KNOX1 is expressed and epigenetically regulated during in vitro conditions in Agave spp
© De-la-Peña et al.; licensee BioMed Central Ltd. 2012
Received: 24 May 2012
Accepted: 23 October 2012
Published: 5 November 2012
The micropropagation is a powerful tool to scale up plants of economical and agronomical importance, enhancing crop productivity. However, a small but growing body of evidence suggests that epigenetic mechanisms, such as DNA methylation and histone modifications, can be affected under the in vitro conditions characteristic of micropropagation. Here, we tested whether the adaptation to different in vitro systems (Magenta boxes and Bioreactors) modified epigenetically different clones of Agave fourcroydes and A. angustifolia. Furthermore, we assessed whether these epigenetic changes affect the regulatory expression of KNOTTED1-like HOMEOBOX (KNOX) transcription factors.
To gain a better understanding of epigenetic changes during in vitro and ex vitro conditions in Agave fourcroydes and A. angustifolia, we analyzed global DNA methylation, as well as different histone modification marks, in two different systems: semisolid in Magenta boxes (M) and temporary immersion in modular Bioreactors (B). No significant difference was found in DNA methylation in A. fourcroydes grown in either M or B. However, when A. fourcroydes was compared with A. angustifolia, there was a two-fold difference in DNA methylation between the species, independent of the in vitro system used. Furthermore, we detected an absence or a low amount of the repressive mark H3K9me2 in ex vitro conditions in plants that were cultured earlier either in M or B. Moreover, the expression of AtqKNOX1 and AtqKNOX2, on A. fourcroydes and A. angustifolia clones, is affected during in vitro conditions. Therefore, we used Chromatin ImmunoPrecipitation (ChIP) to know whether these genes were epigenetically regulated. In the case of AtqKNOX1, the H3K4me3 and H3K9me2 were affected during in vitro conditions in comparison with AtqKNOX2.
Agave clones plants with higher DNA methylation during in vitro conditions were better adapted to ex vitro conditions. In addition, A. fourcroydes and A. angustifolia clones displayed differential expression of the KNOX1 gene during in vitro conditions, which is epigenetically regulated by the H3K4me3 and H3K9me2 marks. The finding of an epigenetic regulation in key developmental genes will make it important in future studies to identify factors that help to find climate-resistant micropropagated plants.
KeywordsEpigenetics In vitro Histone methylation Agave KNOX genes
DNA methylation and histone modifications are important epigenetic mechanisms for gene regulation in eukaryotes [1–3]. In plants, epigenetic mechanisms play an important role in development [4, 5], flowering , pathogen recognition , senescence  and somaclonal variation [9–11]. It has been found that DNA methylation/demethylation is affected by exogenous and endogenous factors in both in vivo and in vitro conditions, such as auxin concentration, temperature and aging [5, 12–14]. DNA methylation patterns can also change, depending on the method of plant propagation .
The use of in vitro plant propagation techniques allows the scale up of crops of agronomic importance [16, 17], maintaining genetic stability among clones. Although clonal plants are usually very stable at the genetic level, epigenetic modifications in DNA [12, 18, 19], as well as histones [20–24], mainly by methylation in lysine 9 and 4 of the histone H3, could be affected and, therefore, induce somaclonal variation . There is evidence of epigenetic changes, although some can be quite stable with time , occurring during in vitro culture. For instance, Valledor et al. found that an increase in plant vigor and rejuvenation is due to DNA methylation. On the other hand, it has been found that the decrease in organogenesis capability of Pinus radiata was related to low levels of acetylation in histone H4 and high levels of DNA methylation . It has been proposed that the changes in DNA methylation patterns in maize and apple are induced by tissue culture during in vitro conditions [27–29], and these methylation patterns play an important role during plant development [4, 5]. Furthermore, not only methylation [30, 31] but also demethylation in the DNA could cause epigenetic alterations that provoke abnormalities during the in vitro process [32, 33]. One of the factors involved in epigenetic changes during in vitro conditions is the exposure to growth regulators, which are widely used in plant tissue culture [34, 35] to promote multiplication and growth.
Plant growth regulators have been involved in the expression of several genes, and some studies have even suggested reciprocal links between growth regulators and homeobox genes . One of the most studied homeobox genes regulated by plant growth regulators is the KNOTTED1-like HOMEOBOX (KNOX) transcription factor group [37–39]. In Arabidopsis thaliana, there are eight KNOX genes that have been divided into two classes. STM, KNAT1, KNAT2 and KNAT6 belong to class I, while KNAT3, KNAT4, KNAT5 and KNAT7 fall into class II . The expression of two, KNAT1 and KNAT6, is altered by cytokinins and auxins [41–43]. Rupp et al. found an increase in the transcription level of KNAT1 in the cytokinin-overproducing mutant amp1, which might occur by acting through the activation of KNAT1. Furthermore, Dean et al. found in A. thaliana that exogenous auxin treatments alter the promoter activity of the gene KNAT6. In the same way, Montero-Cortes et al. found that in somatic embryos from micropropagated coconut plants, the expression of CnKNOX1 was stimulated by gibberellic acid, while in CnKNOX2 the hormone produced a decrease in its expression. Taking all these findings as a whole, it is clear that plant growth regulators have an important impact on KNOX genes.
Studies done in Agave tequilana have shown that KNOX genes are associated with organogenesis during bulbil formation . According to Abraham-Juarez et al., AtqKNOX1, homologous to KNAT1, and AtqKNOX2, homologous to KNAT6, presented an increase in the levels of expression as bulbils mature. The regulation of KNOX genes in Agave has not been clearly understood, but studies in Arabidopsis and maize suggest that chromatin configuration could be an important factor in the regulation of these genes [40, 45, 46]. In humans, homeobox genes, which encode transcription factors evolutionarily conserved, are epigenetically regulated . Therefore, it is possible that, as in animals, plant homeobox genes, such as KNOX, can also be epigenetically regulated.
Although the epigenome is highly dependent on the surrounding environment [48, 49], the epigenetic changes that occur under in vitro conditions are still unknown, as is how these changes impact plants’ development in the field. In order to understand the effect of in vitro conditions on the epigenetic regulation of AtqKNOX1 (KNAT1) and AtqKNOX2 (KNAT6), in this study we provide a detailed epigenetic analysis comparing different Agave plants cultured under different in vitro and ex vitro conditions.
Transfer to ex vitro conditions increases DNA methylation in Agave angustifolia
In vitroconditions induce methylation in H3K9
KNOX expression is induced by in vitroconditions
AtqKNOX1is epigenetically regulated
The H3K4me3 methylation levels in AtqKNOX1 in BM26 were not detected in any condition, which also correlates with its lack of expression found in Figure 5. During in vitro conditions in the Bioreactor, the H3K4me3 levels increased, favoring the expression due to epigenetic regulation of AtqKNOX1 in the clone P20. Considering these results and the ones obtained with gene expression studies, it appears that the AtqKNOX1 expression (Figure 5) is directly correlated with the H3K4me3 levels. Furthermore, we determined the levels of H3K9me2, a mark related to heterochromatin and repressive gene regions, of all analyzed genes (Figure 6). We found that only in BM26 was there an increase in H3K9me2 in AtqKNOX1 in all culture conditions. This result is consistent with the lack of expression of this gene in BM26.
These results could help to develop new strategies to optimize the use of more efficient in vitro conditions in order to guarantee the epigenetic stability of the cultures in the field.
Plant tissue culture has been used for many years to propagate elite plants and for genetic breeding [52, 53]. In Mexico, clonal propagation has been successfully employed to improve revigorization and juvenility in commercial plantations of Agave [16, 50, 54, 55]. It is known that Agave plants cultivated under in vitro conditions for several generations do not contribute to genetic variation among clones , but some phenotypic variations have still been found. One of the explanations for these phenotypic changes under in vitro culture could be epigenetic regulation.
There is small but increasing evidence describing the epigenetic changes during in vitro culture. It has been found that not only the environment during the in vitro culture can change the epigenetic profile of the plant [27–29], but also the epigenetic status of the donor plants; even the organs within the donor plants can determine the later behavior of the explants . For instance, it has been found that in vitro conditions change DNA methylation [27–29] and even this epigenetic mechanism has been related to plant development and rejuvenation [4, 5]. Therefore, the DNA hypomethylation found in BM26 when this clone was changed from T0 to either the Magenta box or the Bioreactor (Figure 3) could be due to stress , which might be related to the increase in the mortality rate observed when these plants were transplanted to soil. Another explanation is that rejuvenation is occurring in this clone during its time in the Magenta boxes or the Bioreactor. Valledor et al. found that there is a relationship between DNA methylation and aging-revigoration in plants, such that aging increases as DNA global methylation increases. In plants, DNA methylation usually increases with aging [26, 56], while in mammals it decreases with time [57, 58]. Therefore, the decrease in DNA methylation observed in A. angustifolia during in vitro culture (Figure 3) could be a mechanism for rejuvenation.
Li et al. reported that several physiological changes related to in vitro culture, such as leaf structural changes, modifications in plant water content and changes in photosynthetic systems, are related to the stress provoked by in vitro conditions, and the stress seems to be related to the content of global DNA methylation. We observed in Agave that the semisolid system in the Magenta boxes generates longer leaves in comparison with the plants cultured in the temporary immersion of the modular Bioreactors (Figure 2). Although we did not observe a significant difference in DNA methylation between Magenta boxes and Bioreactors in the same clone (Figure 3), we observed a difference in histone methylation patterns between plants grown in these two in vitro systems (Figure 4). The genetic expression provoked by stress in plants depends on histone postranslational modifications and DNA methylation . In the case of histone methylation, there are no reports that explain or suggest either the somaclonal variation or the genes affected epigenetically. Although there is information about the role of DNA methylation during in vitro culture, the histone modifications and the changes in chromatin modulation are still unknown. We found that clones genetically and even phenotypically alike have different epigenetic responses to in vitro culture (Figure 4). Moreover, there are no reports of the epigenetic stability of the micropropagated plants once they are ex vitro; so far, it is unknown whether histone modifications are involved. It is known that the epigenetics of an organism can change depending on development , biotic  or abiotic interactions , and even stress exposure . Therefore, the mechanism of stress response due to the exposure to growth regulators during in vitro conditions could be one of the candidates for regulation by epigenetic factors. We found that during in vitro conditions, epigenetic modifications in histones (Figure 4), mainly through H3K9me2, which is very important in the initiation and maintenance of heterochromatin silencing  and in the control of DNA methylation , are affected. However, once the plants were transferred to ex vitro conditions in the field, this histone mark was absent or in present in low amounts, suggesting that plants can change the epigenome-phenotype.
In other in vitro systems, such as the potato, the DNA methylation variation associated with tissue culture protocols has been investigated . It was found that DNA methylation changes occurring among the tissue types are an essential factor contributing to developmental stage differences, as well as tissue-culture-induced variation. Therefore, the hypermethylation found in P159 at SM could be induced by elements of the tissue culture media such as plant hormones, which have been shown to induce methylation changes in plant tissue cultures [12, 19, 65]. There is evidence that the use of the auxin 2,4-D in maize cultures generates changes in the DNA methylation pattern, depending on the concentration . Plant hormones regulate growth and development in plants by controlling the expression of genes involved in these processes.
KNOX genes have been implicated in plant hormone metabolism [66, 67]. Hay et al. found that auxins repress the KNAT1 gene, promoting leaf development in Arabidopsis. Furthermore, it has been proposed that alterations in auxin gradients could result in a failure to down-regulate KNOX expression . Different epigenetic mechanisms have been suggested for the regulation of KNOX genes during organogenesis [68, 69]. In this study, we showed that the AtqKNOX1 gene is epigenetically regulated by H3K4me3 and H3K9me2 (Figure 6). Histone modification is a very complex epigenetic mechanism that so far has not been decoded [70–72]. However, studies in Arabidopsis have revealed that histone H3K9 methylation exists predominately as mono- and di-methylation, while trimethylation in H3K9 is quite rare . There is evidence showing that in plants H3K27me3, H3K9me3 and H3K4me2 are euchromatic marks, while H3K9me2 is more associated with the repression of the transcription [63, 74]. Chromatin changes have become an important key element for development in plants  and histone modification is essential . Changes in KNOX1 gene expression among species could be due to different factors such as diversification of repressors of these genes . Among the main roles of KNOX are the formation of auxin maxima, which provide feedback to repress KNOX expression, allowing leaflet outgrowth [67, 77].
It will be interesting to study the methylation patterns from different generations exposed to in vitro conditions compared to those that were not, to determine whether the plants remember the in vitro exposure through epigenetic marks.
DNA methylation and histone modifications are very important epigenetic mechanisms that can be affected by in vitro conditions. Our studies indicate that under in vitro conditions, DNA methylation is affected in A. angustifolia, but not in A. fourcroydes. In addition, A. fourcroydes presented differential expression of AtqKNOX1 and AtqKNOX2, depending on the in vitro system used. Furthermore, the regulatory expression of AtqKNOX1 was related to the H3K4me3 and H3K9me2 marks. We propose that in vitro conditions change key genes by epigenetic regulation, which could be an important tool to find plants better adapted to overcome climate challenges.
Plant material and growth conditions
Three different in vitro-propagated Agave fourcroydes clones (P20, P21 and P159) and one Agave angustifolia clone (BM26) were used. The media used for plant induction, multiplication and growth of the plants was Murashige and Skoog , at pH 5.7, with some modifications as reported by Robert et al. [16, 55]. Briefly, the plants from each clone were kept for six weeks in Magenta containers filled with 50mL of Murashige and Skoog media with reduced nitrogen, solidified with 1.75g/L of Gelrite (semisolid media) and without growth regulators. All plantlets were then transferred to and maintained in multiplication media supplemented with 10 mg/L BAP and 0.025 mg/L 2,4-D for ten weeks. Sixteen-week-old plants of the same size from each clone were divided as follows: 25 were sampled for analysis (T0) and 100 were cultured in growth medium supplemented with 1mg/L BAP and 0.025 mg/L 2,4-D. At this growing stage, two different systems were used: 50 plantlets were maintained in semisolid growth media in Magenta boxes [Magenta (M)] supplemented with 10 g/L of Agar, and 50 plantlets were cultured in liquid growth medium under temporary immersion in modular Bioreactors [Bioreactor (B)], as described by Robert et al.. After five weeks, 25 plantlets from both in vitro systems (M and B) and from each clone (P20, P21, P159 and BM26) were sampled, and the remaining 25 from M and B were transferred to soil (SM and SB), where they grew for another eight weeks before they were also evaluated (Figure 1B).
Histone isolation and Western blots
Histones from Agave spp. clones (P20, P21, P159 and BM26) were isolated from 0.5 grams of leaf tissue from T0, M, B, SM and SB using sulfuric acid extraction of nuclei proteins followed by acetone precipitation, according to Jackson et al.. Ten micrograms of isolated histones per sample were used for Western blots. The proteins were transferred to nitrocellulose membrane (0.45μm) by electrophoresis for four hours at 265mA. Membranes were blocked with 5% milk and 0.5% Tween in PBS, and probed with various antibodies, as follows: dimethyl-Histone H3 [Lys-4] (Upstate, cat. #07–030), trimethyl-Histone H3 [Lys-4] (Upstate, cat. #04–745), dimethyl-Histone H3 [Lys-9], (Upstate, cat. #07–441) and anti-dimethyl-Histone H3 [Lys-36] (Upstate, cat. #07–274). Di-(m2/H3) and tri-(m3/H3) methylated levels were measured and compared in histones isolated from different samples. The amount of loaded histone H3 in each sample was determined from Western blots using antibodies specific to non-methylated H3 (Upstate, cat. #06-755). Signals from bands obtained with methylation-specific antibodies were normalized against the respective histone H3 amounts (measured as signal intensities of Western-blot bands obtained with anti-histone H3-antibodies). All blots were stripped and reprobed with the histone H3 antibody to demonstrate equal loading. Data from four independent measurements consistently gave the same results.
DNA extraction was done following the method described by Echevarria-Machado et al.. DNA digestion was performed as described Santoyo et al., with slight modifications. Five μg of DNA from P20, P21, P159 and BM26 at T0, M, B, SM and SB were dissolved in 42μL of ultra pure water and mixed with 5μL of 10 X DNA digestion buffer (200 mM acetic acid, 200mM glycine, 50mM magnesium chloride, 5mM zinc acetate, 2 mM calcium chloride adjusted with sodium hydroxide to pH 5.3). The mixture was hydrolyzed with 2μL of DNase I (D2821-Sigma, 10U/μL) and 1μL of Nuclease P1 (N8630-Sigma, 1.25U/μL) overnight at 37°C and then frozen for 10–15 min at 0°C and then incubated at 100°C for five min. Samples were mixed with 5μL of 100 mM NaOH and 2μL Calf intestine alkaline phosphatase (P4879-Sigma, 1U/μL) and incubated for 3.5 h at 37°C and then mixed with 100μl of water and 50μl mobile phase D (see below). Samples were centrifuged at 18,000 × g for 10 min at 4°C, and the supernatant was transferred to a new tube and stored at −20°C until analysis. Forty Âµl of sample was injected to liquid chromatographic system (HPLC, Agilent series 1200), and the bases were separated on a chromatographic column, Luna C18 (250 × 4.6mm, 5μm from Phenomenex) at 40°C. The absorbance was measured using a diode array detector at 286 nm. The separation was realized according to the method described by Lopez Torres et al. with some modifications. Four mobile phases were used: A, deionized water; B, acetonitrile; C, methanol; and D, 50mM ammonium phosphate dibasic, 15mM ammonium acetate adjusted with phosphoric acid to pH 4.1. The gradient program was as follows: 0 to 4 min 80% A, 20% D; 4 to 11 min 78% A, 2% C, 20% D; 11 to 15 min 77% A, 3% C, 20% D; 15 to 15.8 min 35% A, 20% B, 25% C, 20% D; 15.8 to 16 min 30% A, 25% B, 25% C, 20% D at a total flow rate of 1 mL/min. The percentage of global DNA methylation was calculated as follows: concentration of 5-methyl-2’-deoxycytosine (5mdC)/ [concentration of 5mdC + concentration of 2’-deoxycytosine (dC)] × 100. All the analysis was achieved with three biological replicates from different DNA extractions. Statistical comparison was performed by one-way analysis of variance (ANOVA). The significance grade was determined by the test of several means of Tukey (P ≤ 0.01).
Total RNA was extracted from 0.2g leaf tissue of P20, P21, P159 and BM26 from T0, M, B, SM and SB by using the BRL Trizol reagent (Invitrogen) and re-purified with the Qiagen RNeasy Mini Kit, following the manufacturer’s instructions. Reverse transcriptase (RT) reactions were performed in a 20-μl volume containing 2μg of total RNA and 200 units of the M-MLV Reverse Transcriptase (Invitrogen), following the manufacturer’s instructions. cDNA templates for qRT-PCR amplification were prepared from three individual plants for each condition. Each reaction contained 100 ng of cDNA template, 10 pM of each primer and 1× EXPRESS SYBR® GreenERTM pPCR SuperMix Universal (11784-200-Invitrogen). Real-time PCR assays were performed in a Step OneTM Real Time PCR System (Applied Biosystems) under the following conditions: 5 min at 95°C, followed by 35 cycles of 95°C for 40 sec, 62°C for 40 sec and 72°C for 90 sec, and a final cycle of 72°C for 5 min. Transcript levels of AtqKNOX1 and AtqKNOX2 in the samples were normalized to the level of UBIQUITIN (UBQ11) and the data are expressed as the relative expression level. The specificity of the PCR product amplifications was determined by a melting curve analysis. Data obtained from Real-time PCR were used to calculate the relative quantification of the target gene expression and compared to the expression of the UBQ11 using the 2-∆∆ct method . We used the primers reported by Abraham-Juarez et al. to determine gene expression in Agave: AtqKNOX1 (GenBank Accession No. GU980050) forward 5’-gagggcagttcataggtgat -3’, reverse 5’-ttcccacaggagtaggtctc -3’ (190bp); AtqKNOX2 (GenBank Accession No. GU980051) forward 5’- gaatggtggactgctcacta-3’, reverse 5’-cctcagtcgtcgtcatagaa-3’ (225bp) (Additional file 1: Figure S1); and UBQ11 was used as a control 5’-gacgggcgcacccttgcggatta-3’, 5’-tcctggatcttcgccttgacatt-3’ (211bp). Statistical comparison was performed by one-way analysis of variance (ANOVA).
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed as described by De-la-Peña et al.. The antibodies used were anti-dimethyl Histone H3 [Lys9] (Upstate #07-441) and anti-trimethyl Histone H3 [Lys4] (Upstate #05-745). For all ChIP experiments, chromatin was isolated from leaves of P20 and BM26 from T0, M, B, SM and SB conditions. Each immunoprecipitation experiment was independently performed three times with separately isolated biological samples. All PCR reactions were done in 25μl: 5min at 95°C, followed by 38 cycles of 95°C 30 sec, 56°C 30 sec, 72°C 2 min, and 72°C 5 min. Intensities were normalized versus the input sample representing 15% of the DNA used as template. The ChIP primer sequences used were as follows: AtqKNOX1 forward 5’-gagggcagttcataggtgat-3’, reverse 5’-ttcccacaggagtaggtctc -3’; AtqKNOX2 forward 5’- gaatggtggactgctcacta-3’, reverse 5’-cctcagtcgtcgtcatagaa-3’; and UBQ11 was used as a control 5’-gacgggcgcacccttgcggatta-3’, 5’-tcctggatcttcgccttgacatt-3’.
Chromatin immuno precipitation
4-D: 2,4-Dichlorophenoxyacetic acid
High-performance liquid chromatography
Reverse transcription polymerase chain reaction
These studies were supported by the Consejo Nacional de Ciencia y Tecnología (Grants No. 121768 and CB-2012/178149 to CD).
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