A sugarcane R2R3-MYB transcription factor gene is alternatively spliced during drought stress

MYB transcription factors of the R2R3-MYB family have been shown to play important roles in many plant processes. A sugarcane R2R3-MYB gene (ScMYB2) and its two alternative forms of transcript (ScMYB2S1 and ScMYB2S2) were identified in this study. The deduced protein of ScMYB2S1 is a typical plant R2R3-MYB protein, while ScMYB2S2 encodes a truncated protein. Real-time qPCR analysis revealed that ScMYB2S1 is suppressed under PEG-simulated drought stress in sugarcane, while ScMYB2S2 is induced at later treatment stage. A senescence symptom was observed when ScMYB2S1 was injected into tobacco leaves mediated by Agrobacterium, but no symptom for ScMYB2S2. Further investigation showed that the expression levels of 4 senescence-associated genes, NtPR-1a, NtNYC1, NtCAT3 and NtABRE, were markedly induced in tobacco leaves after ScMYB2S1-injection, while they were not sensitive to ScMYB2S2-injection. Moreover, MDA and proline were also investigated after injection. Similarly, MDA and proline levels were induced by ABA and ScMYB2S1, while inhibited by ScMYB2S2. We propose that ScMYB2, by alternatively splicing two transcripts (ScMYB2S1 and ScMYB2S2), is involved in an ABA-mediated leaf senescence signaling pathway and play positive role in respond to drought-induced senescence in sugarcane. The results of this study provide information for further research in sugarcane stress processes.

MYB proteins are elaborately regulated at multiple steps 14,28 . In particular, accumulating evidence illustrates that post-transcriptional control of mRNA modulates the transcription factor activities during plant response to environmental stimuli 28 . Alternative splicing (AS), to regulate gene expression, is one subset of post-transcriptional processes 29 . Through effecting the production of mRNA isoforms with different exonic composition from a single gene, alternative splicing creates multiple mRNA transcripts 30 . Alternative splicing can affect protein function and influence protein diversity when AS events occur within the translated regions of mRNAs 30 . It is also common for AS to result in isoforms that contain a premature termination codon, which subsequently become targets for nonsense mediated decay 30 . By this way, instead of a truncated polypeptide, no protein is produced. Plant leaf senescence is an age-dependent deterioration process and is also triggered by environmental stresses and phytohormones 31,32 . It has been recognized that senescence associated genes (SAGs) are induced by senescence 32,33 . Abscisic acid (ABA) is an important phytohormone and plays a critical role in regulating plant development and responses to various stress signals, such as drought 32 . It is also well-known that ABA promotes leaf senescence 32 .
In the present work, the function of a sugarcane (Saccharum officinarum) R2R3-MYB gene ScMYB2 with its two alternative splicing transcripts, ScMYB2S1 and ScMYB2S2I, were investigated. We utilized real-time qPCR to evaluate the response of the alternative splicing transcripts to drought stress induced by PEG. A visible symptom of leaf senescence, i.e. de-greening, was observed when one alternatively spliced transcript of ScMYB2S1 was transiently over-expressed in tobacco (Nicotiana tabacum) leaves. To further investigate the role of ScMYB2S1 and ScMYB2S2 in senescence, the contents of malonaldehyde (MDA) and proline, and then the expression profiles of 4 previously reported senescence-associated genes (SAGs) are checked in tobacco leaves after ScMYB2S1or/and ScMYB2S2-transiently-transformed. Results suggest that ScMYB2 is involved in the response to drought. The differential expression of two alternatively spliced transcripts during PEG stress could be one kind of drought tolerance molecular mechanism in sugarcane.

Results
Cloning and sequence analysis of ScMYB2. Two rounds of PCR were performed, and gel electrophoresis of the inner PCR products showed one fragment ( Supplementary Fig. S1). The amplicons of about 1,100 bp long were separated and recovered from the agarose gel, which were subsequently used for T-A cloning and transformed into Escherichia coli. Ten randomly positive clones were picked and sequenced, and two R2R3-MYB-like cDNA sequences were obtained, designated as ScMYB2S1 (GenBank Accession Number KM387410) and ScMYB2S2 (GenBank Accession Number KM387411), respectively. ScMyB2S1 had a full length of 1, 066 bp, with an ORF of 687 bp, 5′ UTR (untranslated region) of 40 bp, and 3′ UTR of 339 bp (Fig. 1). The deduced protein of ScMYB2S1 was a typical plant R2R3-MYB protein, containing two MYB DNA-binding domains (R2 and R3 repeats) at the N-terminal. Within the R2 and R3 repeats, the highly conserved tryptophan (W) residues implicated in DNA-binding were spaced by the 19 or 18 amino acid residues, respectively. The first W of R3 repeat in ScMYB2S1 protein was replaced by a methionine (M) (Fig. 1).
Overlapping the full-length sequences of ScMYB2S1, 968-bp long ScMYB2S2 transcript contained an additional 29-bp-sequence inserting into the corresponding location of the ORF, which interrupted the reading frame of a subsequent region behind the start codon and caused frameshift mutation of the sequence. Thus, compared with the amino acid sequence of ScMYB2S1, the first MYB DNA-binding domain (R2) in the amino acid sequence of ScMYB2S2 was missing, which resulted in the residue part starting with the first methionine (M) of R3 repeat, thereafter sharing 100% homology to the ScMYB2S1. Cloning a genomic sequence of the gene was also performed to identify whether the two transcripts, ScMYB2S1 and ScMYB2S2, were produced by alternative splicing of the same gene. The genomic sequence of the ScMYB2 gene (GenBank Accession Number KM387409) displayed at least two alternatively spliced isoforms (Fig. 2): a typical plant R2R3-MYB transcription factor gene ScMYB2S1 and ScMYB2S2 encoding a truncated protein starting at a methionine in the R3 repeat.
The genomic sequence of the ScMYB2 had a highly conserved splicing arrangement with three exons and two introns (126 bp and 76 bp). The 126 bp intron appeared to consist of two short tandem intron-like sequences, 29-bp sequence mentioned above at the 5′ -terminal and the other 97 bp sequence at the 3′ -terminal. All of them conformed to the GT-AG rule (Fig. 2).
Following the methods described by Matus et al. 34 and Lin-Wang et al. 24 , we constructed a phylogenetic tree with ScMYB2S1, ScMYB2S2 and the other 131 Arabidopsis MYB proteins using Mega5.05 software. Figure 3 indicated that ScMYB2S1 and ScMYB2S2 were close to AtMYB48 and AtMYB49, two members from Arabidopsis described as alternative splicing/non-canonical intron subgroup 34 .
Expression profiles of ScMYB2S1 and ScMYBS2 under drought stress. To further examine the function of the alternatively spliced transcripts of ScMYB2, their responses to drought stress were performed. When treated with 25.0% PEG, the expression level of ScMYB2 rapidly decreased at 3 h (Fig. 4) and stayed at the relatively low level during the periods from 3 h to 24 h, but increased in the later periods (48 h and 72 h). By using primers specific for each splice variant, the level of ScMYB2S1 expression decreased steadily after 3 h following the treatment and maintained at a low expression level up to 72 h. In contrast, the expression of the ScMYB2S2 increased dramatically at 48 and 72 h.

Agrobacterium-mediated transient expression in tobacco leaves.
To gain insights into the role of ScMYB2 in sugarcane, transient expression of pGreenII0229-ScMYB2S1, pGreenII0229-ScMYB2S2 and pGreenII0229 (control) were tested for their effect on tissue-cultured tobacco (N. tabacum) leaves via A. tumefaciens injection. The effect of over-expressing ScMYB2S1, ScMYB2S2 or control was recorded at 24 h after injection. The whole leaf over-expressing ScMYB2S1 changed color from green to yellow (Fig. 5a), when compared with control ( Fig. 5c). Meanwhile, there was no obvious change in leaf color when the ScMYB2S2 was over-expressed (Fig. 5b).
Physiological measurement. MDA and proline content in tobacco leaf samples were detected 48 h after ABA-treatment or injection. Figure 6 showed that ScMYB2S1-injection induced both MDA and proline levels obviously, while ScMYB2S2-injection and mixed-injection (with ScMYB2S1 and ScMYB2S2 volume ratio of 1:1) had limited effect on MDA or proline levels. Predictably, both MDA and proline contents were increased significantly after ABA-treatment (Fig. 6).

Expression profiles of SAGs in tobacco leaves.
Real-time qPCR was used to examine the expression profiles of four SAGs, NtNYC1, NtPR-1a, NtCAT3 and NtABRE, in tobacco leaves under different treatments. All these genes were induced after ScMYB2S1 injection, with an expression level about 2.17, 3.80, 5.14 and 2.48 times higher than that of the control, respectively (Fig. 7). Conversely, after injection of ScMYB2S2, the expression of NtNYC1 and NtPR-1a were decreased with 0.81 and 0.75 times, respectively, lower than that of the control (Fig. 7). While at the same time, the expression level of NtCAT3 and NtABRE were about 1.54 and 1.12 times higher than control (Fig. 6). Overall, however, the expression levels of these genes were between the two former situations after mix-injection of ScMYB2S1 and ScMYB2S2 (Fig. 7).

Discussion
In the present study, a R2R3-MYB gene was isolated from sugarcane, designated as ScMYB2, showing to produce two alternatively spliced transcripts: ScMYB2S1 and ScMYB2S2. Sequence analysis showed that the 126-bp-intron of the genomic sequence of the ScMYB2 gene consisted of two short tandem "GT-AG" structures, which provided the structural basis for alternative splicing. Further sequence analysis revealed that ScMYB2S1 was a   functional protein, containing two complete MYB DNA-binding domains (R2 and R3 repeats). ScMYB2S2, with the first MYB DNA-binding domain (R2 repeat) missing in the N-terminal amino acid sequence, might limited its function on directly involved in transcriptional regulation. Similarly, two Arabidopsis R2R3-type MYB genes, AtMYB59 and AtMYB48, both were found to have four distinctively spliced transcripts that encoded either MYB-related proteins or R2R3-MYB proteins 35 . Interestingly, ScMYB2, AtMYB59 and AtMYB48 were found to be within the same phylogenetic subgroup in this study (Fig. 3).
Alternative splicing occurs widely in eukaryote and provides the main source of transcriptome and proteome diversity in an organism 36 . Recent studies suggested that the signal transduction related genes associated with  various stress responses seemed to be particularly prone to alternative splicing in plants and animals 35,37 . The R2R3-MYB transcription factor superfamily has been showed to play an important role in many plant processes including abiotic stress responses 18 . It had been demonstrated that several MYB genes were alternatively spliced, and encoded proteins with regulatory functions 35,38,39 .
Previous studies have shown that alternative splicing also presents in sugarcane MYB transcription factor genes. Prabu et al. 40,41 identified an inducible alternatively spliced R2R3-MYB transcription factor gene ScMYBAS1 (EU670236) in S. officinarum based on a cDNA suppression subtractive hybridization library. Subsequently, semi-quantitative RT-PCR analysis revealed that the alternatively spliced transcripts, ScMYBAS1-2 and ScMYBAS1-3, were constitutively expressed at high level when subjected to water deficit and salt stress treatments, however no sequence information was provided 40,41 . In GenBank the genomic DNA of MYBAS1 (HM136779) from Saccharum hybrid cultivar Co740, and three alternatively spliced variants: MYBAS1V1 (HM136780), MYBAS1V2 (HM136781) and MYBAS1V3 (HM136782), are noted as being induced by water deficit stress based on semi-quantitative RT-PCR analysis. Sequence analysis shows that MYBAS1V1 is only 2 bases different from ScMYBAS1 (Data not showed).
Many members in plant R2R3-MYB family have been reported as abiotic-stress-induced transcription factors [40][41][42][43] . Moreover, over-expressing the R2R3-MYB genes improved the resistance to freezing, drought, and salt stresses in transgenic plants [44][45][46] . In this study, ScMYB2S2, one alternatively spliced transcript of ScMYB2, was induced at the late periods of PEG-simulated drought stress. Contrary to expectation, the expression of ScMYB2S1, the other alternatively spliced transcript of ScMYB2, was suppressed constitutively by PEG-simulated drought stress. This shows that highly specific probes or primers are crucial to identify the expression pattern of a certain gene for alternatively spliced transcripts or allelic genes which are highly homologous to each other.
It was reported that leaf senescence was affected by drought stress 47 . In this study, the de-greening symptom displayed when one alternatively spliced transcript of ScMYB2S1 was transiently over-expressed in tobacco leaves, suggesting the function of ScMYB2S1 might relate to leaf senescence regulation. The stress hormone ABA promotes leaf senescence in an ethylene-independent pathway and induces compatible solutes (e.g. malondialdehyde and proline) accumulation in leaf 48,49 . Within those compatible solutes, MDA and proline levels are commonly known as markers of stress 50 . To further investigate the role of ScMYB2S1 and ScMYB2S2 in leaf senescence, the contents of MDA and proline in tobacco leaves were detected in this study. The results showed that both MDA and proline levels were significantly induced by ABA treatment (Fig. 6). Similarly, the two compatible solutes were also obviously increased after ScMYB2S1-injection (Fig. 6). On the contrary, ScMYB2S2injection had less effect on them (Fig. 6). From those results, we can concluded that it is ScMYB2S1 rather than ScMYB2S2, lead to the accumulation of MDA and proline in tobacco leaves.
Plant senescence is regulated by senescence associated genes (SAGs) 33 . Stress-induced senescence signals are perceived and then transferred via SAGs 51 . To date, a series of SAGs have been identified from various plant species 33,52 . In this study, 4 SAGs genes were selected randomly to investigate whether they were involved in the co-expression network regulating by ScMYB2. Among these four SAGs, pathogenesis-related 1a (PR1a) and catalase (CAT) have been identified as marker gene of biotic stress 51 . Non-yellow coloring (NYC1) gene, encoding the membrane-spanning isoform of Chl b reductase was expressed during leaf senescence in rice 53 , and its function on chlorophyll degradation also has been proved in Arabidopsis 33 . Recent studies have shown that abscisic acid responsive element (ABRE)-binding factor gene (ABRE) also involved in responding to ABA-induced leaf senescence 52,54 . An ABRE-binding factor has been identified as a positive regulator of abiotic stress and ABA signaling both in Arabidopsis and in rice 55 . Collectively, the function of all these four genes was identified to accelerate leaf senescence 33,56 . Previous studies have shown that both endogenous ABA and exogenously applied ABA can induce the expression of SAGs and promote leaf de-greening and senescence in general 52 . The present study showed that these 4 SAGs were significantly induced by ScMYB2S1-injection, while ScMYB2S2-injection didn't have much effect (Fig. 7). We concluded that when transiently over-expressing in tobacco leaf, it is ScMYB2S1 rather than ScMYB2S2 playing an ABA-like function, induced the expression of the four SAGs.
ScMYB2S1 was down-regulated in responding to drought stress during the whole processing period in sugarcane. Combined analysis of its performance in de-greening and promoting both compatible solute contents and the expression levels of SAGs in tobacco (Figs 5, 6 and 7) suggested that ScMYB2S1 could act as a negative regulator and play a positive role in response to drought-induced senescence process. Meanwhile, the expression level of ScMYB2S2 in sugarcane was up-regulated by drought stress at the later stage. It seems that ScMYB2 tends to generate the isoform of ScMYB2S2 by alternative splicing during the later stage. Different with ScMYB2S1, ScMYB2S2 has limited effect on leaf de-greening (Fig. 5), MDA and proline contents (Fig. 6) and the expression of SAGs. It needs to further investigate whether this limited influence is due to its incomplete functional domain. And, recent studies have shown that alternative splicing of some transcription factor genes generates small interfering peptides (siPEPs), which negatively regulates the target transcription factors 28 . Further studies are also needed to substantiate whether the two transcripts are existed an interaction at nucleic acid level or protein level.
In conclusion, a hypothesis is proposed here that ScMYB2, by alternatively splicing two transcripts (ScMYB2S1 and ScMYB2S2), might involve in an ABA-mediated leaf senescence signaling pathway and play positive role in respond to drought-induced senescence in sugarcane. The differential expression of two alternatively spliced transcripts during PEG stress could be one kind of drought tolerance molecular mechanism in sugarcane. The alternative splicing of the transcription factor gene ScMYB2 may be pivotal to the molecular defense mechanism of sugarcane during drought-induced senescence processes. Agrobacterium-infiltrated transient transformation of tobacco was carried out according to Lin-Wang described 24 . For phenotype observation assay, tobacco tissue culture plantlets were used for transiently transformed assay. Approximately 150 μ L of each recombinant Agrobacterium culture was infiltrated at four points into a tender leaf. Tobacco seedlings grown in a greenhouse with environmental control systems were used for physiological measurement and gene expression assay. Two kind of stress treatments were applied to 8-week-old plants before flowering: ABA treatment (sprayed 100 μ M ABA on leaf at 48 h) and Agrobacterium injection (using a suspension of recombinational Agrobacterium introduced into tobacco leaf by direct injection). As for the latter, approximately 300 μ L of Agrobacterium containing the target gene or empty vector control were infiltrated at four points into the leaf. After 48 h treatment, all the leaf samples were collected and divided into three groups and assayed, respectively. One group was fixed in liquid nitrogen immediately and stored in a refrigerator at − 85 °C until RNA extraction. According to the methods from Plant Malondialdehyde (MDA) assay Kit and Proline assay Kit, the other two groups were grinded in buffer solution immediately and then measured, respectively. All of the treatments were repeated independently three times. The data were analyzed with DPS v7.05 directly and the significance difference of the MDA and proline contents were marked using different lowercase in figure. Molecular cloning, sequencing and bioinformatics analysis. A sugarcane R2R3-MYB EST was obtained based on the bioinformatics analysis using the data from the previous RNA-Seq experiments (not yet published). Two nested gene-specific 3′ RACE primers was designed according to the EST sequence information, Scientific RepoRts | 7:41922 | DOI: 10.1038/srep41922 and one pair of gene-specific primer was designed to amplify the given target regions using genomic DNA as a template. Primer sequences are as follows:

Expression profiles of
The real-time qPCR reaction was realized with following conditions: 2 min at 50 °C, 10 min at 95 °C, and then 40 cycles of 94 °C for 15 s, and 60 °C for 60 s. Each assay was repeated by three times. When the reaction was completed, a melting curve was obtained. The 2 −ΔΔ CT method was adopted to analyze the real-time qPCR results 59 . The data were analyzed with DPS v7.05 directly and the significance difference of the gene expression were marked using different lowercase in figure. Binary vectors construction. To study the function of ScMYB2S1 and ScMYB2S2 in tobacco, PCR was performed using the clone containing ScMYB2S1 or ScMYB2S2 as template to obtain the ScMYB2S1 or ScMYB2S2 ORF with matched sites. The primer sequences were MYB2S1F: 5-CCCAAGCTTATGGTGACCGTGAG-3, MYB2S2F: 5-CCCAAGCTTATGTCACCACAAGAA-3 and MYB2SR: 5-CGGAATTCTCACATCATGATTTCT-3′ (Hind III and EcoR I sites are underlined). The 50 μ L PCR reaction mix contained 5.0 μ L 10 × PCR buffer; 4.0 μ L deoxynucleotide triphosphates (dNTPs) (2.5 mM); 2.0 μ L each of forward and reverse primers (10 μ M); 2.0 μ L plasmid DNA (100 ng); and 0.25 μ L Ex-Taq enzyme (5 U/μ L). The ddH 2 O was added as supplement. The PCR amplification program consisted of pre-denaturation for 5 min at 94 °C; denaturation for 30 s at 94 °C, annealing for 30 s at 55 °C, and extension for 45 s at 72 °C for 30 cycles; and final extension for 10 min at 72 °C. The ScMYB2S1 or ScMYB2S2 ORF with Hind III and EcoR I sites was subcloned into intermediate vector pSIM35Scassett (Hind III -EcoR I sites) in the E. coli strain DH5α to generate the putative recombinants. The intermediate vector containing the gene expression cassette of '35S promoter-ScMYB2S1 (or ScMYB2S2)-CaMV polyA' was digested with EcoR V and these fragments were ligated into the equally digested EcoR V pGreen II0229 (Supplementary Fig. S3). A clone with a recombinant plasmid was validated by PCR, double digestion and sequencing, and was termed as pGII0229-ScMYB.

Agrobacterium-mediated transient expression.
A single colony of A. tumefaciens strain EHA105 was cultured overnight in 5 mL LB broth at 28 °C with 250 rpm shaking. Three mL of this culture was shifted to 1,000 mL LB medium and incubated at the same conditions until the OD 600 was about 0.5. The culture was centrifuged at 3,500 × g for 15 min at 4 °C. The pellet was resuspended/washed with 15% glycerol in ultrapure water. After washing, the bacteria were pelleted again. All the steps were performed on ice. This process of washing was repeated three times. After final washing, the pellet was divided into 100 μ L and stored at − 80 °C.
Electroporation was carried out with an electric pulse of 2.0 kV/cm and 400Ω. After each transformation the bacterial cells were resuscitated by suspending in 1 mL of liquid LB media and subsequent incubation at 28 °C with 250 rpm shaking, before spreading on the LB plates. pGII0229, pGII0229-ScMYB2S1 and pGII0229-ScMYB2S2 were used for electroporation, respectively. Screening of positive clones was performed on LB medium containing 50 μ g/mL kanamycin and 35 μ g/mL rifampicin. The plasmid was isolated from the positive clones, and PCR was employed to confirm the transformation. Agrobacterium-infiltrated transient transformation of tobacco was carried out as previously described 24 .