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PROFESSOR PRADEEP KUMAR BURMA (Orcid ID : 0000-0002-6794-591X)

Article type

: Original Article

Regulation of tapetum specific A9 promoter by transcription factors AtMYB80, AtMYB1 and AtMYB4 in Arabidopsis thaliana and Nicotiana tabacum

NeetuVerma and Pradeep Kumar Burma*

Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India

*Corresponding author ([email protected]). Telephone number: 91-9810945226

Running title: Regulation of tapetum specific A9 promoter

Keywords: tapetum specific promoter, MYB transcription factors, positive regulators, negative regulator, regulation, Arabidopsis thaliana, Nicotiana tabacum

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/tpj.13671 This article is protected by copyright. All rights reserved.

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SUMMARY Tapetum specific promoters have been successfully used for developing transgenic-based pollination control systems. Although several tapetum specific promoters have been identified, in-depth studies on regulation of such promoters are scarce. The present study analyzes the regulation of the A9 promoter, one of the first tapetum specific promoter identified in Arabidopsis thaliana. Transcription factors (TFs) AtMYB80, AtMYB1 (positive regulators) identified by in silico analysis were found to upregulate A9 promoter activity following over expression of the TFs both in transient and stable (transgenic) expression assays both in A. thaliana and tobacco. Further, mutations of binding sites of these TFs in the A9 promoter led to loss of its activity. The role of a negative regulator AtMYB4 was also studied by analyzing the activity of A9 promoter following transient expression of RNAi against the TF and by mutating binding sites for AtMYB4 in the A9 promoter. While no changes were observed in case of A. thaliana, the A9 promoter was activated in the roots of transgenic tobacco plants, highlighting the role of these cis- elements in keeping the A9 promoter repressed in the roots of tobacco.

INTRODUCTION Tapetum specific promoters such as A9 from Arabidopsis thaliana (Paul et al. 1992) and TA29 from Nicotiana tabacum (Koltunow et al., 1990) have been successfully used for pollination control for hybrid seed production to enhance crop yield (Mariani et al., 1990, 1992; Jagannath et al., 2001, 2002; Bisht et al., 2004; Ray et al., 2007). These promoters have a restricted pattern of expression in the tapetum, which is a single cell layer surrounding the anther locule and plays a critical role in the development of the male gametophyte. Apart from its importance in biotechnology, these promoters also serve as an interesting model system to understand the

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regulation of plant promoters. In A. thaliana, apart from A9, several other tapetum specific genes like A3, A6, TSM1, CYP703A2, MS2, LTP12, ACOS5, CYP704B1 and LAP5 (Worrall et al., 1992; Hird et al. 1993; Ariizumi et al. 2002; Morant et al., 2007; Zhang et al., 2007; Fellenberg et al. 2008; de Azevedo et al. 2009; Dobritsa et al., 2009; Kim et al. 2010) have been reported. Further, there have been several reports on the identification of transcription factors, which are important for the development of tapetum. These include DYSFUNCTIONAL TAPETUM1 (DYT1) (Zhang et al., 2006), MYB33/MYB65 (Millar and Gubler, 2005), DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1 (TDF1) (Zhu et al., 2008), MYB80 (Higginson et al., 2003; Li et al., 2007; Zhang et al., 2007), ABORTED MICROSPORES (AMS) (Sorensen et al., 2003) and MALE STERILITY1 (MS1) (Wilson et al., 2001). Gene regulatory network involving these transcription factors, has also been proposed for tapetum formation and development (Liu and Fan, 2013). Few studies have also been carried out to identify downstream genes regulated by of some of these transcription factors like AtMYB80, AMS and MS1 (Yang et al., 2007; Xu et al., 2010; Phan et al., 2011) as well as their binding sites on target promoters (Phan et al., 2011; Ferguson et al., 2016; Xiong et al., 2016). Although there is a lot of work on genes involved in tapetal development and their regulatory network, there is a major gap in our understanding on how a tapetum specific promoter is regulated. In the present study, we have used A9 promoter of A. thaliana as a model to understand the transcription factors that activate the promoter in tapetal cells while keeping it repressed in other tissues. The tapetum specific A9 gene was isolated and characterized by Paul et al. (1992). The A9 gene was initially isolated as an anther specific cDNA in Brassica napus and was suggested to be a tapetum specific transcript, as it expressed in stages of anther development when tapetum is This article is protected by copyright. All rights reserved.

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metabolically active and also by in situ hybridisation studies (Scott et al., 1991). This cDNA was used to identify the homologus gene from A. thaliana (Paul et al., 1992). Further, using A9 promoter-reporter (β-glucuronidase, gus) constructs with variable length of promoter (1437 bp, 936 bp and 329 bp), it was shown that a 329 bp region upstream to A9 coding sequence retained both temporal and tapetum specific expression of the gus gene in transgenic tobacco (Paul et al., 1992). Although the promoter has been used to drive tissue specific expression of several genes like barnase (Paul et al., 1992), barstar (Jagannath et al., 2001, 2002), cytotoxic diphtheria toxin A-chain gene (DTx-A) (Guerineau et al., 2003; Lee et al., 2003), LRR receptor kinase EXCESS MICROSPOROCYTES 1 (EMS1) (Feng et al., 2010), no work seems to have been carried out to understand the regulation of the promoter. In the current study, we propose that transcription factor AtMYB80 and AtMYB1 positively regulate the A9 promoter in both A. thaliana and tobacco by interacting with cis- element ''CACCAATCC'' and ''CAACACCTC', respectively. Further mutation of cis- elements which were proposed binding sites for TF AtMYB4 activated the A9 promoter in roots of tobacco.

RESULTS Hypothesis: A9 is regulated by positive and negative regulators An in silico strategy was undertaken to identify putative cis- elements and transcription factors that may regulate A9 promoter. Genes co-expressed with A9 gene were identified from AtGenExpress (Schmid et al., 2005) and ATTED (Obayashi et al., 2008) databases. A total of 17 genes were identified, which are listed in Table S1. 500 bp of the promoter region of A9 and the identified co- expressed genes were analyzed through MEME (Bailey et al., 2007) to identify consensus motifs of 8 to 12 bp lengths present in these promoters. Six motifs were identified in

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this analysis (Table S2). The STAMP web tool (Mahony et al., 2007) was then used to identify transcription factors (TFs), which may bind to these consensus motifs. Four TFs were identified of which, three transcription factors AtMYB80 (Zhang et al., 2007), AtMYB1 (Shinozaki et al., 1992) and AtMYB2 (Abe et al., 2003) are reported as positive regulators, while transcription factor AtMYB4 is reported as negative regulator (Jin et al., 2000; Fornale et al., 2014). For these TFs to function as regulators of A9 promoter, it is expected that the positive regulator should be present in the anther tissue around the time of the expression of A9 gene, while the negative regulator should be absent. AtMYB80 has been reported to express in anthers (developmental stages 5 -10) of A. thaliana and is known to regulate the expression of A6 gene and MS2 genes which were observed to be co-expressed with A9 (Zhang et al., 2007). AtMYB2 is reported to express in basal internodes at late stages of plant development (Guo and Gan, 2011). There is no report on its expression status in the developing anthers. On the other hand, expression of AtMYB4 was reported in all vegetative tissues and buds (Fornale et al., 2014). However, in buds the expression was restricted to the filament and style and no expression was reported in the anthers (Fornale et al., 2014). There is no available literature on the expression pattern of AtMYB1, although the NCBI Ace View database (Thierry-Mieg and Thierry-Mieg, 2006) reports its expression in buds. The expression of these TFs and the A9 gene in different tissues (viz., root, stem, leaf and buds) of A. thaliana was analyzed in the present study by RT- PCR (Figure S1). The amplicons obtained in RT- PCR were also sequenced to validate its correctness. Transcripts for AtMYB80 and AtMYB2 were observed only in the buds, while in case of AtMYB1, in addition to buds a low level of expression was also observed in the leaf tissue. AtMYB4 transcripts were found to be present in all the analyzed tissues. The levels of transcripts of TFs were also quantitated in all

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vegetative tissues and different developmental stages of anthers by qRT- PCR (Figure 1a & b). Apart from TFs, the expression of A9 gene was also analyzed. Transcripts of A9 were observed in anthers of developmental stages 4-6. Tapetum begins to appear in stage 4 of anther development. Stage 5 represents the appearance of microspore mother cells. Microspore mother cells enter meiosis in stage 6 of anther development and tapetum becomes vacuolated (Sanders et al., 1999). The transcripts of AtMYB80 were observed in anther development stages 4-6 and 7-9. Further, the level of transcripts in stages 4-6 was found to be significantly higher than that in the later stages 7-9. Transcripts of AtMYB1 were observed in seedlings, leaf and all developmental stages of anther. Similar levels of transcripts for AtMYB1 was observed in stages 1-3 and 4-6 which was significantly higher than the levels observed in stages 7-9 and 10-12. Transcripts of AtMYB2 were found to be absent in the anther of development stages 4-6 (when A9 expresses) but were observed in anthers of later development stages as well as in the root tissue. The expression of AtMYB4 was observed in all vegetative tissues analyzed as well as in the buds. The level of transcript was observed to be significantly higher in roots as compared to other tissues. In case of buds, it has been demonstrated earlier (Fornale et al., 2014) that its expression is limited to the filament and style parts of flower and no expression is observed in the anthers. Based on the present analysis, TFs AtMYB80 and AtMYB1 are proposed to be positive regulators of A9 as their expression overlap that of A9 gene. On the other hand AtMYB2 expresses at later development stages and this may not be involved in the regulation of A9 gene. Further, AtMYB4 could possibly be proposed as a negative regulator of A9 gene due to its lack of expression in anther tissue (Fornale et al., 2014), and its presence in all other tissues. This hypothesis has been experimentally tested in the present study.

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Ectopic over- expression of positive regulators activate the A9 promoter in A. thaliana and tobacco In order to test whether the TFs, AtMYB80 and AtMYB1 were sufficient to activate the A9 promoter at an ectopic location, a transient expression analysis was carried out using seedlings of A. thaliana. A 1500 bp A9 promoter fragment was used in this part of study. In this strategy, seedlings were co-infected with Agrobacterium strain harboring the reporter construct (A9(1500):gus-intron) and the TF over- expression constructs (35S:MYB80 and 35S:MYB1) expressing TF genes AtMYB80 and AtMYB1 under the control of CaMV35S promoter (Figure 2a& b ). Infection with only 35S:gus-intron or A9(1500):gus-intron construct was used as positive and negative controls, respectively. The activation of A9 promoter was studied by enzymatic assay for GUS activity. Three independent experiments, each with 40 seedlings were carried out, results of which are shown in Figure 2c. It was observed that co- infection with reporter gene and TF constructs led to the activation of the A9 promoter in both the cases. Further the GUS activity (Figure 2c) in case of AtMYB80 (mean value ± SD = 1436 ± 279 pmole MU/min/mg protein) was observed to be significantly higher than that of AtMYB1 (mean value ± SD = 449 ± 37 pmole MU/min/mg protein). Similar results were also observed using a 500 bp long A9 promoter fragment (Figure S2) In order to further validate the above observation, transgenic A. thaliana plants constitutively expressing these TFs under the control of CaMV 35S promoter, were developed with the constructs 35S:MYB80 and 35S:MYB1. The developed transgenic lines are called as AtMYB80OE and AtMYB1-OE lines. Developing transgenic plants with 35S:MYB80 was difficult and only 3 transgenic plants could be generated from the several transformed seedlings obtained. These 3 lines were also phenotypically abnormal (Figure S3), with multiple apical meristems and

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no seed set. The ectopic expression of AtMYB80 was thus detrimental to plant growth and development. In such cases, use of inducible promoters like the dexamethasone inducible promoter (Aoyama and Chau, 1997; Ko et al., 2009) would have helped to circumvent the problem. However, in the present study this was not attempted as enough leaf material was available for the analysis of A9 gene expression. No such abnormality was observed while developing transgenic plants with 35S:MYB1 construct (Figure S3). The expression of TFs and the endogenous A9 gene was analyzed in leaf tissue of 3 independent lines each of AtMYB80-OE and AtMYB1-OE, by qRT- PCR which was repeated three times in each case. Although there was difference in the level of AtMYB1 transcript in the independent AtMYB1-OE lines, the level was significantly higher than that of AtMYB80 in the AtMYB80-OE transgenic lines (Figure 3a). This probably reflects that transgenic lines with high expression of AtMYB80 could not be rescued. A9 gene was observed to be ectopically expressed in the leaf tissue of transgenic lines developed with these two over- expression constructs (Figure 3b and Figure S4). This shows that AtMYB80 and AtMYB1 positively regulate the expression of the A9 gene. Interestingly, the expression of A9 is significantly higher in AtMYB80-OE lines as compared to AtMYB1-OE lines. This was also observed in transient expression experiments performed with these TF over- expression constructs. Thus, it can be proposed that in comparison to AtMYB1, AtMYB80 has a stronger influence on A9 promoter activity. The effect of over- expressing AtMYB80 and AtMYB1 on the activity of A9 promoter was also studied in a hetrologous system viz. N. tabacum by transient assay. Transgenic tobacco plants were developed with the A9(1500):gus-intron construct and the transgenic lines showing no

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leaky gus expression in the leaves and other vegetative tissues, were used for transient assays. Further, these lines showed expression in the anthers of tobacco buds. Leaves of these lines were transfected by Agro infiltration with the TF over- expression constructs and its effect on A9 promoter activity was checked by recording GUS activity in infiltrated leaves (Figure 4). Three independent experiments each with three technical replicates were carried out for each TF. Transient expression of TFs AtMYB80 and AtMYB1 activated the A9 promoter in the leaves of A9:gus-intron transgenic tobacco plants. TF AtMYB80 again showed higher GUS activity (mean value ± SD = 893.5 ± 141.5pmole MU/min/mg protein) in comparison to AtMYB1 (mean value ± SD = 449.5 ± 51.1pmole MU/min/mg protein) (Figure 4). This is similar to the observation made in A. thaliana. Transient expression of both the transcription factors under independent CaMV35S promoter in one construct was also performed to analyze whether both the transcription factors in combination would further enhance the A9 promoter activity. Transient expression of the construct having both the transcription factors did not show any additive effect on reporter gene activity when compared to AtMYB80 transient expression (Figure 4). Mutation in putative binding sites of the positive regulators leads to reduction of A9 promoter activity The 500 bp proximal fragment of A9 promoter has 6 putative binding sites for TFs AtMYB80 and AtMYB1 as identified by in silico analysis. These sites are shown in Figure S5, and hereafter represented as elements 1 to 6. An A9 promoter of 500 bp length promoter was developed where all the six motifs were replaced with a random sequence. The activity of the mutated A9 promoter was analyzed using gus-intron as a reporter gene in the buds of transgenic A. thaliana and tobacco lines. Ten independent transgenic lines were developed in each case. Transgenic This article is protected by copyright. All rights reserved.

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plants developed with WT A9(500):gus-intron, were used as positive control. Histochemical staining for GUS activity showed loss of A9 promoter activity in the buds of independent transgenic plants developed with mutated promoter construct (Figure S6). This was also supported by quantification of GUS activity as well as by measuring the gus transcript level in the buds of A. thaliana transgenic lines (Figure 5a & 5c). A similar observation was made in the case of transgenic tobacco plants developed with mutated A9 promoter (Figure 5b). In order to identify the critical elements for binding of AtMYB80 and AtMYB1 in the identified six elements, a set of six mutated A9 promoters viz. A9m(6), A9m(6+1), A9m(6+5+1), A9m(6+5+4+1), A9m(6+5+4+3+1) and A9m(6+5+4+3+2+1) were developed, each carries a combination of mutated elements (Figure 6a) and cloned upstream to gus-intron gene. The activity of these mutated A9 promoters was analyzed in transient expression analysis where in the promoter- reporter construct and the TF over-expression constructs viz 35S:MYB80 or 35S:MYB1 were simultaneously co- transfected into the leaves of tobacco. A minimum of three biological replicates each with three technical replicates was carried out for each construct and the promoter activity for each construct was compared to the activity of the WT A9 (500 bp) promoter, used as a control. In experiments with 35S:MYB80, the mean GUS activity (± SD) under WT A9 (500 bp) promoter was observed to be 905 ± 55 pmole MU/min/mg protein. This is similar to the GUS activity observed with the 1500 bp length of the WT A9 promoter (Figure 4). Mutation in element 6 [A9m(6)] did not lead to any drop in the observed GUS activity, while mutation in both elements 6 and 1 resulted in a promoter with 26% of activity as compared to the WT A9 (500 bp) promoter (Figure 6b). The mean GUS activity observed in this case was 236 ± 10.6 pmole MU/min/mg

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protein. No significant drop in promoter activity was observed when additional elements were mutated (Figure 6b). In experiments with 35S:MYB1, the mean GUS activity under WT A9 (500 bp) promoter was observed to be 517 ± 15.6 pmole MU/min/mg protein. Mutations in elements 6, 1, 5 and 4 did not lead to any significant decline in GUS activity as compared to WT A9 promoter (Figure 6c). However, the promoter with mutation in element 3 [A9m(6+5+4+3+1)] led to a significant drop (44%) in promoter activity as compared to WT A9 promoter (Figure 6c). Thus, element 1 "CACCAATCC" and element 3 "CAACACCTC" were observed to be critical for the activation of A9 promoter by AtMYB80 and AtMYB1, respectively. Analysis of the identified negative regulator AtMYB4 by loss-of- function strategy To assess the role of the hypothesized negative regulator AtMYB4, a RNAi based construct (Figure 7a) was developed for the down-regulation of AtMYB4 in A. thaliana. However transgenic plants in A. thaliana could not be developed with this construct, suggesting that downregulation of AtMYB4 probably led to lethality of the transformants. The effect of down-regulating AtMYB4 on A9 promoter activity was thus studied by transient expression of the RNAi construct in transgenic A. thaliana seedlings harboring the A9(500):gusintron construct. The Agrobacterium cells carrying the construct AtMYB4 RNAi alone or in combination with Agrobacterium cells harboring the construct 35S:MYB80 were used to infect transgenic A. thaliana seedlings. Infection with empty vector or with 35S:MYB80 alone was used as negative and positive controls, respectively. Activity of A9 promoter was observed by recording the GUS activity. No recordable GUS activity was observed in seedlings infected with AtMYB4 RNAi construct, which was similar to the readings following infection with empty vector control. In case of seedlings co- infected with AtMYB4 RNAi construct and 35S:MYB80 This article is protected by copyright. All rights reserved.

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construct, it was observed that downregulation of AtMYB4 did not lead to any significant increase in the activity of A9 promoter when compared to infection with 35S:MYB80 alone (Figure 7b). These observations do not support a role of AtMYB4 acting as a negative regulator because the gus expression under the control of A9 promoter is expected to increase following knockdown of AtMYB4. The role of negative regulator was also assessed by mutating the putative binding sites for AtMYB4 in the A9 (500 bp) promoter (Figure7c). Three putative sites were replaced by random stretches of DNA to develop the A9(-ve mut) promoter. The activity of the promoter was analyzed in several independent transgenic A. thaliana and tobacco plants. Promoter activity in these lines were checked by histochemical staining for GUS activity in different tissues viz. seedlings, root, stem, leaf and buds. In case of A. thaliana, no ectopic expression was observed supporting our observation with the RNAi strategy. Expression was observed only in anther tissue. However, in case of tobacco, GUS activity was observed in roots of 22 out of the 25 independent transgenic lines developed and analyzed. GUS activity in the roots recorded by the histochemical staining and in vitro assay is presented in Figure S7 & Figure 7d, respectively.

DISCUSSION The A9 promoter was one of the first tapetum specific promoters to be identified in A. thaliana (Paul et al., 1992). The promoter has also been used to drive tapetum specific expression of transgenes in several instances (Jagannath et al., 2001, 2002; Lee et al., 2003; Ray et al., 2007; Dilkes et al., 2008; Feng et al., 2010). However, apart from the initial work of Paul et al.(1992) to delineate the minimum length of the promoter needed for expression in tapetum, to the best of our knowledge no reports are available on how the promoter is regulated.

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In the present study three TFs were identified by in silico analysis, which could probably regulate the A9 promoter. Of these two AtMYB80 and AtMYB1 are reported to be positive regulators, while the third AtMYB4 is a negative regulator. Based on experimental observations involving ectopic over- expression of AtMYB80 and AtMYB1 in transient expression assays carried out in A. thaliana and tobacco as well transgenic plants developed in A. thaliana, the present work demonstrates that these TFs positively regulate the A9 promoter. These evidences are further augmented by the observation that the A9 promoter activity is down-regulated if the putative binding sites for these TFs are mutated. Of the two positive regulators, it is observed that AtMYB80 is more critical as over- expression of this TF gives a significantly higher activity of A9 promoter in comparison to over- expression of AtMYB1. This was observed in all three experimental strategies used in the present work. Further, of the different tissues and stages of anther development analyzed, AtMYB80 was observed to be expressed mainly in the stage when A9 expresses. AtMYB1 on the other hand had a larger window of expression. An earlier study by Zhang et al. (2007) showed that mutation of AtMYB80 gene severely downregulated the expression of A6 and MS2 genes. A6 and MS2 genes are co- expressed with A9. A later study by Phan et al. (2011) based on comparative transcriptome analysis of anthers by microarray between myb80 mutants versus wild type and ChIP array to identify the direct targets of AtMYB80 refuted the claim of Zhang et al. (2007). They suggested that the changes observed in the expression of A6 and MS2 could be consequence of premature tapetal degeneration in myb80 mutant rather A6 and MS2 being the direct targets of AtMYB80 regulator. The present study based on developing ectopic over- expression lines of AtMYB80 shows that along with the A9 gene, both MS2 and A6 are up-regulated in the leaf tissues (Figure S3b). The current work This article is protected by copyright. All rights reserved.

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thus shows that MS2 and A6 in addition to A9 are also targets of AtMYB80 as all these genes are ectopically expressed following over- expression of AtMYB80. Apart from MS2 and A6, the tapetum specific promoter A3 from A. thaliana has been proposed to be regulated by transcription factors (Hird et al., 2000) common to A9. This was based on the observation that presence of A9 promoter duplication caused transinactivation of both A9 and A3 promoters possibly through titrating out common TFs regulating the two. A3 was also identified as one of the genes co- expressed with A9 (Table S1). AtMYB80 and AtMYB1 are examples of R2R3MYB transcription factors. R2R3MYB transcription factors are reported to bind to CA rich cis- regulatory elements (Gubler et al. 1995; Grotewold et al. 1994; Hoeren et al., 1998; Phan et al., 2011). ''CCAA'' forms the core of AtMYB80 binding element (Phan et al.,2011). Analysis of A9 promoter using MEME predicted the presence of six putative elements for the binding of AtMYB80 and AtMYB1. Mutation of these putative binding sites showed that cis- elements "CACCAATCC" (element 1) and "CAACACCTC" (element 3) were found to be critical for the transactivation of A9 promoter by AtMYB80 and AtMYB1, respectively. "CCAATC" sequence present in element 1, was reported to be an important MYB binding element in the promoter of dehydration responsive gene, rd22 (Abe et al., 1997). ATMYB1 has been reported to bind to ciselements with the MYBCORE ''CAA'' (Luischer and Eiseman, 1990; Urao et al., 1993) which is part of element 3 of A9 promoter. Both the elements 1 and 3 lie within the 329 bp promoter region which has been shown to be sufficient for the temporal and spatial expression of A9 promoter (Paul et al., 1992). The element 1 (CACCAATCC) positioned at -57 bp in A9 promoter, is also found to be located in the proximal promoter regions of other tapetum specific genes like A6 and A3 . Element 3 (CAACACCTC) positioned at -290 bp in A9 promoter, is found to be present in MS2 and A3 promoters located at -297 bp and -95 bp, respectively. It was

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found to be absent in A6 promoter. Our observation that over- expression of AtMYB1 in A. thaliana OE lines activated the MS2 promoter in addition to A9 at the ectopic location but did not lead to activation of A6 promoter (Figure S3c), lends further support to our result that AtMYB1 brings out its effect through element 3. The above observations also supports our results that AtMYB80 and AtMYB1 activate the A9 promoter through element 1 and element 3. The transcription factor AtMYB4 has been reported to function as a repressor, particularly of the cinnamate4-hydroxylase (C4H) gene in the phenylpropanoid pathway of A. thaliana (Jin et al., 2000). Orthologs of this gene, AmMYB308 and AmMYB305 from Antirrhinum majus has been reported to negatively regulate the hydroxycinnamic acid biosynthesis in phenylpropanoid pathway in tobacco (Tamagnone et al., 1998b; Preston et al., 2004). Further, AtMYB4 was found to be functionally similar to AmMYB308 as over- expression of these genes in tobacco gives a similar phenotype (Tamagnone et al.,1998a). AmMYB308 is reported to bind to ''GTTAGGT'' elements within the promoters of phenylpropanoid biosynthetic genes (Sablowski et al., 1994).The A9 promoter has one ''GTTTGGT'' elements and two other elements having ''GTT'' core. While mutation of these three elements led to activation of the A9 promoter specifically in the roots of transgenic tobacco plants, no ectopic expression was observed in A. thaliana itself. Thus it seems that a homolog of AtMYB4 or AmMYB308 in tobacco may be involved in keeping the A9 promoter repressed in roots of tobacco. We identified a protein sequence from N. tabacum, annotated as MYB- related protein 308- like (NP_001311732.1) showing 72% of amino acid similarity with AmMYB308 (Figure S8) having near identical DNA binding and repression domains. The difference was observed in the C- terminal extension in

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case of NtMYB308. This protein could possibly be the candidate TF which represses the A9 promoter in tobacco. Further experiments are needed to validate the same. Activation of the A9 promoter in the roots of tobacco following the mutation of elements proposed to bind to MYB308 like protein could also be due to introduction of elements which activate the promoter in the roots. However, this is less likely as the new DNA sequences created by mutation do not match to any known root specific elements reported in the current literature and databases. The A9 promoter is tightly regulated in A. thaliana. This is true for the endogenous promoter or for copy(ies) of the promoter integrated at ectopic location in transgenic plants. This was observed by analyzing the presence or absence of A9 transcripts in different tissues in the first case and recording GUS activity in the later. From the present observations, it seems that this specificity is possibly due to the combinatorial effect of two positive regulators, AtMYB80 and AtMYB1. Of the two regulators, AtMYB80 ectopically activates the A9 promoter at much lower titre that AtMYB1. AtMYB80 also seems to be more crucial for achieving the specificity of A9 promoter as AtMYB80 expresses only at the time of A9 expression. We also evaluated the available microarray data from AtGenExpress to analyze the expression of AtMYB80 and A9 in different tissues of WT A. thaliana. It was observed that the expression pattern of both the genes are identical, although the transcript level varies (Figure 8). Although the present analysis failed to identify a negative regulator for A9, a robust tissue specificity is likely to be achieved by a combinatorial interplay of positive and negative regulators. Some evidences exist in the case of plants where combinatorial interplay of positive and negative elements of the promoters directs the specificity in the gene expression (Groszmann et al., 2010; Simon et al., 2012; Liu et al., 2013). One is the rice tapetum specific promoter OsLTP6 (Liu et al., 2013) and the other is the A. This article is protected by copyright. All rights reserved.

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thaliana ovule specific promoter of TF gene INO (Simon et al., 2012). Although the present observation negates the role of AtMYB4 in regulating the A9 promoter in A. thaliana, the presence of cis- elements important for keeping the A9 promoter repressed in tissues other than the tapetum needs to be evaluated. This could probably be initiated by linker scanning mutagenesis of the A9 and studying its activity at ectopic locations. A tissue specific promoter controlled by the combinatorial action of both the negative and positive regulators is expected to be tightly regulated even when it is integrated at the ectopic location, thus making it an ideal promoter to be used for tapetum specific expression like in case of developing male sterile/ restorer lines for hybrid seed production. EXPERIMENTAL PROCEDURES Plant materials and growth conditions Arabidopsis thaliana ecotype Col 0 and Nicotiana tabacum Cv. Xanthi plants were used in this study. A. thaliana plants were grown in growth chambers at 22°C under a 16/8h light to dark cycle with 50- 70% relative humidity. Tobacco plants were grown in growth chambers at 28°C under 16/8h light to dark cycle with 70% humidity. Transgenic plants were also maintained in vitro on MS medium containing 3% sucrose and 0.6% agar. Development of Binary vectors cDNA of the genes encoding the transcription factors (TF) viz. AtMYB80 and AtMYB1 were synthesized and cloned into pRT101 vector downstream to 35S promoter of cauliflower mosaic virus (CaMV) by homology based recombination (HBR) as described in Verma & Burma, (2014). HBR was carried out using In-Fusion® HD Cloning Kit (Clonetech Laboratories, Inc.). The

35S:TF

expression

cassette

was

then

cloned

(http://www.cambia.org). This article is protected by copyright. All rights reserved.

into

pCAMBIA1300

vector

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RNAi construct of AtMYB4 was also generated by homology based recombination (HBR) and cloned into pCAMBIA1300 vector. A 534 bp region of the AtMYB4 cDNA (from 316 bp to 849 bp) was used for developing the RNAi construct, which did not have any off-targets. In order to study the role of identified cis- elements in the A9 promoter, 7 different mutated A9 promoters of 500 bp length were developed in this study. Mutant A9 promoters were developed by replacing the cis- elements with random sequences. The wild type A9 promoter of 1500 bp and 500 bp and the mutated versions were cloned upstream to an intron containing gus gene (gus-intron) in pRT100 vector. The promoter- reporter cassettes were then mobilized into the binary vector pPZP200N (Bhullar et al., 2003). The details of constructs is given in Table S3. Plant transformation Agrobacterium tumefaciens strain GV 2260 was used for all plant transformations. Genetic transformation of A. thaliana plants was carried out by vaccum infiltration method (Tague and Mantis, 2006). Transformants were selected by placing the T1 seeds on MS medium containing appropriate antibiotic i.e. hygromycin (25mg/l) or kanamycin (50mg/l). Transgenic lines were developed in N. tabacum by Agrobacterium- mediated transformation of leaf disc explants following the protocol of Svab et al. (1995). Transient expression analysis in A. thaliana seedlings Transient expression in A. thaliana seedlings was performed according to the method given by Li et al. (2009) with some modification. In case of positive regulators, A. thaliana seedlings were co- infected with Agrobacterium cells ( atOD600 of 0.5) carrying the over- expression constructs of TFs (35S:MYB80 and 35S:MYB1) and the reporter gene construct (A9:gus-intron). In case of negative regulator, transgenic (A9:gus-intron) seedlings were infected with the Agrobacterium cells harboring the RNAi construct of AtMYB4. The infected seedlings were incubated in plant

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growth chamber at 22ºC for 48 hrs under dark condition. After incubation period, the seedlings were surface sterilized with 0.05% sodium hypochlorite for 10 minutes following by 3- 4 washes in sterilized water. After surface sterilization, the seedlings were placed on 1X MS plates and grown for up to 1 week prior to further analysis. Three independent experiments, each with 40 seedlings were carried out for each construct. The A9 promoter activity was recorded by the expression of the reporter gene gus. Transient expression analysis in tobacco To study the effect of over- expressing TFs on A9 promoter activity in tobacco, Agrobacterium

cells (at OD600 of 1.5) harboring the over- expression constructs of TFs viz. 35S:MYB80 and 35S:MYB1 were infiltrated into transgenic (A9:gus-intron) tobacco leaf halves by applying vaccum of 80 KPa for 20 minutes. The construct having gus reporter gene under CaMV 35S promoter was used as a positive control. Three independent experiments, each with three technical replicates were performed for each construct. The A9 promoter activity was recorded by the expression of the reporter gene gus. To identify the critical elements required for the binding of AtMYB80 and AtMYB1, transient assays were performed by co- infiltrating the tobacco leaves with Agrobacterium cells carrying mutated promoter- reporter constructs and over- expression constructs viz 35S:MYB80 and 35S:MYB1 respectively. For each mutated promoter construct, leaves co- infiltrated with Agro cells harboring the mutated promoter- reporter construct and empty over- expression construct (empty pCAMBIA1300 vector) were used as negative control.

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Analysis of GUS activity Histochemical GUS staining Histochemical staining for GUS activity in transgenic plants was performed as described previously (Jefferson et al., 1987; De Block and De Brouwer, 1992). The buds of transgenic plants were harvested and immediately immersed in the reaction solution composed of 1mM 5bromo-4-chloro-3-indolyl-b-D-glucuronic acid, 50mM sodium phosphate, 1mM ferricyanide, 1mM ferrocyanide and 0.1% Triton X-100, pH 7.0. After 5 min of vacuum infiltration, samples were incubated overnight at 37°C. Chlorophyll of stained tissues was bleached by a series of ethanol washes each with decreasing concentration (from 90% to 50%) for 30 min and pictures of the stained tissues were taken with a stereomicroscope. Quantification of GUS activity Total protein from the tissue was extracted in GUS extraction buffer followed by protein quantification

by

following

Bradford

(1976).

Fluorometric

GUS

assays

using

4-

methylumberrifyl-β-glucuronide (MUG) substrate were performed according to Jefferson et al. (1987). The product 4- methylumbelliferone (MU) of reaction was quantitated at excitation wavelength of 360 nm and emission wavelength of 460 nm. GUS activity was recorded at two time points (after 10 and 20 minutes) for each sample. GUS activity was calculated as pmolMU/min/ mg protein. RT- PCR Total RNA was extracted from vegetative and bud tissues of A. thaliana plants using RNeasy plant kit (Sigma) according to the manufacturer’s protocol. The first stand cDNA was synthesized from the total RNA using QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s protocol and used as a template in subsequent PCR. Semi quantitative PCR This article is protected by copyright. All rights reserved.

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were performed using gene-specific primers as shown in Table S4. The conditions for amplification of cDNA were as follows: 95°C for 2 min; 30 cycles of 95°C for 30 s; primer annealing for 30 s and 72°C for 30 s; one cycle at 72°C for 5 min. Primer annealing temperature was calculated according to the Tm of each primer combination. Real-time quantitative RT-PCR Quantitative RT- PCR was performed on single strand cDNAs using a Power SYBR green PCR master mix supplied by Applied Biosystem. To study the transcript level of each TF and A9 gene in different anther development stages, buds with developing anthers representing stages 1 through to 12 (according to Sanders et al., 1999) were collected in 4 pools (each with three stages). Quantitative PCR was performed with three independent biological replicates. The relative transcript level of each gene was normalized using the internal control EF1α (Czechowski et al., 2005). The primers for quantitative RT-PCR are listed in Table S5. Primer efficiency was checked for each gene. Relative transcript level was calculated by 2 -(∆Ct) method and fold change was calculated by 2-(∆∆Ct) method. Bioinformatics tools and analysis The co- expressed genes were clustered using the co- expression information from the ATTED-II database (Obayashi et al., 2008). The co-expression value ranges from −1.0 to 1.0. A threshold value of 0.80 was set for co- expression analysis. Expression profiles of these genes were also analyzed through AtGenExpress visualization tool (AVT) (Schmid et al., 2005). Common motif prediction was performed using (MEME) tool (Bailey et al., 2007). The structure alignment program (STAMP) was used to calculate the similarity between predicted motifs and known TFBSs (Mahonty et al., 2007). The stamp server transformed the motifs into transcription factors

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based on its transcription factor binding site (TFBS) data. The TFBS information was collected from AGRIS database. Statistical analysis The Student’s t-test program (http://www.graphpad.com) was used for statistical analysis to calculate the quantitative difference between the two groups of experimental data of the qRTPCR analysis and GUS activity assay. The comparison was taken to be statistically significant if P< 0.05). ACKNOWLEDGEMENTS This work was financially supported by the funds from Council of Scientific and Industrial Research (CSIR#38(1368)/13/EMR-II and University of Delhi (R&D Grant), DU-DST PURSE. NV acknowledges CSIR for research fellowship. We thank Dr. Jagreet Kaur for helping A. thaliana transformation and Dr. Surajit Sarkar for microscopy. The authors declare no conflict of interest.

SHORT SUPPORTING INFORMATION LEGENDS Figure S1. Expression analysis of A9 gene and TFs in different tissues of A. thaliana by RTPCR. Figure S2. Analysis of A9(500) promoter activity following co- infection of A. thaliana seedlings with promoter reporter construct , A9(500):gus-intron and TF over- expression (TFOE) constructs (35S:MYB80 and 35S:MYB1).

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Figure S3. Phenotypes of AtMYB80-OE and AtMYB1-OE transgenic plants developed with constructs overexpressing AtMYB80 and AtMYB1 TF genes. Figure S4. Expression analysis of A9 gene, A6 and MS2 genes in WT, AtMYB80 and AtMYB1 over- expression lines of A. thaliana by RT- PCR. Figure S5. A9 promoter sequence with six putative elements identified for the binding of TFs AtMYB80 and AtMYB1. Figure S6. Analysis of A9 promoter activity in the buds of A. thaliana and tobacco transgenic plants developed with A9m(6+5+4+3+2+1):gus-intron construct. Figure S7. Ectopic expression of gus gene in the roots of A9(-vemut):gus-intron transgenic plants. Figure S8. Alignment of amino acid sequences of AmMYB308 and MYB- related protein 308like (NtMYB308) TF proteins. Table S1. List of genes co- expressed with A9 gene. Table S2. Regulatory elements identified by in- silico analysis of A9 promoter. Table S3. List of constructs developed in the study. Table S4. Primer sequences for RT PCR. Table S5. Sequences of primers used in qRT PCR.

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FIGURE LEGENDS Figure 1. Analysis of expression of A9 and TF genes (a) positive regulators - AtMYB80, AtMYB1, AtMYB2 and (b) negative regulator- AtMYB4 in different tissues- root, stem, leaves and buds at different stages of anther development and seedlings of A. thaliana by qRT- PCR. The anthers have been staged as days after initiation of bud formation. Anther stages 1-3, 4-6, 7-9 and 10-12 represent buds, which are ~4-6, 7-10, 10-11 and 11-14 days old, respectively based on

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observations by Smyth et al.,1990 and Sanders et al., 1999. Levels have been represented as fold change relative to the expression of A9 gene at stage 4 – 6. EF1α was used as a reference gene in this and all other qRT-PCR experiments. The Ct value of EF1α was 15.8 ± 0.30 across all samples analyzed. The Ct value for A9 gene at stage 4 – 6 was 18 ± 0.11. Each bar shows the mean ± SD of three independent experiments (each with three technical replicates). In case of positive regulators AtMYB80 and AtMYB1 (a), the expression of TFs in different tissues and anther stages was compared to its expression in anther stage 4 - 6. In case of negative regulator AtMYB4 (b), the expression of TF in different tissues and anther stages was compared to its expression in the root tissue. Means showing significant differences as calculated using Student’s t-test is indicated as * (P < 0.05) and ** (P < 0.01). Figure 2. Analysis of A9 promoter activity following co- infection of A. thaliana seedlings with (a) promoter reporter construct [A9(1500):gus-intron] and (b) TF over- expression (TF-OE) constructs (35S:MYB80 and 35S:MYB1) whose T-DNA segment has been schematically represented. The TF-OE and A9:gus-intron cassettes are cloned in binary vectors pCAMBIA1300 and pPZP200N, respectively (c) A9 promoter activity observed in seedlings coinfected with A9:gus-intron and TF-OE constructs. Infection with A9:gus-intron and 35S:gusintron constructs served as negative and positive controls, respectively. Three independent experiments each with 40 seedlings were carried out. The number above a set of bars represent the mean GUS activity ± SD. The difference in GUS activity following co-infection with AtMYB1 and AtMYB80 OE constructs was observed to be significant (**;P value < 0.005). Figure 3. Ectopic expression of A9 gene in leaf tissue of independent transgenic lines of AtMYB80-OE and AtMYB1-OE plants. (a) Relative transcript level (ΔCt) of TF AtMYB80 and AtMYB1 genes in three independent transgenic lines of AtMYB80-OE and AtMYB1-OE. Each

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bar represents the mean ± SD of three replicates. The transcript level of AtMYB1 was observed to be significantly higher than that of AtMYB80 (**; P< 0.005). ( ) Relative transcript level (ΔCt) of A9 gene in leaves of AtMYB80-OE and AtMYB1-OE plants. Transcript levels of A9 gene was observed to be significantly higher in AtMYB80-OE transgenic lines as compared to AtMYB1OE lines (*; P< 0.05). Unt- Untransformed Figure 4. Analysis of A9 promoter activity by transient expression of AtMYB80 and AtMYB1 over- expression constructs into the leaves of transgenic [A9(1500):gus-intron] tobacco plants. Leaves transfected with empty vector and a 35S:gus-intron construct were used as negative and positive controls, respectively. Three independent experiments each with three technical replicates were carried out. Each bar represents the average GUS activity ± SD of three technical replicates. GUS activity observed following transfection with 35S:MYB80 was found to be significantly higher than that with 35S:MYB1 (*; P value