 0.5 M Na2EDTA; 10 μL Triton X-100; 1 mL 1 mM K4Fe(CN)6⋅3H2O; 1 mL 1 mM K3Fe(CN)6; 2 mL methanol and supplemented with 240 μL H2O) at 37 °C overnight. After staining, the tissue was transferred to absolute ethanol. Root, leaf, and silique measurements Root lengths were measured 5 days after seed germination and recorded every 24 h for three consecutive days. The primary root length of at least 20 plants of the selected OE and RNAi lines and the wild type were acquired for each data set. Silique length and width, and leaf length (L) and width (W), as well as petiole length (P) were measured. Each leaf was collected from the same level of the rosette of greenhouse-grown plants and then scanned. The images were analyzed using ImageJ 1.41 software. Statistical analysis All experiments were carried out in triplicate and repeated three times for each experimental data set. Data were analyzed via SPSS 11.5 software. p values lower than 0.05 were considered statistically significant. Results are expressed as means ± standard deviation (SD).
Results Generation of stable transgenic plants from the three model species M. truncatula, L. japonicus, and A. thaliana was of crucial importance in elucidation of HAC1 function and tissue localization. After genetic transformation of wild-type M. truncatula and L. japonicus, stable T0 lines were obtained and verified by PCR for the presence of the selectable marker gene. Three to five independent transgenic T1 lines with modified expression levels of HAC1, and HAC1-GUS/GFP promoter reporters were used for all further analyses. Five independent transgenic T1 lines were selected for phenotypic characterization. To facilitate selection of transgenic plants, the seeds produced from the primary transformants of A. thaliana were
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sown on selective medium with 50 mg/L Km. In the homozygous T3 plants, HAC1 transcript levels were evaluated and three to five independent lines selected for further analyses. Expression analysis of 20-day-old T1 seedlings of OE M. truncatula lines displayed enhanced transcript levels of HAC1. In T1 OE 1 and OE 3, expression of HAC1 was increased from 6 to 9.5 times (р ≤ 0.01), while in OE 2, HAC1 transcripts increased about 15 times (Fig. 1a). In all knockdown plants, HAC1 transcript abundance was lower as compared to the wild type (р ≤ 0.001) (Fig. 1b). The expression level of HAC1 in 90-day-old plants with overexpression (OE 2 line) and downregulation (RNAi 1 line), grown in a hydroponic system, did not change significantly with age (р ≤ 0.01) (Fig. 1g). The HAC1 transcripts in the nodules of T1 OE and RNAi lines showed higher levels in the OE line of M. truncatula (p < 0.05) and lower abundance in the knockdown line (p < 0.01) compared to control. Evaluation of the expression of HAC1 in 20-day-old seedlings of T1 OE and RNAi lines of L. japonicus showed higher
transcript levels in the OE 2 line (р ≤ 0.05) and reduced levels in all knockdown lines (Fig. 1c, d). The transcript level in 90-dayold leaves and stems of the OE 2 and RNAi 4 lines, grown as hydroponic cultures, remained unchanged (Fig. 1h). In L. japonicus, the HAC1 transcript level in nodules from the OE line was significantly higher than the transcript level in leaves and stems (p < 0.01) and twice higher than the level in nodules from the M. truncatula OE line. Approximately 10 times lower transcript abundance was detected in the nodules of the knockdown line (p < 0.01) compared to the OE line, and 5 times lower (p < 0.05) compared to the wild-type control (Fig. 1h). HAC1 was heterologously overexpressed in A. thaliana, and monitoring the transcript abundance in three homozygous lines, OE 1, OE 2, and OE 3, showed three times higher expression than in the control (р ≤ 0.05) (Fig. 1e). Endogenous level of HAC1 transcripts in the three RNAi lines, RNAi 4, RNAi 5, and RNAi 10, was also evaluated. Only line RNAi 10 showed reduced transcript levels (р ≤ 0.001) (Fig. 1f).
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Fig. 1 Transcript levels of HAC1 in a M. truncatula T1 OE lines and wild type (WT); b M. truncatula T1 RNAi lines and WT; c L. japonicus T1 OE lines and WT; d L. japonicus T1 RNAi lines and WT; e A. thaliana T3 OE lines and WT; f A. thaliana T3 RNAi lines and WT; g M. truncatula
leaves, stems, and nodules from mature transgenic OE and RNAi lines and WT; and h L. japonicus leaves, stems, and nodules from mature transgenic OE and RNAi lines and WT
Different functions of the HAC1 gene in three model plants
Transcriptional reporters of the three model species M. truncatula, L. japonicus, and A. thaliana allowed detection of HAC1 localization in different plant tissues and organs. In T1 plants of M. truncatula, GUS expression was detected at different stages of somatic embryogenesis: globule, torpedo, and cotyledon (Fig. 2a–c); in nodes (Fig. 2d) and young internodes regions (Fig. 2e); and also at the base of the petiole (Fig. 2f) and vascular system of primary root (Fig. 2g). In L. japonicus, GUS expression was observed in stem nodes (Fig. 2h), leaf bases (Fig. 2i), young petioles (Fig. 2j), and nodule vasculature (Fig. 3a). Expression of GUS reporter gene in A. thaliana plants was seen in stem nodes (Fig. 2k), petal vascular tissue (Fig. 2l), and on the top of the stamen stalk (Fig. 2m). Confocal microscopy observations revealed GFP fluorescence in the nuclei and cytoplasm of root cells (Fig. 4a, b) and in trichome nuclei and root meristem of A. thaliana
Fig. 2 Expression of GUS reporter for beta-glucuronidase activity in HAC1 transcriptional reporter plants of M. truncatula: a–c different stages of somatic embryogenesis, d stem nodes, e young internodes, f petiole base, g primary root; L. japonicus: h young node, i petiole base, j young petiole; and A. thaliana: k stem nodes, l petals of young flower buds, and m stamen stalk. Scale bars = 0.5 cm
(Fig. 4c, d). GFP activity was also detected in the roots and nodules of L. japonicus (Fig. 3c). The function of HAC1 gene was studied by assessment of morphological parameters of М. truncatula OE and RNAi lines, grown either in vitro, in vivo (greenhouse), or as hydroponic cultures. The root system of in vitro-grown М. truncatula OE lines was characterized by primary roots in a circular arrangement around the stem and forming more secondary branches (Fig. 5a), as compared to controls (Fig. 5b). Roots of in vitro RNAi lines were similar to the control roots but thicker (Fig. 5c). In vitro RNAi lines had a dwarf phenotype with shortened and thick stems (Fig. 5c), thick roots covered with long root hairs (Fig. 5e), and small leaves, covered with many trichomes (Fig. 5d). М. truncatula OE lines grown in greenhouse conditions (Fig. 6a) had a large well-developed aerial part and more
I. Boycheva et al. Fig. 3 Expression of GUS and GFP reporters in nodules of HAC1 transcriptional reporter plants of L. japonicus: a GUS activity in nodules, b negative control for green fluorescence, and c GFP activity in nodules
extensively branched stems (Fig. 6a, left) compared to the control (Fig. 6a, right). Hydroponically grown plants showed highly branched root system with a pronounced spindle shape (Fig. 6c) and a higher number of small nodules, as compared to the wild type (Fig. 6b). M. truncatula RNAi lines grown in greenhouse conditions possessed shorter aerial part with small leaves. Most of the flowers turned black and fell off; some plants were able to form small pods (Fig. 7a). Hydroponically grown plants formed nodules in the upper part of the root system that was considerably less branched (Fig. 7b). In vitro OE lines of L. japonicus possessed short branched stems and thickened roots, which was opposite to the unbranched stems and long thin roots in wild-type plants. Hydroponically grown OE lines of L. japonicus had largesize nodules usually located in the upper part of the root system, while the control plants formed smaller and dispersed nodules (Fig. 8a, b, e, f). Downregulation of HAC1 resulted in plants with smaller leaves and nodules located in the middle part of the root (Fig. 8c, d). A reduced nodule number was noticed in both L. japonicus OE and RNAi lines, as compared to the control plants. To examine T4 homozygous A. thaliana OE 2 and RNAi 10 lines, morphometric analyses were done, and the following
Fig. 4 Expression of GFP marker gene in A. thaliana HAC1 transcriptional reporter plants: a cytoplasm and nucleus, b nucleus, c trichome, and d root tip. Scale bars: a = 20 μm; b and c = 50 μm; d = 200 μm
parameters quantified: root growth dynamics, width and length of the leaf blade, petiole length, and silique length and width. Statistical analyses of root growth dynamics showed higher root growth rate of OE 2 line at 24 h (р ≤ 0.05 ) and 48 h (р ≤ 0.001), compared to the wild-type (Fig. 9а). In contrast, the RNAi 10 line showed reduced primary root length at 48 h (р ≤ 0.001) and 72 h (р ≤ 0.001), as compared to the control (Fig. 9a). The OE 2 line possessed significantly smaller leaf blade width (р ≤ 0.05) and length (р ≤ 0.01) and petiole length (р ≤ 0.05), compared to the wild type (Fig. 9b). In contrast, in the RNAi 10 line, the same parameters were very similar to those of the wild type (Fig. 9b). The silique length of the OE 2 line was similar to the wild type, while the silique width was significantly reduced (р ≤ 0.05) (Fig. 9c, d). In the RNAi 10 line, the silique length and width were significantly increased (р ≤ 0.01), as compared to the control (Fig. 9c, d). Microscopic analyses of leaf epidermis of A. thaliana with modified expression of HAC1 showed that RNAi 10 produced
Fig. 5 Phenotype of in vitro M. truncatula plants: a roots in T1-HAC1OE 2 line, b roots in WT plant, and c–e in vitro T1-HAC1-RNAi 1 line. Scale bars: a, b, d = 1 cm; c, e = 2 cm
Different functions of the HAC1 gene in three model plants Fig. 6 Morphology of M. truncatula plants grown in greenhouse conditions: a plant height and architecture in T1HAC1-OE 2 line (left) and WT (right), b root system of WT plants, and c root system of T1HAC1-OE 2 line. Scale bars = 5 cm
bigger leaves with larger epidermal cells (Fig. 10a, c; ESM 1c) and a decreased epidermal cell number (Fig. 10b), compared to the wild type and OE 2 line. On the other hand, OE 2 line formed smaller leaves with an increased epidermal cell number, compared to the wild type and RNAi 10 line (Fig. 10a, b; ESM 1a, b). In mature first leaves, DNA ploidy levels were assessed by flow cytometric measurements. Line RNAi 10 formed leaves with a significant increase in 8C and 16C cells, as compared to line OE 2. The ploidy level was between 2 and 3 times higher than in wild-type leaves (Fig. 10d). This observation correlated well with the larger epidermal cell size detected by microscopic observations (ESM 1c). In OE 2, the ploidy distribution was similar to the control with the only exception of almost doubled number of cells with 16C nuclei.
Fig. 7 Phenotype of M. truncatula T 1 -HAC1-RNAi 1 plants: a greenhouse flowering plant and b root system with nodules
A. thaliana root tips from seedlings of OE 2, RNAi 10, and wild type were synchronized by treatment with 1 mM HU (Cools et al. 2010). After 8 h of synchronization, corresponding to S phase, root tips were harvested. Transcript levels of the S-phase marker gene (CYCA3;1) and two core histones H2B and H4 were evaluated by qRT-PCR. In HAC1 knockdown line (RNAi 10), a higher accumulation of H2B and H4 transcripts was detected, as H2B showed more distinct increase. In the OE 2 line, transcript levels of the tested genes were equal to the control, which imply that HAC1 is probably involved in acetylation of histones H2B and H4 (Fig. 10e). We also tested whether the acetyltransferase inhibitor curcumin is effective in plant cells. L. japonicus OE and wild-type seedlings were treated with curcumin at a concentration of 80 and 100 μM for 24 and 48 h. Expression analyses of collected root tips displayed that the transcript level of HAC1 in wild-type root meristem was reduced after treatment with 100 μM curcumin at time points 24 and 48 h. On the other hand, in the OE line, HAC1 transcript level was also reduced but showed the same values at the two time points analyzed (Fig. 10f). In order to test if the homeotic genes AP1 and AP3 could be possible targets of HAC1, we evaluated their transcript levels in the flowers of A. thaliana transgenic and wild-type plants. Results showed higher levels of AP1 transcripts in the flowers of OE 2 line, as compared to wild-type plants. In the flowers of the knockdown RNAi 10 line, the transcript level of AP1 was considerably lower than in the control and OE 2 line. The transcript level of AP3 was similar to the control (Fig. 10g). Deviations in flower morphology, such as the number and
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Discussion
Fig. 8 Phenotype of L. japonicus hydroponically grown plants: a, b roots with nodules in T1-HAC1-OE 2 line; c, d roots with nodules in T1-HAC1RNAi 4 line; e, f roots with nodules in WT. Scale bars: a, c, e = 5 cm; b, d, f = 1 cm
shape of petals (Fig. 11b, c), were observed in knockdown plants of the line RNAi 10, grown in greenhouse conditions, compared to control flowers (Fig. 11a).
The studied gene encoding HAC1 from M. truncatula belongs to the р300/CBP family and participates in one of the major posttranslational modifications of histone, histone acetylation. By Agrobacterium-mediated genetic transformation, stable transgenic plants with HAC1 overexpression, RNAimediated downregulation, and endogenous promoter fusion to the marker genes GUS and GFP were generated for the model legumes M. truncatula and L. japonicus and for A. thaliana as a referent plant. The transgenic plants were grown under in vitro conditions, as hydroponic cultures and in greenhouse conditions. Experiments were performed with T1 stable transgenic plants of M. truncatula and L. japonicus and T3 homozygous progeny of A. thaliana. Transcript analyses of T 1 transgenic seedlings of M. truncatula showed high levels of HAC1 expression in the OE lines and very low levels in the lines with HAC1 downregulation. T1 seedlings of L. japonicus with heterologous overexpression of HAC1 showed considerably less pronounced overexpression compared to M. truncatula. On the contrary, the nodules from mature L. japonicus plants with HAC1 overexpression had significantly higher transcript levels than the nodules of M. truncatula OE line. The high abundance of the HAC1 transcripts in the nodules of L. japonicus OE line corresponded to the observed intense GFP signal and GUS staining, which was not detected in the nodules of M. truncatula. In the referent plant A. thaliana, the HAC1 transcript levels in the three selected lines (OE 1, OE 2, and OE 3) were significantly higher; however, reduced HAC1 transcripts were detected in only one of the tested lines (line RNAi 10) with downregulation. A possible explanation for the observed expression profiling results could be the used RNAi strategy for obtaining A. thaliana plants with HAC1 downregulation. Most knockdown A. thaliana plants are generated via T-DNA or amiRNA inhibition strategies (Schubert et al. 2004; Eamens et al. 2014). Additionally, in Arabidopsis, there are five p300/CBP HAT homologues (HAC1, HAC2, HAC4, HAC5, and HAC12) that are highly related and could be functionally redundant (Deng et al. 2007). This suggestion is supported by the fact that a much stronger flowering phenotype is observed in hac double mutants (Han et al. 2007). The expression of the reporter genes was detected in different stages of indirect somatic embryogenesis of M. truncatula but was not observed during embryogenesis of L. japonicus. GUS and GFP activities were detected in the nodules of L. japonicus but were not observed in the nodules of M. truncatula. The observed difference in gene reporter activity in the nodules of the model legumes could be associated with different types of the nodules formed: indeterminate nodules in M. truncatula and determinate in L. japonicus. This can contribute to different transcriptional and metabolic activities (Oldroyd et al. 2011). On the other
Different functions of the HAC1 gene in three model plants Fig. 9 Morphometric analyses of A. thaliana plants—T3-HAC1OE 2 line, T3-HAC1-RNAi 10 line, and WT: a dynamic of root growth, b leaf growth (W leaf width, L leaf length, P petiole length), c length of siliques, and d width of siliques
a
b
c
d
hand, in all three model species, the signal appeared in the young plant tissues and organs, where the cells were actively dividing. Confocal imaging of the subcellular localization of HAC1 revealed GFP fluorescence within the cell nuclei and cytoplasm. This finding is a direct experimental validation of the predicted subcellular localization of HAC protein members, determined using the online tool WoLF PSORT, which concludes that HAC1 is located mainly in the nucleus and cytoplasm (Cemanovic et al. 2014). The investigated HAC1 gene plays an essential role in multiple plant developmental processes, including root growth, cell differentiation, and leaf and floral organogenesis. We characterized growth of A. thaliana plants, where HAC1 was disrupted by the insertion of T-DNA element into the gene. Reduced root growth was observed in the RNAi 10 line, compared to the OE line and control. The line with downregulation was characterized by large-size leaves developing larger epidermal cells that contained almost doubled 4C and 8C values, compared to the control. Mutations in AtHAC1 led to multiple defects in plant development, such as decreased growth, short primary root, and reduced fertility, which were observed also by Deng et al. (2007). The results acquired using stable transgenic plants with HAC1 overexpression and downregulation displayed a number of deviations from the control. Transgenic M. truncatula OE lines possessed a spindle-like root system with multiple secondary branches (Fig. 6c), while the L. japonicus OE lines had short and thickened roots (Fig. 8a). Both model legumes
with HAC1 overexpression, grown as hydroponic cultures, formed many root nodules; however, their localization patterns were substantially different. The root system of M. truncatula had small nodules along the entire length of the root system, while L. japonicus formed large nodules that were localized in the upper part of the root system (Fig. 8a, b). The high shoot of M. truncatula OE lines was in contrast with the short and branched stems of L. japonicus OE lines. The phenotypic deviations from the control observed in both legumes with HAC1 overexpression confirmed the participation of HAC1 gene in plant growth and development. In the M. truncatula lines with HAC1 downregulation, nodules were localized in the upper part, while in L. japonicus, most of the nodules were concentrated in the middle part of the root system. Transgenic A. thaliana plants with knockdown of HAC1 possessed larger leaves and epidermal cells; however, their number was almost unchanged, in comparison with the wild type (Fig. 10a–c). The same observation has been reported for the Arabidopsis mutant gcn5-2, where leaf epidermal cells are larger and do not have the regular arrangement of the wildtype cells (Servet et al. 2010). The same authors have further shown that the Arabidopsis HAT gene AtGCN5/HAG1 plays a key role in many plant development processes, such as meristem function, cell differentiation, leaf and floral organogenesis, and responses to light and cold. It has been demonstrated that HAC1, belonging to the р300/CBP gene family, is involved in the acetylation of H2B and H4 histones during the G1/S cell cycle transition
I. Boycheva et al.
a
b
d
e
f
g
c
Fig. 10 Functional analyses of transgenic and control plants of A. thaliana (T3-HAC1-OE 2 line, T3-HAC1-RNAi 10 line, and WT) and L. japonicus (T1-HAC1-OE 2 line, T1-HAC1-RNAi 4 line, and WT): a leaf area in A. thaliana plants, b epidermal cell number in A. thaliana leaves, c epidermal cell size in A. thaliana leaves, d
histogram of DNA content in A. thaliana leaves, e relative transcript level of S-phase marker genes (CYCA3;1, H2B, and H4) in root tips of A. thaliana, f relative transcript level of HAC1 gene after treatment of L. japonicus seedlings with 100 μM curcumin, and g relative transcript level of homeotic genes AP1 and AP3 in A. thaliana flowers
(Jasencakova et al. 2000; Fu and Kurzrock 2010). Our results confirmed that H2B and H4 transcript levels in A. thaliana with knockdown of HAC1 showed slightly higher accumulation during S phase, as compared to the lines with overexpression and wild-type plants (Fig. 10e). It seems that in A. thaliana with HAC1 knockdown, the accumulation of both
core histones during S phase probably interferes with the replication process and completion of the mitotic phase, thus leading to endoreduplication, which probably underlies the observed high number of large epidermal cells of higher ploidy (8C, 16C; Fig. 10d). In OE lines, the size of epidermal cells was similar to that of wild-type plants (Fig. 10c). Based on the
Fig. 11 Deviation in flower morphology in A. thaliana T4HAC1-RNAi 10 line: a flower from WT plant and b, c abnormal flower morphology in the knockdown line
Different functions of the HAC1 gene in three model plants
transcriptional analyses, currently, we can only speculate that the studied HAC1 is involved in the acetylation of the H2B and H4. Curcumin is a specific inhibitor of the histone acetyltransferases HATs belonging to the p300/CBP family. It has been shown that curcumin application strongly suppresses acetylation of histones H3 and H4, mediated via p300/CBP HAT activity (Fu and Kurzrock 2010). The effect of curcumin as histone acetyltransferase inhibitor is studied in animals and humans, and these analyses are related to neoplastic, neurological, cardiovascular, pulmonary, and metabolic diseases (Aggarwal and Sung 2009). A number of reports have shown the effect of curcumin on human and animal cells, addressing problems in the field of medicine. This study asked the question if curcumin could be used as an active inhibitor of HATs in plant cells, and what is the effective dose and appropriate treatment time. Different studies have used various concentrations and exposure times, as the efforts aimed at reducing or eliminating the toxic effects of curcumin treatment on the cell. Specificity of plant cells that are surrounded by cell walls could be related to a higher tolerance to curcumin that requires a longer treatment time. Results from our experiments demonstrated that this inhibitor reduces the expression of the histone acetyltransferase HAC1. A number of data confirm that molecular targets like p300/cAMP-response element-binding protein (CREB) could be affected by curcumin and their expression upregulated or downregulated (Aggarwal and Sung 2008). Biological activities of curcumin on histone acetyltransferases p300/CBP are manifested by a direct inhibition of p300 and downregulation of p300 expression (Marcu et al. 2006; Fu and Kurzrock 2010). The tested two concentrations and two time points showed that the concentration of 100 μM for 24 and 48 h treatment could reduce HAC1 transcript level in the wild-type root meristem more effectively after the longer treatment period, while the transcript level in the OE line was reduced after 24 h treatment but remained unchanged after 48 h treatment (Fig. 10f). Choudhuri et al. (2005) have also found that curcumin less effectively downregulates the expression of cyclin D in cell systems with overexpression. To gain further insights into the different aspects of curcumin effects on plant cells, additional experiments are required. It has been shown that possible targets of HAC1 are genes involved in plant organ development (Sundstrom et al. 2006; Deng et al. 2007; Han et al. 2007). We compared transcript levels of AP1 and AP3 genes in A. thaliana with HAC1 overexpression, knockdown, and wild-type plants. Results showed higher transcript levels of AP1 in the HAC1-OE flowers compared to the wild type and low transcript level in the flowers of the line with HAC1 downregulation. The obtained results suggest that AP1 could be a possible target gene of HAC1. The AP3 transcript levels in both lines with modified expression were similar to the control, indicating that AP3 could not be a
target gene of the investigated HAC1 (Fig. 10g). The results from the transcript analyses correlate well with the observed disturbances in flower morphology of the A. thaliana knockdown line, such as a modified petal shape or absence of petals (Fig. 11b, c).
Conclusion Based on the generated stable transgenic lines of the model plants M. truncatula, L. japonicus, and A. thaliana, and tracking the activity of GUS and GFP transcriptional reporters, we confirmed the expression of the HAC1 gene in different plant organs, where actively dividing cells are located. Transgenic lines with modified HAC1 expression showed a wide range of morphological deviations in plant architecture, which confirmed the role of the gene in plant development. It could be suggested that HAC1 participates in the acetylation of the two core histones H2B and H4 during S phase of the plant cell cycle, which was confirmed by accumulation of their transcripts in the knockdown line. A possible role of HAC1 in the processes of cell proliferation and differentiation could be hypothesized. Our results demonstrated that the histone acetyltransferase inhibitor curcumin could be used to modify histone acetyltransferase expression in plant cells. Acknowledgments This study was supported by a grant from the National Science Fund of the Ministry of Education and Science of the Republic of Bulgaria, project Do 02-268 (Integrated functional and comparative genomics studies on the model legumes Medicago truncatula and Lotus japonicus, acronym IFCOSMO). The authors are grateful to Kety Krastanova for her valuable technical assistance. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.
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