Overexpression of BcHsfA1 transcription factor from Brassica campestris improved heat tolerance of transgenic tobacco

Heat shock proteins (HSPs) are a type of conserved molecular chaperone. They exist extensively in plants and greatly contribute to their survival under heat stress. The transcriptional regulation factor heat shock factor (HSF) is thought to regulate the expression of Hsps. In this study, a novel gene designated BcHsfA1 was cloned and characterized from Brassica campestris. Bioinformatic analysis implied that BcHsfA1 belongs to the HsfA gene family and is most closely related to HsfA1 from other plants. Constitutive overexpression of BcHsfA1 significantly improved heat tolerance of tobacco seedlings by affecting physiological and biochemical processes. Moreover, the chlorophyll content of transgenic tobacco plants was significantly increased compared with wild type after heat stress, as were the activities of the important enzymatic antioxidants superoxide dismutase and peroxidase. BcHsfA1 overexpression also resulted in decreased malondialdehyde content and comparative electrical conductivity and increased soluble sugar content in transgenic tobacco plants than wild-type plants exposed to heat stress. Furthermore, we identified 11 candidate heat response genes that were significantly up-regulated in the transgenic lines exposed to heat stress. Together, these results suggested that BcHsfA1 is effective in improving heat tolerance of tobacco seedlings, which may be useful in the development of new heat-resisitant B. campestris strains by genetic engineering.


Introduction
Changes in the natural environment such as heat, drought, and salinity can cause plant growth retardation and even death [1]. Plants have therefore evolved a series of defense responses to adverse environment, in which the accumulation of heat shock proteins (HSPs) plays an important role in sustaining homeostasis [2]. Heat shock transcription factors (HSFs) have been shown to regulate the expression of HSPs [3] in response to physical and chemical stress, and to co-adjusts other important signaling pathways [4]. PLOS  HSFs exist extensively in plants, with 21 Hsfs in Arabidopsis thaliana and 34 in soya. They play a critical role in cell homeostasis under various conditions [4], and can be divided into three categories: HsfA, HsfB and HsfC [4][5][6]. HsfA regulates Hsp expression following exposure to abiotic stress, especially heat [5].
Heat and oxidative stress both induce plants to produce HSPs, and the biosynthesis of these stress proteins can be considered an adaptive mechanism in which mitochondrial protection is essential [7]. Similarly, the chloroplast HSP protein protects the chloroplast against damage caused by oxidative stress and heat stress [8]. Heat stress was shown to induce an oxidative stress reaction in Salmonella typhimurium causing the up-regulation of many antioxidant enzyme genes [9], while the overexpression of catalase (CAT) and superoxide dismutase (SOD) genes increased thermotolerance in Saccharomyces cerevisiae [10]. Furthermore, the heat-shock cis element also contributed partially to the induction of the ascorbate peroxidase (APX) gene under oxidative stress [11].
As a traditional vegetable in many countries worldwide, plants of cabbage family are very sensitive to high temperatures [12], which can severely affect both quality and yield. Therefore, the development of new heat-tolerant cabbage varieties is warranted. In the present study, we cloned a gene designated BcHsfA1 from young seedlings of the traditional non-heading Chinese cabbage Brassica campestris 'Suzhouqing', which has comparatively strong heat tolerance [13]. We also identified its biological function. Our findings provide new insights into the breeding of novel cabbage varieties that show resistance to hot temperature by genetic engineering.

Plant materials
Seeds of B. campestris 'Suzhouqing' were germinated for 48 h in darkness at 25˚C in pots filled with a moist substrate of peat moss. After germination, the seedlings were incubated in a green house and regularly irrigated with tap water. Nicotiana tabacum seeds were sterilized and sown on Murashige and Skoog (MS) medium in vitro kept at 4˚C for 2 days before germination [14] at 25˚C. All the photoperiods were 16 h light /8 h dark.

DNA sequence analysis
Coding sequences were predicted using the opening reading frame (ORF) finder. The nucleotide sequence and deduced protein amino acid sequence were submitted to NCBI and the latter was analyzed with online ExPASy Proteomics Tools (http://au.expasy.org/tools/); multipe sequence alignments were performed using ClustalX software [15]. A phylogenetic tree was compiled using MEGA5.0 software combined with CLUSTAL W alignments [16].

Expression pattern of BcHsfA1 under heat stress
Heat stress was administered by exposing B. campestris seedlings to 42˚C for 0, 0.5, 1, 2, 3, 4 and 5h. Leaves were then harvested for RNA extraction using the TRIzol reagent (Invitrogen, Waltham, MA) and treated with DNaseI (Promega, Madison, WI) according to the manufacturers' protocols. Purified RNAs were used for real-time quantative (RT-q) PCR analysis. Quantification of gene expression was performed using the comparative Ct method [17][18]. Data represented the average of three independent experiments.

Construction of BcHsfA1 overexpression vectors
Single-strand cDNAs were synthesized from 5μg of total RNA with an oligo (dT) 17 primer through reverse transcription reactions according to the manufacturer's protocols (Power-Script TM , Clontech, Palo Alto, CA). PCR was performed using the primer pairs BcHsfA1-ORF F and BcHsfA1-ORF R (S1 Table). PCR products was inserted into the cloning vector pMD18-T (TaKaRa, Bio, Shiga, Japan) for sequencing as described before [19][20]. The vectors pMD18T-BcHsfA1 and pCAMBIA2300 + were double-digested with NcoI and BstE II. After purification, the gus gene from pCAMBIA2300 + was replaced by the BcHsfA1 coding region to construct the recombinant pCAMBIA2300 + -BcHsfA1 (S1 Fig). BcHsfA1 gene was regulated by the cauliflower mosaic virus (CMV) 35S promoter. The blank vector pCAMBIA2300 + lacking BcHsfA1 was used as a control. Each of the above-constructed plasmids and the disarmed Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA2300 + -BcHsfA1 plasmid were used for plant genetic transformation.

Plant transformation and PCR assay of transgenic plants
Tobacco transformation was carried out as previously described [21]. Transgenic plants were selected on 1/2 MS medium supplemented with 100 μM kanamycin [21][22]. Genomic DNA was extracted from seedlings of transgenic plants using the modified CTAB method [23]. Prime pairs BcHsfA1-35S-F23 and BcHsfA1-QR were used for PCR detection of the transgenic tobaccos (S1 Table).

Expression profile analysis of transgenic plants byRT-qPCR
Expression profiles of BcHsfA1 in the transgenic tobacco were analyzed by RT-qPCR using total RNA extracted from the leaves of transformants and wild-type tobacco plants [20,24]. The primer pair BcHsfA1-real-F/BcHsfA1-real-Rwas used to examine the transcription of BcHsfA1, and NtActin-real-F and NtActin-real-R amplified the reference gene (S1 Table).

Heat tolerance profile in transgenic tobacco
Transgenic tobaccos and wild-type (WT) seedlings grown in a greenhouse were exposed to 42˚C as described previously [25], then leaves were harvested at 0, 1, 2 and 3h to determine physiological and biochemical indexes. Leaves treated with heat stress for 3h were collected to detect the expression profiles of two antioxidative-related genes, SOD and peroxidase gene (POD), three heat stress defense genes, late embryogenis abundant protein 5 (LEA5), and early response to drought 10C and 10D (ERD10C and NtERD10D), and six heat stress marker genes (NtHSP17.6, NtHSP18.2, NtHSP70, NtHSP82, NtHSP90 and NtHSP101) using RT-qPCR as described above.
The amount of chlorophyll (Chl) was determined according to the method of Wang et al. [26]. Total soluble sugar was extracted from leaves by using 80% hot ethanol and the content was determined as reported by Irigoyen et al. [27][28]. MDA concentrations were measured by the thiobarbituric acid (TBA) reaction according to Wang et al. [29]. For the analysis of POD and SOD enzyme activities, the crude enzyme extract were prepared as reported by Zhang and Kirkham [30]. SOD and POD enzyme activity was measured as described previously [31][32]. Determination of the comparative electrical conductivity was conducted by the deflation method [33].

Statistical analysis
Each experiment was repeated three times, and standard deviation was reported. Statistical significance was analyzed by the one sample t test with SPSS software version 11.5 (SPSS Inc, Chicago, IL).

Cloning and sequence analysis of BcHsfA1
BcHsfA1 cDNA (1464bp) was cloned using the homologous primer pairs BcHsfA1-ORF F and BcHsfA1-ORF R. It encoded a protein of 487 amino acids, with a deduced molecular mass of 73.2 kDa and an isoelectric point of 5.54. BLAST search in the GenBank database showed that BcHsfA1 shared high homology sequences from B.napus and B.rapa.
Sequence domain prediction revealed that BcHsfA1 protein contained conserved domains including a DNA-bind domain, an oligomerization domain consisted of hydrophobic repeat regions A/B indispensable for oligomerization, a nuclear localization signal (NLS) recognized by the NLS receptor, an activator motif characterized by aromatic, hydrophobic and acidic amino acid residues associated with transcriptional activation and a leucine-rich nuclear exportsignal (NES) for receptor-mediated nuclear export in complex with the NES receptor [4][5][6] A phylogenetic tree was constructed based on the deduced amino acid sequences of BcHsfA1 and other HSF proteins. As showed in Fig 1, HSF proteins were divided into three groups: A, B and C types, and BcHsfA1 was classified into A family.

BcHsfA1 responses to heat stress
To determine the transcription characteristics of BcHsfA1 exposed to heat stress, seedlings of B. campestris were incubated at 42˚C for heat treatment with 0, 0.5, 1, 2, 3, 4 or 5 h. BcHsfA1 expression was rapidly triggered at 0.5h and peaked at 1 h with a 22.5-fold increase (Fig 2). The accumulation of BcHsfA1 then reduced sharply, and stabilized 4h later.

Overexpression of BcHsfA1 improved the growth of transgenic plants under heat stress
To study the biological function of BcHsfA1, pCAMBIA2300 + -BcHsfA1 was overexpressed in N.tabacum to produce T 0 transgenic tobacco plants. PCR analysis identified a clear band at 1464bp for transgenic but not non-transgenic plants ( S3 Fig). This suggested that BcHsfA1 had been successfully introduced into the transformants.
RT-qPCR analysis was conducted to determine the transcription of BcHsfA1 in transgenic plants. Total RNA was extracted from transgenic seedlings (line 1, 2, 3, 6, 7, 8 and 11) and was subjected to RT-qPCR using the transcription level of line 3 as a reference. BcHsfA1 exhibited a varied expression pattern in different transgenic plants (Fig 3). Line 11 showed the highest BcHsfA1 transcription, whereas line 3 showed the lowest. Line 1, 2, 6, 7 and 8 showed moderate BcHsfA1 transcription.
Three transgenetic tabocco lines (2, 7 and 11) showing high BcHsfA1 expression were selected for subsequent heat stress analysis. Under heat stress (42˚C), the transgenic tobacco lines showed more growth and less damage than WT (S4 Fig), suggesting that BcHsfA1 overexpression improve the heat tolerance of transgenic plants.

Overexpression of BcHsfA1 improved the Chl biosynthesis capacity in transgenic plants under heat stress
The plant Chl content is representative of its photosynthetic efficiency. In this study, total Chl, Chl a and Chl b decreased both in BcHsfA1 transgenic lines and WT under elongated heat stress (42˚C) from 0 to 3 h (Fig 4). However, BcHsfA1 transgenic lines showed higher contents of total Chl, Chl a and Chl b than the controls, and the extent of Chl decrease was slower than in WT.

Overexpression of BcHsfA1 increased antioxidative enzyme levels and alleviated cell damage under heat stress
WT plants showed higher foundation levels of MDA in their leaves than transformants at 0h under heat stress (Fig 5A). In general, the MDA content increased in leaves of both transgenic and WT plants following heat treatment. However, there was no significant difference between transformants and WT after 1h of treatment (P<0.05). After 2h of heat stress, transgenic line 7 and 11 showed significant lower MDA content than WT, and all three transgenic lines (2, 7 and 11) had significant lower MDA level than WT after 3h of heat stress (P<0.05). This implies that BcHsfA1 improved the tolerance to heat stress in transgenic tobacco plants.
The MDA content reduction in transgenic plants could be explained by the activation of antioxidant enzymes. Therefore, we next determined the activities of SOD and POD.    Transgenic plants showed significantly higher SOD activity than WT at both basal levels (0h) and under heat stress (1, 2, and 3h) (Fig 5B). SOD activity also increased from 0 to 2h heat treatment in both transgenic plants and WT, then declined. All three transformants (line 2, 7 and 11) showed significant higher POD activity than WT from 0 to 3h heat treatment ( Fig  5C), and the activity level increased in transformants and WT plants after heat stress although there was no significant difference among 1, 2 and 3h in transgenic plants.These results suggested that BcHsfA1 overexpression conferred higher SOD and POD activities to protect plants by reducing the lipid peroxidation.
The content of soluble sugar and comparative electrical conductivity are effective indicators of plant heat tolerance. We observed that soluble sugar contents increased significantly in leaves of both WT and transgenic plants from 0 to 3h of heat stress (42 o C) (Fig 6A). Prior to heat stress, only line 11 had a significantly higher soluble sugar content than WT. However,  after heat stress (1, 2, and 3h), the soluble sugar content was significantly higher in all transgenic lines than WT. As shown in Fig 6B, the comparative electrical conductivity also increased significantly from 0 to 3h heat treatment in both WT and transformants. Moreover, the comparative electrical conductivity of all transgenic lines (lines 2, 7 and 11) was significantly lower than that of WT both before (0h) and after heat stress (1, 2, and 3h), suggesting that less cell membrane damage occurred in transgenic tobacco plants than WT after heat stress.

Overexpression of BcHsfA1 upregulated the expression of heat stress responsive genes
Next, we used RT-qPCR to examine the expression of antioxidative-related genes including NtSOD and NtPOD. Heat stress was found to upregulate NtSOD and NtPOD expression in all tobacco plantstested, but the three transgenic tobacco lines 2, 7, and 11 showed significantly higher (P<0.05) NtSOD and NtPOD expression than WT (Fig 7A and 7B). These expression profiles were consistent with the enzyme activity of SOD and POD in transgenic tobacco lines.
We also measured the expression profiles of three heat stress defense genes (NtLEA5, NtERD10C, and NtERD10D), and six HSP genes (NtHSP17.6, NtHSP18.2, NtHSP70, NtHSP82, NtHSP90, and NtHSP101). Compared with controls, the expression of all genes was  significantly up-regulated in the three transgenic tobacco lines 2, 7, and 11 under heat stress (Fig 7C-7K), indicating that BcHsfA1 acts as a positive regulator in response to heat stress.

Discussion
In this study, we cloned and characterized a novel Hsf gene from B.campestris. Sequence analysis revealed that the predicted amino acid sequence of BcHsfA1 is very similar to that of other plant HsfA1s, in particular AtHsfA1. The deduced BcHsfA1 protein exhibited motifs and domains that are conserved with HsfA1s from other plants such as A. thaliana and B.napus [34][35], suggesting that those domains play important roles in maintaining the HSF function [36][37][38]. Additionally, BcHsfA1 contained a NLS at its C-terminal [36,[39][40], indicating that it might be located in the nucleus.
The analysis of BcHsfA1 transcription showed that it responded to heat stress, which is in accordance with earlier findings in other plants [41][42], and implied that BcHsfA1 may be involved in heat resistance of B. Campestris. Previous reports also suggested that HSFs could be induced under thermal or other abiotic stresses [43], while Hsf overexpression conferred tolerance to heat and other abiotic stresses in plants [3,37,42,44]. To understand the function of BcHsfA1, we overexpressed it in tobacco under the control of the 35S CMV promoter and measured various physiological and biochemical indexes.
Environmental stress such as heat and cold damage can affect Chl biosynthesis, resulting in a lower accumulation [45]; therefore, the Chl content can be used as a heat tolerance indicator. In this study, Chl a, Chl b and total Chl was significantly increased in transformants compared with WT after heat stress (Fig 4), suggesting that the higher Chl content might play a vital function in heat tolerance. These results were similar to those of other studiesin wheat, fine fescue and Arabidopsis [46][47][48].
Membranes are the site of primary physiological injury in the plant response to heat stress [49], and damage to membranes causes leakage which induced Chl spillage and degradation in the stoma. Hence, the extent of membrane damage can be evaluated by determining solute leakage [48, 50-51]. In the present study, electrolyte leakage was reduced in transgenic tobacco plants (Fig 6B), which was consistent with previous reports on sweet potato, yeast, Arabidopsis, and potato [52-54]. Moreover, the soluble sugar content was also significant higher in transformants than in WT. This could maintain the osmotic pressure of the cell to avoid excessive water loss under heat stress [55].
When plants were exposed to detrimental environmental conditions such as heat, cold, salt, or drought, reactive oxygen species (ROS) including superoxide (O 2 -), hydrogen peroxide (H 2 O 2 ) and the hydroxyl radical are produced and accumulate [45,56]. Plants ROS cytotoxicity occurs through enzyme deactivation, the breakage of important cellular components such as cell membranes through oxidative damage, and inhibition of photosynthesis [57]. To prevent this, ROS accumulation is alleviated by inducing activities of ROS-scavenging enzymes [58]. SOD is a key enzyme of the antioxidant defense system, forming the first defense level against superoxide radicals. SOD-catalyzed O 2 dismutation renders H 2 O 2 as a reaction product, which in turn is removed by POD, CAT and APX activities [58]. In this study, BcHsfA1overexpressing lines showed much higher SOD and POD activities when exposed to high temperatures ( Fig 5). Moreover, as an indicator of membrane peroxidation, the MDA content of transgenic tobacco was lower than that of WT. These increased SOD and POD activities of transformants reflect an active and effective antioxidant response that might be involved in maintaining a lower MDA content, therefore helping plants to cope with the heat stress [59-61].
In conclusion, this study indicated that BcHsfA1 plays an important role in improving the heat resistance of transgenic tobacco plants. These results will be useful in understanding how plants respond to environmental stresses, and how tolerance to these abiotic stresses can be enhanced by genetic manipulation.