It has been well documented that upon exposure to abiotic stresses transcript levels of a multitude of genes are altered. Numerous studies have shown that ABFs/AREBs play significant roles in regulating these stress-related genes via interaction with ABRE cis-element in their promoters, suggesting that ABFs/AREBs are tightly involved in plant response to various adverse environment conditions. In this work, a gene encoding ABF from P. trifoliata was isolated by RT-PCR in combination with bioinformatics approach based on ESTs deposited in the public database. Sequence multiple alignment demonstrated that PtrABF showed high degree of sequence identity with ABFs of other plants retrieved from the database at amino acid level. A highly conserved bZIP domain consisting of a basic region responsible for DNA binding and three heptad leucine repeats related to TF dimerization  was observed near the C-terminus of PtrABF, indicating that PtrABF encoded a bZIP family protein. Apart from the bZIP domain, it also contained four highly conserved regions at the N or C-terminus (C1, C2, C3 and C4). Within these regions, several serine (S) and threonine (T) residues or consensus sequences are present, consistent with the structure of ABFs from other plants [11, 12]. These conserved residues have been suggested as phosphorylation sites of different kinases, such as calmodulin-dependent protein kinase II (R/KXXS/T, position 28-31) and casein kinase II (S/TXXD/E, position 36-39), cGMP-dependent protein kinase (K/RXXXS/T, position 49-53), implying that activation of PtrABF might be regulated by protein phosphorylation, as has been reported in other AREBs/ABFs [12, 15, 24, 25]. Presence of these common characteristics demonstrated that PtrABF cloned in our study shared striking sequence similarity with other ABFs and may have a biological function same as or similar to them in abiotic stress response. The phylogenetic tree revealed relationship between PtrABF and ABFs from other plants, in which PtrABF was closely related to ABFs from dicots, including the ABFs of Arabidopsis thaliana. Although most ABFs in the tree remain to be characterized, in planta functions of ABFs of Arabidopsis have been determined, in which ABF3 and ABF4 act as activators of ABA/stress response. In addition, overexpression of ABF2, ABF3 has been shown to affect multiple stress tolerance, implying that PtrABF might function in the same way as ABFs of Arabidopsis.
An important feature of plant ABFs is the induction of their transcript levels by abiotic stresses [8, 11, 12, 16]. QRT-PCR analysis demonstrated that steady state mRNA levels of PtrABF were induced by ABA, dehydration and low temperature. Expression patterns of PtrABF were largely similar to ABF4/AREB2 that has been shown to be induced by ABA, salt, cold and drought . However, it has to be mentioned that PtrABF was not induced by salt, different from ABF4. In Arabiopsis it has been shown that although ABFs were all induced by ABA, they are differentially regulated by various stresses and have been suggested to play various roles in stress response . ABF family members might have specificity in their role under different stresses apart from existence of function redundancy, in which ABF3 and ABF4 played essential roles in germination control and ABA/stress response, whereas ABF2 was more closely implicated in seedling growth regulation and glucose response [8, 13]. Induction of PtrABF by both dehydration and low temperature seems to indicate that this gene may participate in response to these cues.
Compared with low temperature, dehydration caused more profound induction of PtrABF mRNA abundance, which compelled us to do in-depth work on elucidation of the potential role of this gene for enhancing dehydration and drought tolerance. To this end, transgenic tobacco plants were produced via Agrobacterium-mediated transformation of PtrABF under the control of CaMV 35S promoter. The two selected transgenic lines exhibited better phenotypic morphology, concomitant with less water loss, lower electrolyte leakage and higher chlorophyll content than wild type under either dehydration or long-term water stress, suggesting that overexpression of PtrABF conspicuously conferred tolerance to these adverse conditions. Apart from the water stress used herein, the transgenic plants also exhibited enhanced tolerance to low temperature treatment in comparison with WT (data not shown). Our work agreed with earlier reports, in which overexpression of ABF family members has been shown to render tolerance to multiple stresses in the same transgenic line [13, 19–21, 26], implying that ABFs class transcription factors hold great potential for genetically manipulating stress tolerance.
Despite the fact transformation of ABF genes led to improvement of abiotic stress tolerance, the physiological mechanism underling the tolerance remained largely unknown. This stimulated us to carry out more work to find out physiological difference between the transgenic plants and WT under stress. We put special emphasis on comparing their ROS levels because it has been well accepted that in biological systems ROS accumulation is related to physiological perturbation and ROS levels can reflect the degree of damage to cellular components . Histochemical staining by DAB and NBT clearly demonstrated that under dehydration and drought conditions the two transgenic lines accumulated remarkably less O2- and H2O2 than WT. As ROS level during stresses greatly relies on the homeostasis between generation and removal , accumulation of less ROS in the transgenic lines seems to indicate that scavenging systems in these plants might work more effectively compared with WT. In order to detoxify stress-induced ROS, plants evolve a complex antioxidant system, in which several enzymes play essential roles, leading to scavenging ROS and protecting the cells against oxidative stress [27, 28]. Of the enzymes, SOD provides the first line of defense against ROS by catalyzing the dismutation of O2- to oxygen and H2O2, which was then scavenged by coordinated action of CAT and POD . In our study, activities of SOD, POD and CAT in the two transgenic lines were not profoundly different from those of wild type under well-watering conditions although they were slightly higher in the transgenic lines, which sounds reasonable because under normal conditions ROS production remained at low levels and oxidative stress was not serious . However, under water stress, activities of the three enzymes were significantly higher in the transgenic plants than WT, implying that the transgenic plants had more robust detoxifying system to eliminate ROS produced during stress, which is consistent with the dramatic reduction of ROS level and ROS-associated membrane damage (lower electrolyte leakage). It is noticed that activation of antioxidant enzymes was consistent with the upregulation of the three genes, suggesting that these enzymes may be regulated at transcriptional levels. Induction of genes involved in ROS scavenging has been previously reported when TF was ectopically expressed , suggesting that the TF might transcriptionally regulate the expression of genes related to oxidative reactions. Our work suggested that deployment of a better ROS-scavenging system might be an integral part of defense against drought in the transgenic plants expressing PtrABF.
To cope with unfavorable environmental constraints plants modulate the expression of a large spectrum of stress-responsive genes, constituting an important molecular basis for the response and adaptation of plants to stresses [22, 31, 32]. In order to understand regulatory function of PtrABF and to explain the enhanced drought tolerance at molecular levels, transcript levels of nine stress-responsive genes were monitored before and after drought treatment, including five genes encoding functional proteins (NtADC1, NtADC2, NtSAMDC, NtERD10C and NtLEA5) and four encoding regulatory proteins (NtAREB, NtCDPK2, NtDREB and NtERF), which or whose homologues in other plants have been shown to be involved in abiotic stress response. RT-PCR analysis showed that steady-state mRNA levels of these genes were higher in the transgenic plants compared with those of WT in the absence of water stress, in line with earlier reports in which overexpression of a TF resulted in extensive alteration of transcript levels of an arsenal of related genes [33, 34]. Although expression levels of all of the tested genes were upregulated by drought, they were still higher in the transgenic plants than in WT, indicating that these genes were more intensely induced in the transgenic lines. NtADC1, NtADC2 and NtSAMDC are genes involved in biosynthesis of polyamines, which are low-molecular-weight polycations and have been shown to be important stress molecules [35, 36]. Polyamines function in stress adaptation by acting as osmoticum regulator or membrane stabilizer through binding to macromolecules like proteins, nucleic acids and phospholipids of plasma membrane [37, 38]. More drastic induction of these genes implied that the transgenic plants might synthesize higher levels of polyamines to prevent them from lethal injury and maintain better growth under water stress . On the other hand, polyamines have been also proposed to act as free radical scavengers , and the larger induction of the polyamine biosynthetic genes agreed with lower ROS accumulation in the transgenic plants after drought stress. NtLEA5 and NtERD10C encode hydrophilic late embryogenesis abundant (LEA) proteins that are assumed to play critical roles in combating cellular dehydration . Higher expression levels of these genes suggested that the transgenic plants might provide more chaperones for various substrates and maintain membrane integrity or efficiently bind water, which are important strategies for plants to sustain growth during drought [41, 42]. Induction of these functional genes to higher levels suggested that the transgenic plants might synthesize more protective compounds (polyamines) or proteins (LEA), which, along with others that were not identified herein, provided better adaptive or defensive niches against water stress, leading to alleviation of cellular damage when they were subjected to drought. Upregulation of the above genes suggests that they might be transcriptionally regulated by PtrABF through binding of the cis-element ABRE in their promoter. Although we could not provide evidence for this speculation, ABRE has been discovered in the promoter of most genes encoding LEA proteins  and in AtADC2 and AtSAMDC2 of Arabidopsis . In addition to the functional genes, it is noted that the NtCDPK2 and NtDREB were also induced to higher levels in the transgenic lines with or without drought stress, while NtERF and NtAREB were only slightly induced upon water stress. Induction of their mRNA to higher levels raised the possibility of interaction between them and PtrABF in order to orchestrate well-defined stress tolerance machinery that functions in protection of the plants against adverse environment. Interestingly, the expression patterns of NtCDPK2 and NtDREB were largely consistent with those of the functional genes before and after water stress. NtCDPK2 and NtDREB are important regulatory molecules involved in signal transduction or transcriptional regulation during stress conditions [4, 5, 44, 45]. These genes may act as the intermediates between PtrABF and the aforementioned functional genes. In this case, PtrABF might function to facilitate transcriptional upregulation of these endogenous regulatory genes, which in turn activated their downstream target genes, including those mentioned above. In the future, extra work is needed to decipher the connection between these genes so as to gain more insight into the molecular mechanisms underlying PtrABF function in water stress tolerance.