Overexpression of OsNAC 6 transcription factor from Indonesia rice cultivar enhances drought and salt tolerance

Drought is a major constrain in crop production that reduce growth and cause yield loss of up to 70%. Transcription factor plays a major role in cellular regulation and physical changes of plants as a response to stress. A number of transcription factors, such as CBF/DREB, NAC, zinc finger protein are regulators during stress. The Oryza sativa NAC6 (OsNAC6) gene is one of the transcription factor in rice that can regulate gene expression during stress conditions. Thus, pCambia 1305 harboring OsNAC6 chimaeric gene with CaMV 35S promoter was introduced into rice zygotic embryo using Agrobacterium tumefaciens mediated transformation to regenerate transgenic rice overexpressing the transgene. As many as 39 putative transgenic lines in which 21 lines possitively harbored hpt gene have been regenerated. The positive identification of hpt in the regenerated transgenic rice indirectly indicated integration of the targeted OsNAC6 since both transgenes were part of the same T-DNA. Further analysis indicated the presence of 1-3 copies of transgene integration in the genome. The expression of OsNAC6 transgene in the transgenic rice line#C.73, C.83 and C.91 were higher than wild type non-transgenic one. Further analysis indicated those three transgenic lines carrying OsNAC6 transgene exhibited higher tolerance against drought and salinity stresses. Moreover, three known stress-associated regulatory genes (AP2, Zincfinger protein and MYB) were up-regulated in those three transgenic lines. These findings demonstrated that OsNAC6 might be a candidate of stress-responsive NAC regulatory gene that can be used to develop either drought or salt tolerant tolerant transgenic plants.


Introduction
Rice (Oryza sativa L.), a major staple crop to Indonesian and to other developing coutries in the world.Dehydration stress is a major constrain in rice production in certain areas or seasons.Such dehydration stress may be initiated by either drought or high salinity.Therefore, availability of dehydration stress tolerance variety is highly desirable.Dehydration stress tolerance is a character controlled by multi-genes, making the development of drought tolerance transgenic crops a challenging task.One need to understand the physiology and molecular processes associated with dehydration stress responses to be able to develop crop with higher tolerance to dehydration stress.
A number of transcription factors (TFs) are associated with dehydration stress responses in plants.The TFs control expression of a number of target genes and may associate with a number of pathways.Therefore, modifying complex metabolic pathways in plants can be done by transforming a single TF (Hussain et al. 2011).Interaction among transcription factors with the cis-acting elements of the target genes regulate the expression of the target gene and eventually direct a number of cellular activities (Xiong et al., 2005).
The NAC is a drought inducible gene, initially studied in Arabidopsis.Improved stress tolerance in Arabidopsis could be achieved by over-expressing the Arabidopsis thaliana (At) NAC genes (ANAC019, ANAC055, and ANAC072).In these NAC gene expressing transgenic Arabidosis, there were a number of altered expression of genes induced by drought, salinity, and low-temperature stress (Tran et al., 2004).The function of NAC gene in drought tolerance response was further supported by Hu et al. (2006).In their study, transgenic rice overexpressing SNAC1 were more tolerance to drought stress during flowering in the field and yielded more seeds than the control nontransgenic one (Hu et al. 2006).Hu et al. (2006) also demonstrated that the function of SNAC1 gene is also associated with plant responses to cold and salt stresses (Hu et al., 2006).The NAC6/SNAC2 and OsNAC10 genes originated from Oryza sativa (Os) have also been reported as transcription factors regulating the expression of genes associated with stress responses (Nakashima et al., 2007;Jeong et al., 2010).These two genes belong to the ATAF gene sub-family (Kikuchi et al., 2000;Ooka et al., 2003).Expression of the AtNAC2 gene from Arabidopsis was induced by both salt stress and by abscisic acid.The function of AtNAC2 is associated with tolerance to salt stress and with root development (He et al., 2005).
Regeneration of transgenic rice has been proposed as an alternative route for developing drought tolerant rice genotype.Drought tolerance is a character regulated by many genes.Shinozaki and Yamaguchi (2007) have identified the two gene groups associated with response of plant to drought.In group one includes genes that protect plant cells under dehydration stress, such as genes encoding osmotin, detoxification enzymes, cell recovery and structural adaptation proteins.The second group includes one dealing with those that regulate signal transduction and dehydration stress inducible gene, such as one coding for protein kinases and TFs (Shinozaki and Yamaguchi, 2007).
The TF proteins have an important function in regulating gene expression and physical changes of plants as a response to stresses.A number of the known TFs include CBF/DREB, NAM, ATAF, CUC, zinc finger protein regulators during stress (Huang et al., 2009).Overexpression of three NAC genes from Arabidopsis thaliana, such as ANAC019, ANAC055 and ANAC072 in transgenic plant has increased stress tolerance and changed gene expression induced by drought, salinity and low temperature (Tran et al., 2004).Therefore, these three genes might actually play a major role in stress tolerance responses.Either OsNAC6 or OsNAC10 gene was also reported to encode protein during drought stress (Jeong et al., 2010;Nakashima et al., 2007).The expression of stressinduced SNAC-A domain carrying gene such as RD26, Arabidopsis ATAF, and rice SNAC (SNAC1, OsNAC6 and OsNAC5) were also reported as associated with increasing drought and salinity tolerance (Nakashima et al., 2012).
Rice (Oryza sativa) NAC6 gene (OsNAC6) has been isolated from drought tolerance upland rice cv.Batutegi and the appropiate transgene is constructed.Overexpression of OsNAC6 was done by introducing the transgene into Indica rice cv.Ciherang, the most widely planted rice variety in Indonesia and regenerating transgenic rice lines.This research was conducted to evaluate drought and salt stress responses of the regenerated transgenic Indica rice cv.Ciherang overexpressing OsNAC6.

Rice cultivars
Seedlings of upland rice (Oryza sativa L.) cv.Batutegi and lowland rice cv.Ciherang were obtained from Muara Experimental Station, Indonesian Center for Rice Research (ICRR), Bogor, West Java, Indonesia.Rice cv.Batutegi is more tolerant to drought stress than Ciherang.However, Ciherang is more commonly planted rice variety by farmers in Indonesia.

Transgene construct and transformation
Plasmid pCAMBIA 1305 was obtained from CAMBIA-Australia.The complete cDNA of OsNAC6 was generated by PCR amplification using gene specific primer pairs developed based on NAC cDNA sequences available in the GenBank DNA Database (Accession No. B028185.1).The total cDNA was generated from total mRNA isolated from upland rice (Oryza sativa) cv.Batutegi and it was used as template.The resulted PCR amplified product was sequenced to determine its identity and the correctly identified full length OsNAC6 cDNA fragment was fused into appropiate site of pCAMBIA 1305 overexpression vector.The transgene construct of pCNAC6 (Figure 1) was introduced using Agrobacterium-mediated transformation method into rice cv.Ciherang and a number of rice transgenic lines were regenerated.Hygromycin phosphotransferase gene (hpt) in the overexpression vector allows transformed cells expressing the transgenes to be selected out of the non-transformed cell populations using selective medium supplemented with hygromycin.As part of this rice transformation system, a selective and toxic hygromycin that interferes with the cellular metabolism of non-transformed cells is applied to population of putatively transformed ones.

Drought and salt tolerance assay
The rice seeds derived from selected transgenic rice cv.Cihearng -lines#C.72,C.83 and C.91 were germinated on Yoshida medium (Yoshida et al., 1973) containing hygromicin (100 mg/l) at room temperature for 7 days.For drought and salt tolerance assay, hygromycin resistance-transgenic rice seedlings growing in the selective medium were grown in Yoshida medium containing either nos nos RB LB 100 µM abscisic acid (ABA), 20% polyethylene glycol (PEG) or 200 mM sodium chloride (NaCl) for 14 days in a culture room at room temperature.Wild type (WT) rice cv.Ciherang of the same stage used as control were parallelly grown under the similar conditions.After 14 days, the length of the seedling root and leaf were recorded.

Quantitative real-time RT-PCR (qRT-PCR)
For the gene expression analysis, seedling leaves of WT and transgenic rice were harvested.The leaf samples were collected at 0 and 24 hours after stress treatments.Total RNA was extracted from collected rice leaves using RNA isolation kit (the Trizol reagent of Invitrogen).After DNasetreatment, cDNA was synthesized from total RNA aided by M-MLV reverse transcriptase (Invitrogen).Real-time RT-PCR was performed using SYBR Premix with the Eco Ilumina real time PCR System.The PCR amplification conditions were as follow: one cycle of 5 minutes denaturation at 95°C, followed by 40 cycles of amplification steps of 5 seconds denaturation at 95°C; 30 seconds primer annealing at 60°C, and 30 seconds primer elongation at 70°C; and one cycle of final extension at 70°C for 5 minutes.Relative transcript levels was determined by using actin rice gene expression as internal reference.
Table 1.Primer sequences used for quantitative real time RT-PCR of the respective genes.

Results and Discussion
Drought, salt, cold, and biotic stresses are major constrains in rice production.Developing stress tolerance rice cultivars has been a major research areas in a number of institutions.One of the route undertaken to develop stress tolerant rice cultivars are regenerating transgenic lines expressing a number of stress induced genes (AP2/ERF, bZIP, NAC, MYB, MYC, Cys2His2 zinc-finger or WRKY) (Zhang et al., 2004;Umezawa et al., 2006).
As many as 39 plantlets were regenerated after Agrobacterium -mediated transformation to introduce OsNAC6 transgene into rice genome.PCR analysis was also conducted using specific primer for hpt gene.Based on the PCR results, twenty one of the regenerated plantlets were positively harbored hpt transgene (Figure 2), indirectly indicating that OsNAC6 has also been integrated in the genome of the regenerated plantlets.The resulting PCR (+) for the hpt gene is an indirect indicator of integration of the other gene that was in one T-DNA structure into the putative transgenic rice genome (Zaidi et al., 2006).
The results of Southern blot analysis (Table 2  and Figure 3) indicate that the evaluated putative first generation (T1) of transgenic rice integrated a range of 1-3 copies of hpt transgenes.The duplicate Southern blot analysis resulted in the same estimate of transgene integration number.The presence of different number of integrated hpt transgenes indicate that the evaluated putative transgenic lines have originated from independent transgenic regeneration events.Since the hpt transgene was in the same T-DNA structure as the OsNAC6 transgene; therefore, the presence of hpt could be used as indirect evidence for the presence of OsNAC6.In such case, it could be assumed that the OsNAC6 transgene might have also been integrated in the putative rice transgenic line in a range of 1-3 copies.Putative T1 transgenic lines C.72 and C.83 integrated 1 copy of the inserted hpt transgene, while genotype C.91 integrated 2 copies (Table 2 and Figure 3).The above southern blot results also indicated that there were some putative T1 transgenic lines showed negative result.Those genotype showed positive result when analysed for PCR using hpt gene specific primers.Such cases may indicate that the analyzed samples might have low qualities and or quantities of DNA for southern analysis.However, the quality and the quantity of the DNA were high enough for PCR analysis.
Expression pattern of OsNAC6 in rice seedling stage was analyzed using quantitative real time RT-PCR.Results of the real time RT-PCR indicated the expression level of OsNAC6 was generally higher in young transgenic leaves than that of non-transgenic rice cv.Ciherang (Figure 4).There was no significant increased in OsNAC6 expression in ABA treated transgenic rice cv.Ciherang at 0 and 24 hour after treatment.However, slight increase in OsNAC6 expression was observed at 24 hour after treatment of ABA in WT rice cv.Ciherang (Figure 4  ABA is the most important phyto-hormones involved in growth and development of plants and in a number of stress adaptation (Schroeder et al., 2001;Shinozaki and Yamaguchi-Shinozaki, 2000;Verslues et al., 2006).High cellular ABA levels leads to synthesis of storage proteins in seeds, promote seed desiccation tolerance, dormancy (Finkelstein et al., 2002(Finkelstein et al., , 2008) ) and inhibit seed germination.ABA is also reported to control lateral root formation and seedling growth (Xiong et al., 2006).It also associated with the reduction of water transpiration through promotion of stomatal pore closure (Hetherington, 2001;Kim et al., 2010).Moreover, ABA controls the expression of a number of stress-responsive genes.Based on those, ABA is aptly called as a stress hormone (Hoth et al., 2002;Nemhauser et al., 2006;Seki et al., 2002).
Although the OsNAC6 is a stress induced regulatory gene, in this study, transgenic rice cv.Ciherang overexpressing OsNAC6 transgene did not response to the at 24 hours after treatment.Increased in expression of OsNAC6 was not observed at 24 hours after ABA treatment among evaluated transgenic rice cv.Ciherang.Such failure in observing an increased in OsNAC6 expression in the ABA treated transgenic rice cv.Ciherang might be due to the use of 35S CaMV promoter in the OsNAC6 transgene construct (Figure 1).The 35S promoter is a constitutivestrong promoter, therefore, slight increase in native OsNAC6 gene epression because of ABA treatment may not be observable in the transgenic line.This was supported by the data shown in Figure 4.A.ABA treatment to the WT rice cv.Ciherang could only induce a slight increase in OsNAC6 native gene expression.
The OsNAC6 gene is a regulatory gene for many other stress-responsive TFs, such as AP2, Zincfinger protein, and MYB, respectively.In this experiment, expression level of those genes were evaluated in either the WT or the transgenic rice cv.Ciherang over expressing OsNAC6 transgene.Result of the evaluation indicated that the expression levels of the AP2, Zincfinger protein and MYB, were up-regulated in transgenic lines (Figure 5).The AP2, Zincfinger protein and MYB are regulatory genes for many plant stress responses.The AP2/EREBP is the largest plant TF family and they play an important role for regulating responses for abiotic or biotic stresses and for a number of plant developments (Ohto et al., 2009).A number of NAC protein are also involved in plant responses to stress (Delessert et al., 2005).In this study, we again demonstrated the function of a novel rice NAC gene -OsNAC6 associated with stress responses.Analysis of OsNAC expression in the regenerated rice transgenic showed that the evaluated OsNAC6 transgene was associated with both tolerance to drought and salinity stresses in seedlings of rice young leaves and roots (Figure 6).
The effect of OsNAC6 overexpression in transgenic rice grown under PEG and salt stress was also investigated.As shown in Figure 6.A. and 6.B., transgenic rice cv.Ciherang line#C.72,C.83 and C.91 showed better growth, longer roots and leaves length than those of WT.Such data indicated that constitutive expression of OsNAC6 in transgenic rice cv.Ciherang increased rice tolerance to salt and drought stresses.Such data also indicated that constitutive expression of OsNAC6 might be used to improve drought and salt tolerance in rice.
In this experiment, level of drought tolerant for the evaluated non-transgenic and transgenic rice cv.Ciherang was calculated based on the recovery rate A B C of rice seedlings after drought treatment.The drought stress arrangement was applied to 14 days old rice seedlings as follow: no watering for up to 7 days followed by normal watering for 7 days of recovery periods in the greenhouse.At the end of recovery period, recovery rate of the evaluated rice lines was recorded.
The three transgenic rice cv.Ciherang line#C.72,C.83 and C.91 showed higher survival rate than the wild type one (Figure 7), indicating that constitutive expression of OsNAC6 increased the tolerance of the rice transgenic lines against the stresses.The recovery rate of the WT rice cv.Ciherang and the transgenic rice cv.Ciheranglines #C.72, C.83 and C.91 were 33.3%, 65.5%, 74.4% and 67.8% (Figure 7).

Figure 1 .
Figure 1.Structure of pCNAC6 construct, carrying both OsNAC6 and hpt transgenes in its T-DNA.This construct was used for rice cv.Ciherang genetic transformation mediated by Agrobacterium.35S: 35S CaMV promoter; OsNAC6: OsNAC6 coding sequence; hpt: hygromycin-phosphotransferase coding sequence; nos: nopaline synthase terminator; LB and RB: left and right border of the T-DNA structure.
.A.).Both NaCL (200 mM) and PEG (20%) treatment induced slight increased in OsNAC6 expression in both WT and transgenik rice cv.Ciherang (Figure 4.B and 4.C), indicating that the expression of OsNAC6 in rice is affected more by either NaCL or PEG treatment than by ABA.

Figure 4 .
Figure 4. Expression pattern of OsNAC6 in young leaf of transgenics rice cv.Ciherang were detected by quantitative real-time RT-PCR.The experiments were repeated twice.Error bars are standard deviations of two technical repeats.Wild type (WT) rice cv.Ciherang was used as non-transgenic control.Expression of OsNAC6 in young rice leaves in response to (A) ABA (100 uM), (B) NaCl (200 mM), and (C) PEG (20%) treatments.

Figure. 5 .
Figure. 5. Expression analysis of three stress-responsive genes in the transgenic rice cv.Ciherang line#C.72,C.83 and C.91. Wild type (WT) rice cv.Ciherang was used as non-transgenic control.Error bars are standard deviations of two technical repeats.(A) AP2, (B) Zincfinger protein, and (C) MYB are stress-responsive genes, respectively.

Figure 6 .
Figure 6.Representative samples of rice seedling growth in response to drought and salinity stress of transgenic rice cv.Ciherang lines#C.72,C.83 and C.91 overexpressing OsNAC6.Wild type (WT) rice cv.Ciherang was used as nontransgenic control.Error bars are standard deviations of two technical repeats.The observations were conducted at 14 days after treatment.(A) PEG (20%) and (B) NaCl (200 µM).

Figure 7 .
Figure 7. Drought tolerance assays of transgenic rice overexpressing OsNAC6.Left to right -performance of wild type (WT) rice cv.Ciherang as non-transgenic control and transgenic rice cv.Ciherang line#C.72,C.83 and C.91. Error bars are standard deviations of two technical repeats.(A) under normal condition and (B) after 7 days of drought stress, followed by 7 days of recovery periods.(C) Survival rates of the WT and the three transgenic rice lines after 7 days of drought stress, followed by 7 days of recovery periods.

Table 2 .
Estimation of number of hpt transgene integration in the genome of the first generation (T1) putative transgenic rice cv.Ciherang as indicated in Figure3.