The ALDH7 promoter of Acacia nilotica L is a moisture stress inducible promoter

Plant Gene 10 (2017) 1–7

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Plant Gene journal homepage: www.elsevier.com/locate/plantgene

The ALDH7 promoter of Acacia nilotica L is a moisture stress inducible promoter☆ Ajay Panchbhai a,b, Bharat Char a, Arun S. Kharat b,⁎ a b

Mahyco Research Center, Maharashtra Hybrid Seeds Co. Ltd., Dawalwadi, Jalna-Aurangabad Road, P.O. Box 76, Jalna 431 203, Maharashtra, India. Department of Biotechnology, Dr. Babasaheb Ambedkar Marathwada University, Subcampus Osmanabad, 413 501 Maharashtra, India.

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Article history: Received 8 November 2016 Received in revised form 22 February 2017 Accepted 3 March 2017 Available online 06 March 2017 Keywords: ALDH7 promoter Acacia nilotica moisture stress GUS and Glycine max

a b s t r a c t The most popular CaMV 35S promoter used for the construction of transgenic plants is a constitutive promoter. The use of conditionally expressed promoters would be a better option as the energy towards expression would be accounted for when the need for expression is indicated. The Acacia nilotica has been known to sustain severe moisture stress. The ALDH7 gene has been reported to be an antiquitin. The PCR amplicon for the ALDH7 promoter from A. nilotica was obtained and sequenced from both the ends to decipher nucleotide sequence. Nucleotide sequence analysis of the A. nilotica ALDH7 promoter indicated the presence of a response element. The nucleotide analysis using PLACE indicated presence of the ACGT, drought and salinity stress response element whereas NNPP indicated existence of seven transcriptional start sites and TSSP indicated presence of two promoters. The PCR amplified ALDH7 promoter of A. nilotica was cloned into a binary vector carrying a promoter-less GUS cassette. Thus, the GUS gene could be expressed under the influence of the A. nilotica ALDH7 promoter. Transgenic tobacco plants expressing the GUS gene under the A. nilotica ALDH7 promoter were constructed. Using histochemical staining, the GUS gene expression levels in transgenic tobacco plants during moisture stress and under irrigation were studied. Histochemical staining experiments demonstrated that the GUS gene in transgenic plants was expressed only during moisture stress and remained unexpressed while irrigation was maintained. In contrast, transgenic tobacco plants expressing the GUS gene under the CaMV 35S promoter were found to be constitutive. The histochemical studies also indicated that the GUS expression was stable at least for two generations. Studies of the gene indicated that transgene segregation was in agreement with that of a monogenic trait described by Mendelian inheritance. The ALDH7 promoter of A. nilotica is a conditionally expressible promoter, expressed during moisture stress and suppressed during irrigation. © 2017 Published by Elsevier B.V.

1. Introduction Plant genetic engineering has contributed substantially to the understanding of gene regulation and plant development, with the generation of transgenic organisms for widespread usage in agriculture. It has also helped to increase the potential uses of crops for industrial and pharmaceutical purposes (Potenza et al., 2004). The most widely used promoter for expression of a foreign gene (transgene) in dicot plants is the cauliflower mosaic virus (CaMV) 35S promoter (Ow et al., 1986). Due to limited availability, the feasibility of promoters Abbreviations: ALDH, Aldehyde Dehydrogenase; GUS, Glucuronidase; CaMV 35/s, Cauliflower Mosaic Virus 35S promoter; promF, Promoter Forward primer; UTR-R, Upstream Reverse primer; PCR, Polymerase Chain Reaction; TSSP, Transcription Start Site Program; NNPP, Neural Network Promoter Prediction; PLACE, Plant Cis; bp, Base pair; DAS, Days After Sowing. ☆ Area: Environmental and Stress ResponseRegulation of Gene Expression ⁎ Corresponding author at: Department of Biotechnology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad Subcampus Osmanabad, MS, 413 501, India. E-mail address: offi[email protected] (A.S. Kharat).

http://dx.doi.org/10.1016/j.plgene.2017.03.001 2352-4073/© 2017 Published by Elsevier B.V.

introducing multiple traits to generate value-added plants is a great challenge. Some of the challenges include the ability of gene expression to maintain stoichiometry, and in other cases the over expression of traits has been documented to lead to gene silencing (Atkinson et al., 1998). Attaching a number of traits to the CaMV35S promoter provides a site for homologous recombination and thus can sometimes lead to inactivation of the transgene -for review see, Bhullar et al. (2003). Many laboratories around the globe are looking towards plant genetic engineering as a future resource to serve various aspects of biotechnology. Some examples include the following: to boost agroeconomy by introducing desired traits in plants, to improve nutritional value of nutraceuticals, to express molecules for human use and consumption without further processing, and to generate vaccines against various human diseases (Beyer et al., 2002; Cardi et al., 2010; Chakraborty et al., 2000;Daniell et al., 2005). The demand calls for a search for novel alternative promoters. Indeed, a variety of promoters are required at different steps in plant genetic engineering, from basic discoveries, to formulation of concepts and answering questions, to the development of economically viable crops and plant commodities.

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All these uses would address legitimate concerns that have been raised about the safety and containment of transgenic plants in the environment. While reconnoitering drought sustained plant species, the A. nilotica was one of the obvious choice. This was primarily due to the fact that it has been known to grow well on infertile, barren land (Phillips and Riha, 1993; Rasanen and Lindstron, 2003; Sprent et al., 2010). Lipid peroxidation is one of the consequences of environmental stress in plants causing the accumulation of highly toxic, reactive aldehydes. One of the processes that helps to detoxify these aldehydes is their oxidation into carboxylic acids in a process catalyzed by NAD(P)+-dependent 3aldehyde dehydrogenases (ALDHs) (Stiti et al., 2011). The plant ALDH genes are represented in 11 ALDH families: ALDH2, ALDH3, ALDH5, ALDH6, ALDH7, ALDH10, ALDH11, ALDH12, ALDH18, ALDH19, and ALDH21 (Rodrigues et al., 2006). These ALDH genes are known to be involved in the antioxidant defense system, along with drought and salt stress tolerance, by catalyzing osmoprotectants (Kirch et al., 2004; Kirch et al., 2005). In addition to stress-associated ALDH, members of the family ALDH7 are also designated antiquitin. Literature survey indicated that the ALDH7 gene could be one of the drought response candidate genes. The physiological function of antiquitin is believed to be related to the regulation of turgor pressure or response to general stress (Rodrigues et al., 2006). An alternate promoter to the CaMV 35S promoter that may drive transgene expression conditionally would be of great importance. The expression of a transgene would be best triggered only under indicated physiological conditions and remain turned off at other times. The Acacia species are known to withstand severe drought but have never been exploited to understand the mechanisms or the genes responsible for the species' ability to withstand and flourish with a scant water supply or even under drought conditions (Sprent et al., 2010). Thus, in this study, we aimed to investigate whether or not the ALDH7 promoter from A. nilotica plays a role in moisture stress tolerance. The observations described here demonstrate that the ALDH7 promoter of A. nilotica is a moisture stress inducible promoter that is stable for at least two generations.

Fig. 1B now has gus expression under the control of the A. nilotica ALDH7 promoter. The deletion constructs; DF1, DF2 and DF3 were designed from the wild type ALDH7 promoter. To construct deletions, the potential translational start site was regarded as + 1 and forward primers were designed with a HindIII restriction site within promDF3-F (5′AAGCTTAGTGCGTTTTTATGACCTCAAC-3′), promDF2-F (5′-AAGCTTGTAC ACGTACGAGTCTGGAC-3′) and promDF1-F (5′-AAGCTTCAACCACAATC CCTCTCTGAG-3′). Forward primer binding positions were designed in to obtain a PCR amplicon with UTR-R at − 1 to − 1019 for DF3, − 1 to − 908 for DF2 and − 1 to − 754 for DF1 deletion constructs. The amplified fragments were cloned upstream of the GUS reporter gene in vector pC2301. Fig. 1B depicts the structural organization and co-ordinates of the deletion construct, which were named pC2301DF3-GUS, pC2301-DF2-GUS and pC2301-DF1-GUS. 2.3. Generation of transgenic tobacco lines Tobacco (Nicotiana tabacum L., cv Petit Havana) was transformed following the modified leaf disk method as described by Gallois and Marinho (1996). Tobacco leaf disks were immersed in liquid culture of Agrobacterium tumefaciens strain LBA4404 carrying the transformation constructs pC2301-ALDH7-GUS, pCAMBIA 2301(GUS under CAMV 35S), pC2301-DF1-GUS, pC2301-DF2-GUS, and pC2301-DF3-GUS. During transformation, the antibiotic Kanamycin was used for plant selection. The plant tissues were co-cultivated with Agrobacterium on regeneration medium for 2 days. Co-cultivated leaf disks were then transferred to regeneration medium supplemented with cefotaxime to kill bacteria and a selective agent (Kanamycin) against untransformed plant cells. Two rounds of Kanamycin selection (50 mg/l) on regeneration medium was performed before the plantlets were rooted. Rooted plantlets were hardened and transferred to soil. GUS gene expression was assayed using histochemical staining methods as described by Sun et al. (2010). 2.4. Molecular & Functional characterization of transgene

2. Methods 2.1. Isolation and characterization of ALDH7 promoter Genomic DNA of Acacia nilotica was extracted following the method of Edwards et al. (1991). To clone the ALDH7 promoter from A. nilotica PCR primer pair; ALDH7 promF (5′-AAGCTTGTA TCT CGA GCA TAA TTA CTT CC-3′)and UTR-R (5′-CCATGGTTTCCTGCGAAGATCCACCCAG-3′) were designed on the basis of nucleotide sequence information for the ALDH7 gene of Glycine max L. (NCBI). An amplicon of approximately 1.2 kb was cloned into pGEM-T Easy vector (Promega, Madison USA following the TA cloning strategy. The pGEM-T-ALDH7 clone was then processed to decipher nucleotide sequence information from both strands and was submitted to GenBank (accession number: KC206084). The existence of promoter motifs within this sequence was analyzed with the help of the Transcription Start Site Prediction (TSSP), Plant Cis-Acting Regulatory DNA Elements (PLACE) and Neural Network Promoter Prediction (NNPP) software tools. 2.2. Construction of the promoter-reporter plasmids (pC2301-ALDH7-GUS and deletion derivative) The ALDH7 promoter fragment from pGEM-T-ALDH7 was excised with the use of HindIII and NcoI restriction endonucleases and cloned at the same sites of a pCAMBIA 1301(Cambia, Australia) vector to obtain a pC1301-ALDH7 clone. The GUS expression under the CaMV 35S promoter from pC2301 was swapped by substituting the HindIII and BstEII restriction fragment of pC2301 with that of the pC1301-ALDH7 clone. The chimeric construct pC2301-ALDH7-GUS depicted in Fig. 1A and

The presence of the ALDH7 promoter in putative transgenic lines was confirmed by PCR amplification. In PCR reactions, 100 ng purified genomic DNA was used as a template with primer pair ALDH7 promF and UTR-R, and the wild type ALDH7 promoter. Primer pairs promDF1-F with UTR-R and promDF3-F with UTR-R were used to amplify DF1 and DF3 deletion derivatives, respectively. The PCR amplicons from different lines were subjected to electrophoresis along with an appropriate control. Stability of the transgene was tested by performing self-crossing with T0 transgenic plants to obtain T1. Seeds collected from T1 were sown in the greenhouse to obtain seedlings. The GUS expression in 25–30 day old seedlings was performed using a histochemical staining assay. In brief; T1 seedlings were divided into two halves. From the 30thDAS, one of the halves was subjected to moisture stress for 5 to 6 successive days while the remaining half received appropriate irrigation. The GUS score were identified for both groups of plants. 2.4.1. The GUS histochemical analysis The reporter activity for GUS placed under ALDH7 or CAMV 35S promoters was performed on transgenic plants following GUS histochemical staining as described by Sun et al. (2010). Briefly the plant tissues were incubated at 37 °C overnight in 100 mM sodium phosphate buffer (pH 7.0) containing 0.1% Triton X-100, 10 mM EDTA, 1 mM X-gluc and 0.5 mM potassium ferricyanide. The stained tissues were then washed several times with 70% ethanol to bleach the chlorophyll and observations were recorded. 2.4.2. Moisture stress The T1 seedlings containing wild type ALDH7 promoter were equally divided in two parts. One part was subjected for moisture stress by

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Fig. 1. A GUS expression cassettes in the transformation vectors; A, GUS driven by the ALDH7 promoter; B, GUS driven by the CAMV 35S promoter B Construction of the promoter-reporter plasmids: A, Full length ALDH7 promoter linked to the GUS reporter gene. B, A set of 5′ deletion promoter fragments fused to the GUS reporter gene. The number stands for the nucleotide position from the translational initiation site, ATG (A as +1).

preventing irrigation from the 30th DAS for 5 to 6 successive days. Other half seedlings received irrigation as per schedule and were considered as irrigated control. 3. Results 3.1. Cloning and in silico characterization of the A. nilotica ALDH7 promoter Despite great ability to fix nitrogen and sustain under sever moisture stress A. nilotica has not been well exploited by plant molecular biologists (Rasanen and Lindstron, 2003; Sprent et al., 2010). We sought to address whether there exists a promoter that may serve as an alternative promoter to the CaMV 35S promoter during the generation of transgenic plants. The ALDH7 promoter homologue of Glycine max was amplified via PCR ona A. Nilotica genomic DNA template using the primer pairs promF and UTR-R, which were designed based on the nucleotide information from the G. max ALDH7 promoter. A 1.2 kb sized PCR amplicon was cloned by TA cloning into a pGEM-T Easy vector that was then sequenced, and the sequence information was submitted to GenBank. KC206084 is the unique accession number that was given to the A. nilotica ALDH7 upstream regulatory sequence bearing within it the promoter and response element. When KC206084 was used to probe the nucleotide database using the www.ncbi.nlm.nih.nlm.gov/ blast search engine, no significant homology was found with sequences in the database suggesting that the promoter sequence is likely to be novel. The ALDH7 putative promoter sequence was used to identify existing promoters using the Transcription Start Site program (TSSP), Web signal scan program (PLACE) and Neural Network Promoter Prediction (NNPP) program. The TSSP uses files with selected factor binding sites from RegSite DB (Plants) developed by Softberry Inc. With the help of this algorithm predicted two independent promoters in the ALDH7 sequence located one at 653 and other at 1080 co-ordinates. Data shown in Fig. 2 indicates that the NNPP program identified seven potential transcription start sites are located at co-ordinates; 40–90,

274–324, 379–429, 768–818, 1036–1086 and two overlapping transcriptional start sites at co-ordinates 576–626 and 618–668. All of these TSS are highlighted in green color while overlapping sequence in cyan, Fig. 2. Interestingly, potential cis-acting regulatory DNA element search carried with PLACE indicated presence of the ACGT a moisture stress response motif located at four coordinates; 320–323, 709– 712, 971–974 and 1080–1083, shown as red alphabets in Fig. 2. These in silico analyses suggested that the cloned fragment may carry a functional promoter.

Fig. 2. TSS and Moisture Stress Response Element Prediction: Nuclear Network for Promoter Prediction identified seven Transcriptional Start Sites (TSS) highlighted in green color. The overlapping nine nucleotides within 4th and 5th TSS are highlighted in cyan color. A moisture stress response ACGT element identified with PLACE is shown as red nucleotides. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.2. Construction & Characterization of transgenic tobacco plants expressing GUS under the ALDH7 promoter The transformation of tobacco plants with Binary plasmid pC2301ALDH7-GUS construct was performed as described in the Methods. Eight independent primary transformed lines were selected. Independent transformations were performed for the truncated ALDH7 promoter pC2301-DF1-GUS, hereafter referred to as DF1, and pC2301-DF3GUS, here after referred to as DF3. Eight independent primary tobacco transformed lines with the DF1 construct, four independent lines with the DF3 construct were obtained. Five primary transgenic tobacco lines were constructed with pC2301, which served as an internal control as the GUS expression is under the CAMV 35S constitutive promoter. Transgenic lines with full length A. nilotica ALDH7, DF1 and DF3 and pC2301were grown in the presence of kanamycin for selection. Integration of the corresponding promoter was confirmed with PCR. Eight independent transgenic lines from T0 generation seedlings containing the ALDH7 promoter were used to isolate genomic DNA, and PCR amplification was performed with the promF and UTR-R primer pair. It is evident from Fig. 3A (top and bottom panels) that every transgenic line was able to amplify the desired insert size. Interestingly, the insert was absent from the escape tissue regenerated line. Consistent with Fig. 3A, when T1 seedlings with DF1 transgenic lines were used as the DNA template, each of the eight independent transgenic lines was able to PCR amplify the insert of the size of DF1, (see Fig. 3B). This observation demonstrates that integration of the transgene was successful and stable as it successfully penetrated into the T1 generation. The PCR analysis of the transgenic line obtained with DF3 as a transgene amplified the amplicon corresponding to the size of the control (data not shown) indicating existence of the transgene. Segregation analysis was performed by obtaining the T1 generation. Neomycin phosphotransferase II (NPT II) expression was assayed by germinating T1 seeds on media containing Kanamycin 50 mg/l. All lines carrying the ALDH7 and CAMV 35S promoters segregated for the nptII gene in the T1 generation. Germination frequency with Kanamycin for transgenic plants containing the ALDH7 promoter was found to be in the range of 59% to 79%, while the frequency without kanamycin ranged from 92% to 94%. Studies on the nptII gene segregation with the Kanamycin marker suggested that the trait followed Mendelian segregation similar to a monogenic trait (data not shown). 3.3. Histochemical studies on GUS expression Histochemical studies to determine GUS expression were conducted on T1 generation seedlings. Plantlets from the T1 generation expressing GUS under the CAMV 35S and ALDH7 promoters were subjected to analysis for GUS expression. Data presented in Fig. 4A shows that the GUS gene expression under CAMV 35S promoter is semiconstitutive, level was enhanced upon imparting moisture stress. It is evident from Fig. 4B that none of the plantlets with the ALDH7 promoter could express GUS either prior to subjecting them to the moisture stress test or during irrigation. The GUS expression under ALDH7 promoter was seen in 62% plantlets only after subjecting the plants to moisture stress for three consecutive days, shown in Fig. 4C. Interestingly, when GUS expression was tested in the T1 plantlets with the CAMV 35S promoter, it was found that 64% of the plantlets exhibited GUS expression prior to being subjected to moisture stress, and 66% of plantlets during moisture stress exhibited GUS expression Fig. 4C. Similar findings in connection with GUS expression were also noted in the T2 generation, see Fig. 4D. These observations indicated the CAMV 35S promoter could express GUS gene unconditionally. Data presented in Fig. 4D shows that either prior to moisture stress or during irrigation none of the transgenic lines with the ALDH7 promoter in the T2 generation could express GUS. However, after subjecting the plants to moisture stress for 3 consecutive days, GUS expression was seen in 68% of the plantlets shown in 4D. These observations demonstrates that the GUS gene expression

Fig. 3. Molecular characterization of transgenic lines for existence of the transgene: A (top and bottom panel): PCR analysis of T0 primary transformants with the ALDH7 promoter: Amplification was performed using ALDH7 promF and UTR-R primers. M, 1Kb DNA ladder; W, reaction mix without DNA; FL-1, FL-2, FL-3, FL-4, FL-5, FL-6, FL-7, FL-8, eight independent transgenic lines; R, regenerated escape shoots; NC, DNA from nontransgenic tobacco plant; PC, Plasmid DNA (control) carrying the ALDH7 promoter. B PCR analysis of T1seedlings obtained with the DF1 promoter. PCR amplification was performed using promDF1-F and UTR-R primers. M, Molecular weight marker; NC, DNA from non-transgenic tobacco plant; W, Reaction mix without DNA; P, Plasmid DNA; 1 to16, DNA of GUS expressing T1 seedlings from eight DF1 lines in duplicates.

under ALDH7 promoter was conditional, induced during moisture stress and reversed upon restoring irrigation. Studies extended with transgenic lines containing the CaMV 35S promoter in the T2 generation showed that before moisture stress 68% of plantlets were able to exhibit GUS expression, while 70% of the plantlets did so during moisture stress, Fig. 4D. Thus GUS expression in the transgenic plants with the ALDH7 promoter was not only induced during moisture starvation but also repressed upon restoring water supply, see Fig. 4B. Studies were also conducted on transgenic plants expressing the GUS gene under deletion derivatives of ALDH7, such as DF1 and DF3. Observations made on plantlets from theT1 generation for the plants with DF1 or DF3 ALDH7 deletion derivatives showed that the expression of the GUS gene was present only upon subjecting the plantlets to moisture stress (see Fig. 5A to F), similar to the expression of this gene in the wild type ALDH7 promoter bearing transgenic plants. After establishing that the ALDH7 promoter and its derivatives were able to display conditional expression, histochemical staining for glucuronidase was performed on seedlings obtained from T1 and T2

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Fig. 4. Histochemical staining for characterization of the GUS gene expression prior to and during moisture stress: A. Transgenic plant leaf expressing the GUS gene under CAMV 35S promoter, B. Transgenic plant leaf expressing the GUS gene under ALDH7 promoter, C. The GUS expression studied from T1 seedlings. The T1 plantlets expressing the GUS gene before and during moisture stress. The black histogram shows the number of transgenic plantlets expressing the GUS gene under the ALDH7 promoter, the light gray histogram shows the number of transgenic plantlets expressing the GUS gene under the CaMV 35S promoter. Line bars on the histogram denote standard error bars. From each transgenic line, 30–50 plantlets were considered for studying expression. D. The GUS expression studied from T2 seedlings. The T2 plantlets expressing the GUS gene before and during moisture stress. The black histogram shows the number of transgenic plantlets expressing the GUS gene under the ALDH7 promoter, the light gray histogram shows the number of transgenic plantlets expressing the GUS gene under the CaMV 35S promoter. Line bars on each histogram denote standard error bars. From each transgenic line, 30–50 plantlets were considered for studying expression.

Fig. 5. Functional staining of the ALDH7 promoter and it's derivatives from transgenic Seedlings. Histochemical staining for glucuronidase was performed on primary root or leaf tissue from transgenic line seedlings with the ALDH7, DF1 or DF3 promoter in the T1 and T2 generations. Arrows indicate staining. A: wild type ALDH7; primary root B: wild type ALDH7 leaf cut tissue, C: DF1 promoter,T1 generation, primary root, D: DF1 promoter, T1 generation, leaf cut tissue, E: DF3 promoter, T2 generation, primary root, F: DF3 promoter, T2 generation, leaf cut tissue,.

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generations expressing GUS under the ALDH7, DF1 or DF3 promoters. Data presented in Fig. 5A, B, C, D, E & F demonstrate that positive staining is found in the primary roots and leaf cuts (denoted by black arrows) of the transgenic plants from T1 and T2 generations. The presence of this transgene in T2 generations is another indicator of stability, penetrance and expressivity of the conditional expression of the ALDH7 promoter. As the DF1 retains only 754 bps, exhibited conditional expression of GUS within transgenic lines suggests that the conditional switch or response element along with the promoter is located between the − 1 to −754 bp coordinates. 4. Discussion Plant Molecular Biology has been generously applying the CaMV 35S constitutive promoter. This study aimed to identify an alternative conditionally expressed promoter that can withstand moisture stress. For this purpose we chose A. nilotica, primarily because the plant is known to flourish well even with a scant water supply or in drought conditions, suggesting that there exists a mechanism that allows the plant to survive under moisture stress. To the best of our knowledge, this is the first report providing the idea that A. nilotica could be used in generating moisture stress tolerant transgenes. The ALDH7 family members are also regarded as antiquitin, whose physiological function is to regulate turgor pressure or respond to general stress. To the best of our knowledge, no other reports are available documenting existence of ALDH family members in A. nilotica. To amplify the ALDH7 promoter, primers were designed based on nucleotide information for ALDH7 from G. max. The PCR amplified 1233 bp fragment represents a homologue ALDH7 promoter of G. max. Obtained from A. nilotica. Molecular characterization of the transgene in T1/T2 generations along with functional studies on ALDH7 and DF1 during moisture stress indicated that the integration of the reporter cassette was stable and it was found to follow Mendelian segregation principles for a monogenic trait. Conditional expression of the ALDH7 promoter demonstrated by the response to moisture stress is the strength of our article. When probed with nucleotide sequences within the GenBank search engine, it was evident that nucleotide information for KC206084 was unique and contained a novel sort of moisture response element along with potential two transcriptional start sites within allowing conditional expression. Earlier studies on potato and Solanum americanum (Siebertz et al., 1989) used GUS reporter cassette successfully. In their reports the authors were able to show GUS activity in various tissues of transgenic plants. Histochemical staining experiments performed on transgenic tobacco plants in this study were in agreement and exhibited GUS expression in different plant parts. The histochemical staining results in Fig. 4 and Fig. 5 shows that expression is noticeable in the primary roots and leaves of transgenic plants from T1 and T2 generations, demonstrating stability and expressivity of the trait. In our efforts to determine minimal regions of the promoter in the subject, deletion derivatives DF1, DF2, and DF3 were constructed from wild type ALDH7 promoter. In one of the studies similar to those performed earlier, nine DNA fragments containing different 5′-deleted series of the TsVP1 promoter region were amplified by PCR and characterized in transgenic Arabidopsis (Sun et al., 2010). A new 130 bp cis-acting element involved in the salt stress response was identified in the TsVP1 promoter and it was found that deletions retaining + 1 to - 537 and + 1 to − 327 promoter sequences demonstrated basal GUS expression in the vascular tissue, both in the shoots and roots (Sun et al., 2010). Studies reported by Siebertz et al. (1989) documented that sequences between − 111 and − 571 showed a slightly higher activity. The addition of further upstream sequences (− 571 to −1022) enhanced the level of expression. In the present study, deletion derivative DF1showed expression of GUS comparable to that of the wild type ALDH7 promoter. Histochemical staining demonstrated that the core region of ALDH7 promoter and moisture response element is located within the − 1 to − 754 bps. Identified promoter positions at

coordinates 653 and 1080 bp using TSSP corresponds to −580 (TATA box at −594) and −153 bp (TATA box at −191), respectively, which were retained in the DF1 construct. Promoter prediction tools such as PLACE and TSSP were reported to predict the gene regulating elements successfully, shown in Fig. 2 (Higo et al., 1999; Sun et al., 2010; Zhou et al., 2010). The PLACE identified presence of ACGT a moisture and salinity stress response motif located at four coordinates; 320–323, 709–712, 971–974 and 1080–1083, shown as red alphabets in Fig. 2. The erd1 gene of A. thaliana has been known to upregulate by both moisture stress and etiolation. In their studies (Simpson et al., 2003) showed that there exist two different cis-acting elements in the erd1 gene promoter encoding ClpA homologous protein. One of these two elements was ACGT, which was found to exist at four co-ordinates of ALDH7 promoter. The erd1 like gene from soya bean roots was found to be drought inducible Neves-Borges et al., 2012. The erd1 gene was found to be involved in identifying coping strategy to drought in the case of Eutrema salsugineum MacLeod et al. (2015). The erd1 gene has been shown to be upregulated in various plant system and it carries ACGT motif within the promoter, which has been detected to exist in ALDH7 promoter. This may suggest that the ACGT motif from ALDH7 is likely be playing some role in moisture stress inducibility. Studies reported in this article indicate that the cloned ALDH7 upstream regulatory sequence carries a promoter and a moisture stress response element. The moisture stress response element drives a conditional switch so that the promoter is turned on as soon as plants experience moisture stress, allowing the conditional expression of transgene under moisture stress. In addition to molecular stacking, conditional expression of transgene would be metabolically affordable as energy would not be spent when the transgene's expression is not desired. Competing interests The authors have no competing interest to declare. Acknowledgements The authors would like to express sincere thanks to the Mahyco Research Centre for providing the facilities where this work was performed. Administrative support from University and Subcampus authorities is gratefully acknowledged. Critical readings/ comments by laboratory members and two anonymous peer reviewers are gratefully acknowledged. There is no conflict of interest to declare. References Atkinson, R.G., Bieleski, L.R.F., Gleave, A.P., Jannsen, B.J., Morris, B.A.M., 1998. Post-transcriptional silencing of chalcone synthase in petunia using a geminivirus-based episomal vector. Plant J. 15, 593–604. Beyer, P., Al-Babili, S., Ye, X., Lucca, P., Schaub, P., Welsch, R., Potrykus, I., 2002. Golden Rice: introducing the beta-carotene biosynthesis Golden Rice: introducing the betacarotene biosynthesis pathway into rice endosperm by genetic engineering to defeat vitamin A deficiency. J. Nutr. 132 (3), 506S–510S. Bhullar, S., Chakravarthy, S., Advani, S., Datta, S., Pental, D., Burma, P.K., 2003. Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping. Plant Physiol. 132, 988–998. Cardi, T., Lenzi, P., Maliga, P., 2010. Chloroplasts as expression platforms for plant-produced vaccines. Expert Rev. Vaccines 9, 893–911. Chakraborty, S., Chakraborty, N., Datta, A., 2000. Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus. Proc. Natl. Acad. Sci. U. S. A. 97, 3724–3729. Daniell, H., Chebolu, S., Kumar, S., Singleton, M., Falconer, R., 2005. Chloroplast-derived vaccine antigens and other therapeutic proteins. Vaccine 23, 1779–1783. Edwards, K., Johnstone, C., Thompson, C.A., 1991. Simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 19, 1349. Gallois, P., Marinho, P., 1996. Leaf disk transformation using Agrobacterium tumefaciensexpression of heterologous genes in tobacco. Plant gene transfer and expression protocols. Methods Mol. Biol. 49 (1), 39–48. Higo, K., Ugawa, Y., Iwamoto, M., Korenaga, T., 1999. Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res. 27, 297–300.

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