Arabidopsis thaliana ecotype Col-0 was used in all of the transformation and promoter analysis in this study. The plants were cultivated under 16 h light/8 h dark photoperiod with 300 Es⁻¹m⁻² illumination intensity, at 22 ± 1°C. The seeds were stratified at 4°C for 4 days prior to growth.

The tobacco (Nicotiana tabacum L. cv Bright Yellow 2, BY2) was cultivated in a modified liquid Murashige and Skoog (MS) medium (Zhou et al., 2014) at 28°C with 120 rpm shaking avoiding light and maintained by weekly dilution (V/V = 1/10) of cell.

The pre-existing 513 bp DYT1 promoter-driven GFP expression construct, designated as DYT1^513bp::GFP (Shumin et al., 2015), was used as PCR template in this study. The primers to generate site mutations of the constructsDYT1^TTCC::GFP, DYT1^CGCC::GFP, DYT1^CTTC::GFP and DYT1^CTCT::GFP; CTCC flanking site mutation constructDYT1^TCTCCT::GFP were designed and synthesized respectively. Novel 5′ end primers of truncation constructs DYT1^489bp::GFP, DYT1^497bp::GFP and DYT1^505bp::GFP were designed and synthesized, respectively (Figure 1). The PCR products were obtained and cloned into pCAMBIA1300 to make reporting constructs according to the report of Zhou (Shumin et al., 2015).

Transgenic plants were generated via floral-dip transformation. The positive transgenic seedlings were screened on MS medium containing 25 mg/L hygromycin (Clough and Bent, 1998). At least 10 independent transgenic T1 generation lines for each construct were observed in this study.

The transformation of BY2 suspension was carried out according to the report of Zhou (Zhou et al., 2014). BY2 cell suspension was co-cultivated with the Agrobacterium GV3101 strain harboring transgenic construct in liquid medium without antibiotics avoiding light at 28°C for 48 h, so that the final concentration of cell suspension was approximately OD₆₀₀ = 0.6. The resulted BY2 cell suspension was enriched by centrifuge and plated on MS solid medium containing 50 μg/ml hygromycin and 100 μg /ml vancomycin, and incubated at 28°C avoiding light. Two weeks later, grown-up antibiotics-resistant callus were subjected to amplified liquid cultivation, and the resulted BY2 cell suspension was used for genotyping and fluorescence observation. At least 10 independent original antibiotics-resistant callus were observed for each construct. The pre-existing transgenic callus of cauliflower mosaic leaf virus 35S promoter-driven GFP expression 35S::GFP was used as a positive control (Zhou et al., 2014).

Total RNA was extracted from the transgenic BY2 cell suspension and performed reverse transcription according to Zhou et al. (2014). Then GFP cDNA fragment was PCR amplified with GFP specific (GFP RT-F&R) primers with the sequence listed in Table 1.

Anthers were stripped and collected from transgenic plants flower bud just around male meiosis (anther stage 4–9) on a microscopy slides. Added one drop of sterile water on the anthers and covered a slide carefully without squeezing. Then the sample was observed and photographed under Zeiss LSM-710 confocal microscope (Zeiss, Germany) and Leica DM2500 fluorescence microscope. As for semi-quantification of the fluorescence intensity, randomly 10 sites on fluorescence images were selected and the intensity was measured and normalized by the SMART software. Statistics of at least 15 anthers per line, 10 independent T1 generation transgenic lines were counted for each construct transformation. As for BY2 cell suspension, at least 100 cells per callus ancestor were observed, and 10 callus were counted for each construct transformation.

Previous studies showed that the 513 bp length DYT1 promoter could faithfully regenerate the temporal and spatial profile of native DYT1 expression (Shumin et al., 2015). The GFP signal of transgenic DYT1^513bp::GFP firstly appeared in the secondary parietal cell and microsporocyte of stage 4 Arabidopsis anther. Then the GFP expression increased significantly and reached its peak preferentially in the tapetum of stage 5 and 6 anthers. Subsequently, the GFP signal rapidly weakened at stage 7 and disappeared at stage 8 (Figures 2A,G). The “CTCC” cis-element locating at −468 bp from the TSS is particularly important for the correct expression of the DYT1 gene. The deletion of “CTCC” completely knocked out GFP expression (Shumin et al., 2015). To investigate the function of each nucleotide in the “CTCC” cis-element, a series of modifying constructs based on DYT1^513bp::GFP with site mutations in or around the “CTCC” sequence were made, and transformed into Arabidopsis, respectively (Figure 1). The transgenic plants were identified by PCR using nucleotide specific primers (Table 1) and restriction endonuclease digestion assay (Supplementary Figure 1). The site mutations of the two flanking nucleotides of the “CTCC” cis-element (5′ end from “C” to “T,” and 3′ end from “C” to “T,” respectively), and the first and third nucleotide substitutes from “C” to “T” in the “CTCC” imposed no effect on the expression pattern of GFP (Figures 2B,C,E,G). On the contrast, however, “G” replacing “T” in the “CTCC” resulted in weak expression of GFP in the connective and epidermis tissues in addition to the tapetum and PMC (before stage 6, then microspore at stage 7 and 8; Figures 2D,H). Furthermore, “T” replacing the final “C” resulted in strong GFP expression in all tissues of stage 4–8 anthers (Figures 2F,H). Thus, the “T” and final “C” of the “CTCC” cis-element were suggested to play predominant roles in controlling the tissue specificity and appropriate intensity of the gene expression.

The previous study had elucidated that beside the core motif “CTCC,” the −481 to −513 bp region of DYT1 promoter was also indispensable for appropriate expression. To uncover finer structure within this region, in addition to original 481 and 513 bp truncated DYT1 promoter-driven GFP reporter constructs, 489, 497, and 505 bp truncated DYT1 promoter-driven GFP reporter constructs were made and transformed into Arabidopsis, respectively. As a result, both 505 and 497 bp DYT1 promoter gave rise of the identical expression pattern as the 513 bp DYT1 promoter (Figures 3A,B,E), suggesting as short as 497 bp DYT1 promoter was sufficient to recapitulate appropriate DYT1 expression in Arabidopsis anther. On the other side, in DYT^489bp::GFP transgenic plants, GFP exhibited an ectopic and weaker expression losing the tapetum-preferential pattern, similar to that of the 481 bp DYT1 promoter. The detectable green fluorescence was distributed not only in the tapetum and PMC (before stage 6, then microspore at stage 7 and 8), but also in the connective and epidermis tissues (Figures 3C,D,F), suggesting that the sequence from −489 to −497 bp in DYT1 promoter was essential for tapetum-preferential expressing pattern, and as short as 497 bp DYT1 promoter sequence was sufficient for tissue-specific expression.

As mentioned before, the flanking −489 to −497 bp region seemed to play as a restriction element to limit DYT1 expression with certain spaces so that DYT1 expression exhibited as a specific spatial profile. Then one more question was brought up whether there was other region in 513 bp DYT1 promoter imparting the species specificity. In order to test such possibility, the series of truncated DYT1 promoter-driven GFP reporting constructs were transformed into tobacco BY2 cell suspension. In DYT1^497bp::GFP, DYT1^489bp::GFP and DYT1^481bp::GFP transgenic BY2 cell suspension, weaker GFP expression comparing with that of 35S::GFP transgenic cells was found (Figures 4A,D–F,M). However, in DYT1^513bp::GFP and DYT1^505bp::GFP transformed cell lines, no GFP signal could be detected (Figures 4B,C,M). Thus, 505 bp DYT1 promoter sequence was sufficient for restricting the gene expression in A. thaliana rather than in other species such as tobacco BY2 cell suspension.

Furthermore, all site mutations within “CTCC” based on DYT1^513bp::GFP gave rise of ectopic GFP expression in BY2 cell suspension, suggesting that the “CTCC” cis-element participated in determining species specificity. However, the substitutes of the “T” and final “C” generated stronger ectopic expression than the other two nucleotides (Figures 4I,K), suggesting the “T” and final “C” also contributed in determining species specificity more than the other two “C” nucleotides (Figures 4H,J), though not so exclusively as in determining tissue specificity in Arabidopsis. Consistent to the results obtained from Arabidopsis study (Figure 2), the mutations of “CTCC” flanking nucleotides had no effect on the driven gene expression (Figure 4G), further supporting “CTCC” itself was a four-nucleotides motif. Unlike site mutations, the “CTCC” deletion DYT1^513bpΔcis:: GFP generated little GFP fluorescence either in Arabidopsis anther (Shumin et al., 2015), or in BY2 cell suspension (Figures 4L,M), adding complexity to the function of intact “CTCC.” One explanation is that in addition to controlling spatial expression pattern of the driven gene, “CTCC” as a whole is also important for gene expression activation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.