Role of Soybean GmbZIP132 under Abscisic Acid and Salt Stresses

Identifying and cloning of GmbZIP132 from soybean (Glycine max)

Using the Arabidopsis bZIP proteins as queries, 130 bZIP genes were identified by searching the 56 147 soybean unigenes identified in our previous study (Tian et al. 2004). The expression patterns of these genes under drought, cold, salt, and exogenous ABA treatments were examined using reverse transcription-polymerase chain reaction (RT-PCR). Several bZIP genes inducible by various stress treatments were cloned. Of particular interest was one gene named GmbZIP132. Database Basic Local Alignment Search Tool (BLAST) analysis identified several homologous cDNA sequences sharing 20.0% to 73.9% amino acid identity with GmbZIP132 (Figure 1). Among them are three homologous Arabidopsis bZIP sequences (AtbZIP20, AtbZIP45 and AtbZIP47). They belong to the group D bZIP transcription factors, which participate in two different processes: defense against pathogens, and development (Jakoby et al. 2002). GmbZIP132 contains two protein domains: PD388003 and PD004114, as defined by ProDom (Servant et al. 2002). Protein domain PD388003 has been annotated as a tumor-related protein-like domain because it was found in the protein sequence (accession number BAA05470) from tobacco hybrid Nicotiana glauca×Nicotiana langsdorffii, which shows tumorous cell growth. PD004114 is known as DNA-binding nuclear transcription factor bZIP activator coiled coil regulation leucine domain, which is present in the group D bZIP transcription factors.

The expression of GmbZIP132 was monitored by RT-PCR. Its expression was strongly induced by salt and drought treatments, and weakly induced by cold. It was slightly induced for the first 3 h of ABA treatment and came back to the pre-treatment level (Figure 2A). Expression of GmbZIP132 was also investigated in different soybean organs including cotyledons, roots, stems, leaves and flowers. The results showed that the GmbZIP132 mRNA accumulated in an organ-specific manner with the strongest expression in cotyledons and stems, comparatively lower expression in roots and flowers, and barely detectable expression in leaves (Figure 2B).

DNA-binding specificity of GmbZIP132 protein

One of the characteristics of bZIP proteins is that they can bind ABRE or G-box (Jakoby et al. 2002). Therefore, the binding ability of GmbZIP132 protein was examined by analyzing its binding ability to three known bZIP binding elements in the yeast one-hybrid assay system (BD Clontech, Mountain View, CA, USA). The GCN4-like motif (GLM; 5′-GTGAGTCAT-3′) (Onate et al. 1999), ABA responsive elements (ABRE; 5′-CCACGTGG-3′) (Jakoby et al. 2002) and the prolamin box (PB; 5′-TGAAAA-3′) (Onate et al. 1999), and their corresponding mutant sequences mGLM (5′-GTGAAAAAT-3′), mABRE (5′-CCAAAAGG-3′) and mPB (5′-TGACGT-3′), were synthesized and inserted into the plasmid pHIS2 containing the reporter gene HIS3. A minimal promoter was present downstream of the cis-elements but upstream of the HIS3 gene. Reporter pHIS2 plasmids harboring different elements plus the reporter gene HIS3, together with the effector plasmid pAD-GmbZIP132 were transformed into yeast cells. Growth of the transformants on 3-AT medium indicates the activation of the HIS3 gene and binding of the GmbZIP proteins to the corresponding cis-elements. Figure 3 shows that all transformants grew well on the yeast extract-peptone-dextrose plus adenine (YPAD) medium. But only transformants harboring the effector plasmids pAD-GmbZIP132 and the reporter plasmid containing the GLM, grew well on the 3-AT medium, indicating that this protein could bind to the sequence GLM; a GCN4-like motif.

Functional analysis of transgenic Arabidopsis plants overexpressing GmbZIP132 under ABA, salt, and freezing treatments

The expression of GmbZIP132 under salt, drought, cold and ABA treatments suggested that it might be involved in the tolerance of plants to abiotic stresses. To characterize the function of GmbZIP132 in planta, GmbZIP132 in the sense orientation was inserted into a binary vector pBIN438 under the control of the 2 × CaMV 35S promoters and transformed into Arabidopsis using the vacuum infiltration method. Two homozygous transgenic lines with high target gene expression (Figure 4A) were used for further analysis. While growing on Murashige and Skoog (MS) agar plates or in the soil, neither transgenic lines showed a meiotically stable abnormal phenotype compared with wild-type plants grown under identical conditions (data not shown). Therefore we examined the performance of these two transgenic lines under various abiotic stresses.

Since ABA plays an important role in abiotic stresses and the expression of GmbZIP132 was weakly induced by exogenous ABA treatment, the ABA sensitivity of transgenic plants overexpressing GmbZIP132 was first examined. The seeds of all of the transgenic plants and wild type (WT) plants were germinated in the medium containing various concentrations of ABA. Figure 4B shows that the germination rates in transgenic plants were significantly higher than that in WT plants under the concentrations above 0.5 μM ABA. The water loss rates of both transgenic lines were also higher than those of the WT plants (Figure 4C). Three-day-old seedlings after germination were transferred to MS medium containing different concentrations of ABA. The root lengths were measured after 7 d, and the root lengths of transgenic plants were longer than those of WT under the concentrations of 1 and 5 μM ABA (Figure 4D). These results implied that GmbZIP132 might reduce ABA sensitivity of transgenic plants.

Next, the salt, drought and freezing tolerances of the transgenic plants were examined. The seeds of all of the transgenic and WT plants were germinated in the medium containing various concentrations of NaCl. Figure 5A shows that the germination rates were significantly higher in transgenic plants than in WT plants under the concentrations over 100 mM NaCl. Two-week-old seedlings from the MS medium were transferred to soil, and treated with increasing concentrations of NaCl or high salt by sitting pots in 600 mM NaCl. No differences in average plant height (Figure 5B) and survival rate (Figure 5C) were observed between transgenic and WT plants. No significant difference between transgenic and WT plants in tolerance of transgenic plants to drought treatment was observed either (data not shown). These results implied that GmbZIP132 might improve salt tolerance of transgenic plants at the germination stage, but not at the seedling stage.

Many studies have shown that accumulation of proline in plants can improve salt and freezing tolerance of plants (Khedr et al. 2003; Vannini et al. 2004). We thus measured the proline contents in 16-day-old WT and transgenic plants under normal conditions. Figure 6 showed that proline accumulation of GmbZIP132 transgenic plants was higher than that of WT plants.

Finally, some genes related to abiotic stresses were examined to see whether they were regulated by GmbZIP132 in transgenic plants. As shown in Figure 7, though expressions of rab18, COR15a and RD28 were not changed, expressions of rd29B, ICK1, DREB2A, RD17, P5CS, and ERD10 were upregulated in the transgenic plants. This result indicated that GmbZIP132 could regulate or interact with these abiotic-related genes.

Growth of soybean seedlings and various treatments

Soybean (Glycine max L. Merr.) plants were grown on vermiculite under a photoperiod of 16 h/8 h (light/dark) at 25 °C for 2 weeks and subjected to stress treatments. For drought treatment, plants were dehydrated on Whatman No. 3MM paper at 25 °C and with a RH (relative humidity) of 70% for various times. For ABA and high salt treatment, plants were immersed with their roots in the solution of 100 μM ABA and 150 mM NaCl for various periods of time. Cold treatment was carried out under dim light by continuously exposing plants to 4 °C for different times. After treatments, plant leaves were harvested, frozen in liquid nitrogen, and stored at −80 °C for RNA isolation. Various organs from mature soybean plants were also collected for RNA isolation.

RNA isolation and RT-PCR analysis

RNA isolation from soybean plants and transgenic Arabidopsis seedlings was carried out as described (Zhang et al. 1995). Total RNA (5 μg) from each sample was digested with DNase I (Promega, Madison, WI, USA) to get rid of any DNA contamination and then used to synthesize the first-strand cDNA using the cDNA synthesis kit according to the manufacturer's instructions (Promega). One μL of the cDNA mix was used as a template in a 25 μL PCR reaction volume to examine expressions of genes. The PCR condition was 94 °C, 3 min and 30 cycles of 94 °C, 30 s; 56 °C, 1 min; 72 °C, 1 min, with a final extension of 10 min at 72 °C. PCR products were separated on 1% agarose gel containing ethidium bromide and were photographed. Tubulin gene was amplified as a sample loading control. The asparagine synthetase gene from soybean, whose transcript was dramatically induced by salinity, osmotic stress and exogenous ABA, was amplified as a positive control (Hughes et al. 1997; Wang et al. 2005).

Soybean bZIP132 gene cloning

Soybean GmbZIP132 was cloned by RT-PCR from soybean cDNAs using gene specific primers: 5′-CGCGGATCCAGCAACAATGAACAACCCTA-3′; and 5′-CGGGGTACCGCAGCCTAATAGCGAAGGAC-3′. PCR products were cloned into pMD18-T vector (TaKaRa, Otsu, Shiga, Japan) and confirmed by sequencing.

DNA binding ability of the GmbZIP132 by yeast one-hybrid system

Three copies of each cis-element (GLM, mGLM, ABRE, mABRE, PB or mPB) were synthesized (Sengon, Shanghai, China) and annealed. The annealed double-strand including SacI and MluI sites at the two ends were double-digested with restriction enzymes SacI and MluI, and cloned into the pHIS2 reporter plasmid containing the nutritional reporter gene HIS3. The sequence of the insert was confirmed by sequencing. A minimal promoter was present downstream of the cis-elements but upstream of the HIS3 gene.

The coding region of GmbZIP132 was cloned into the DNA-activation domain vector pAD to generate the pAD-GmbZIP132 construct. The pAD-GmbZIP132 was used as effector plasmids. Transformation and screening analyses were carried out according to the manufacturer's instruction (BD Clontech). Both the effector plasmids and the reporter plasmids were transformed into yeast cells (Y187) and the transformants were selected on SD/-Trp-Leu. The transformed yeast cells were dropped onto SD/-Trp-Leu-His plus 10 or 30 mM 3-AT to examine the cell growth. For negative control, pAD-GmbZIP132 and pHIS2 without cis-elements were co-transformed into yeast cells (Y187) and the growth of the transformants was examined on SD/-Trp-Leu-His plus 10 or 30 mM 3-AT medium. The sequence of each original and mutant element was: GLM (5′-GTGAGTCAT-3′) (Onate et al. 1999), ABRE (5′-CCACGTGG-3′) (Jakoby et al. 2002), PB (5′-TGAAAA-3′) (Onate et al. 1999), mGLM (5′-GTGAAAAAT-3′), mABRE (5′-CCAAAAAGG-3′) and mPB (5′-TGACGT-3′).

Generation of the transgenic Arabidopsis plants

The coding region of the GmbZIP132 was cloned into the BamHI and KpnI sites of the plant expression vector pBin438 and confirmed by sequencing. The gene was controlled by the 35S promoter plus a Ω translation enhancer. The resulting plasmids were transformed into Agrobacterium tumefaciens strain GV3101 and further transformed into Arabidopsis plants by the vacuum infiltration method. Homozygous transgenic lines expressing higher GmbZIP132 gene were used for further analysis. Primers used for plasmid construction were: 5′-CGCGGATCCAGCAACAATGAACAACCCTA-3′; and 5′-CGGGGTACCGCAGCCTAATAGCGAAGGAC-3′.

Stress treatments of the transgenic Arabidopsis plants

Seeds (>100) of the transgenic Arabidopsis plants were planted in triplicate on filter papers saturated with different concentrations of ABA or NaCl, and incubated at 4 °C for 4 d before being placed at 22 °C for germination under a photoperiod of 16 h/8 h (light/dark). Germination (emergence of radicals) was scored 3 d later. The experiment was repeated three times independently. Results from one set of experiments are shown.

For NaCl treatment in soil, 14-day-old seedlings of WT and transgenic seedlings from MS agar medium were moved into potted soil for additional growth of 7 d. Then the plants were watered with 100 mM, 150 mM, and 200 mM NaCl, and lasted for 4 d. The inflorescence height was measured after the treatments. The 14-day-old seedlings were also grown in pots for 7 d and then treated with high salt by placing the pots in a tray containing 600 mM NaCl. After 14 d, the survival of the plants was examined. The experiments were repeated independently three times. Results from one set of experiments are shown.

For freezing tolerance assay, 14-day-old WT and transgenic seedlings from the MS agar medium were grown in potted soil for 7 d, and the plants were subjected to −6 °C for 2 d. After that, the plants were placed under normal conditions. The survival of the seedlings was scored after 7 d. The experiments were repeated independently three times. Results from one set of experiments are shown.

To measure the root length under ABA treatment, 3-day-old seedlings after germination were transferred into the plates containing MS medium with various concentrations of ABA, and then the plates were vertically placed under normal conditions. After 7 d, the root length was measured. Each experiment was repeated three times.

Water loss measurements

For water loss measurements, leaves were detached from plants at the rosette stage and weighed immediately on a weighed paper. The leaves, together with the paper, were then placed at 22 °C, under continuous light and with an RH of 50%. The weight was measured at designated time intervals. There were three replicates for each transgenic line. The percentage loss of fresh weight was calculated based on the initial weight of the plants. The experiment was carried out three times independently. Results from one experiment are shown.

Determination of proline contents

Free proline content in plants was determined following the methods of Bates et al. (1973). Briefly, 2-week-old seedlings (100 mg) were homogenized in 1 mL sulfosalicylic acid (3%) using a mortar and the mixture was mixed and centrifuged at 17 950g for 15 min at 4 °C. The extract (200 μL) was transferred to a new eppendorf tube, and 200 μL of acid ninhydrin and 200 μL of acetic acid were added. The reaction mixture was boiled in a water bath for 1 h, and placed at 4 °C for 30 min. Then 800 μL of toluene was added to the mixture. The tube was vortexed for 15 s and then 700 μL of the toluene phase was taken for the measurement of absorbance at 520 nm on spectrophotometer. Each data point represents the average of three plant samples. Two independent experiments were carried out. Results from one experiment are shown.

Statistic analysis

The data were subjected to statistic analysis, and analysis of variance was carried out using the SPSS 12.0 program. The bars indicate SD. * indicates that the differences between the transgenic line and the Col-0 plants are significant (P < 0.05). ** indicates that the differences between the transgenic line and the Col-0 plants are highly significant (P < 0.01).