BAG2 and BAG6 expression during Arabidopsis development
To determine expression patterns of the BAG2 and BAG6 genes, we generated Arabidopsis transgenic lines expressing the GUS reporter gene under control of the promoter fragments of the BAG2 and BAG6 genes, respectively (ProBAG2:GUS and ProBAG6:GUS) and analyzed the expression of the GUS reporter gene in various tissues. Strong GUS staining was observed in most tissues throughout the ProBAG2:GUS and ProBAG6:GUS transgenic plants (Fig. 1 A-T). In 7-day-old seedlings, ProBAG2:GUS activity is strongly detected in cotyledons, hypocotyl, and root vascular tissues, with the strongest signal at the lower part of the hypocotyl (Fig. 1A and B), while ProBAG6:GUS expression is strongly detected in all of the tissues of the whole seedling (Fig. 1 K and L). In 2-week-old plants and bolting plants, both ProBAG2:GUS and ProBAG6:GUS are highly expressed in rosette leaves and cauline leaves (Fig. 1 C-E and M-O), with ProBAG6:GUS showing additional GUS activity in stems (Fig. 1P). In flowers, both ProBAG2:GUS and ProBAG6:GUS are expressed in sepals, anther filaments, pollen grains, and style (Fig. 1 F-H and Q-R), with ProBAG6:GUS showing additional expression in ovules (Fig. 1R). In siliques, both ProBAG2:GUS and ProBAG6:GUS are expressed in young siliques with ProBAG6:GUS showing stronger GUS signals (Fig. 1I and S), while in old siliques both genes expression are very weak (Fig. 1J and T).
BAG2 and BAG6 expression response to abiotic stress and plant hormones
We further investigated whether BAG2 and BAG6 expression is regulated by abiotic stressors or plant hormones. The results show that ProBAG2:GUS expression is strongly increased by mannitol, salt (NaCl), heat and ABA treatments (Fig. 2A-B; Fig. S2 A) and its expression is also slightly increased in response to other stressors and hormones such as PEG, SA and ACC, respectively. Similarly, ProBAG6:GUS showed significant enhanced GUS staining in response to JA, ABA, mannitol, salt, and heat treatment, while showed slight increase in response to ACC, PEG and SA (Fig. 2C-D; Fig. S2 B-D). These results are similar to a previous report that had analyzed AtBAG genes response to abiotic stressors such as cold, heat, mannitol, and salt and hormones such as ABA, ACC, MeJA and SA by reverse transcription quantitative PCR (RT-qPCR) [1]. Together, these results suggest that AtBAG2 and AtBAG6 genes are involved in plants response to environmental stress.
Loss-of-function of BAG2 and BAG6 in Arabidopsis improves tolerance to ABA and drought Stresses
To explore the function of these genes, transfer DNA (T-DNA) insertion mutants were isolated for BAG2 and BAG6 within the fourth exon (SALK_030285, bag2; Fig. 3A) and within the first exon (SALK_004760, bag6; Fig. 3C), respectively. These mutants were crossed to generate the bag2 bag6 double mutant. The bag2 and bag6 mutants had been reported previously [6,12]. RT-PCR analyses revealed that weak BAG2 transcript was detected in the homozygous bag2 mutant (Fig. 3B; Fig. S3A-B) and no BAG6 transcript was detectable in the homozygous bag6 mutant (Fig. 3D; Fig. S3C-D), indicating that the bag2 mutant is a knockdown allele and the bag6 mutant is a null allele. Under normal growth conditions, the bag2 and bag6 single and bag2 bag6 double mutants exhibited slightly larger rosette size than the WT (Fig. S4).
To study the response of bag2 and bag6 single and bag2 bag6 double mutants to the plant stress hormone abscisic acid (ABA), these mutants and wild type (WT) seeds were sown on Murashige and Skoog (MS) medium supplemented with different concentrations of ABA to compare the germination and greening rate between WT and mutants lines. Without ABA, the WT and mutants showed similar germination rate and greening rate, but when ABA was applied, higher germination and greening rate were observed in the bag2 and bag6 single and bag2 bag6 double mutants (Fig. 4). When compared germination and greening rate on 0.75 μM ABA, we found a significant difference i-e WT germination rate (72%) and greening rate (46%) while bag2 75% and 84%, bag6 79% and 88% and bag2 bag6 81% and 91%, respectively (Fig. 4B-C). Similarly, when compared on 1 μM ABA, WT exhibited 57% seeds germinated and after 7 days 29% greening rate were found but bag2 single mutant exhibited 65% germination while 49% greening rate were observed, bag6 also showed more germination and greening rate than WT (i-e 68% and 55%, respectively) (Fig. 4B-C). Interestingly bag2 bag6 double mutant germination rate was more than each single mutant and WT which is 75% and 71%, respectively (Fig. 4B-C). Finally, when compared on 2 μM ABA, WT exhibited 37% germination and 24% greening rate, while bag2 exhibited 50% and 36%, bag6 55% and 36% and bag2 bag6 62% and 49%, respectively (Fig. 4B-C). Thus these mutants appeared to enhance the ABA tolerance of Arabidopsis during germination and the early vegetative growth period.
To study drought stress tolerance, 21-day-old WT and mutants plants were used, and 9 plants per pot were sown and 12 pots were prepared for each genotype (108 plants in total for each genotype). After withholding water for 14 days, almost all the WT and mutants plants were wilted and near to die (Fig. 5A). Then the plants were re-watered for three days. On third day a significant difference were noticed between WT and mutants plants (Fig. 5A); bag2 showed 43%, bag6 showed 44%, bag2 bag6 showed highest survival rate i-e 73% while WT showed less survival rate i-e 29 % (Fig. 5B). To further verify these results, we performed water loss experiment with different time interval. Consistently, the water loss in mutants plants rosette leaves was slower than that of the WT (Fig. 5C). These results suggest that BAG2 and BAG6 play a negative role in Arabidopsis response to ABA and drought treatments.
Loss-of-function of BAG2 and BAG6 in Arabidopsis compromises tolerance to heat stress
Our ProBAG2:GUS and ProBAG6:GUS expression results and previously reported RT-qPCR results showed that BAG2 and BAG6 genes were upregulated during heat stress [1,13]. So here in this study we have studied the effect of heat stress on bag2 and bag6 single and bag2 bag6 double mutants. The results showed that the bag2 and bag6 single and bag2 bag6 double mutants are sensitive to heat stress when compared to WT seedlings (Fig. 6). The survival rates of these mutants were one third less than WT (Figs. 6B). Also we measured chlorophyll content in mutants and WT on control untreated seedlings (22 ℃) and 45 ℃ heat treated seedlings. Significant difference was observed (Figs. 6C). Total chlorophyll levels decreased as consequence of the heat treatment both in WT and mutants, but the mutants retained less chlorophyll contents (Fig. 6C). These results suggest that BAG2 and BAG6 play a positive role in Arabidopsis response to heat stress.
Stress related genes are upregulated in the bag2 and bag6 mutants
To further investigate the role of BAG2 and BAG6 in plants response to ABA, heat and drought treatment, we performed RT-qPCR analysis on several stress-related genes such as RD29A, RD29B (stress related), and NCED3 (ABA biosynthesis genes). We found that the expression of RD29A and RD29B genes was upregulated in bag2 and bag6 single mutants and bag2 bag6 double mutant compared to WT when treated with ABA (Fig. 7A-B), while expression of the ABA biosynthesis gene NCED3 was slightly upregulated in these mutants compared to WT (Fig. 7C). These results further support the heat, ABA and drought phenotypic data.
ROS accumulations in WT and mutant plants
The accumulation of H2O2 and superoxide anion (O2-) was detected using 3,3-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining in seedling shoots and roots. Upon 20 μM ABA treatment, the cotyledon and root of the mutant plants displayed lighter color than that of the WT, which clearly indicates lower accumulation of O2- and H2O2, respectively in the mutant seedlings (Fig. 8A-B), whereas upon heat stress treatment, the mutants exhibited higher accumulation of O2- than the WT (Fig. 8C). Finally DAB staining was also applied to drought stressed rosette leaves of WT and mutant plants. We got the same results that leaves of mutant lines accumulated lower H2O2 than that of the WT (Fig. S5). Combined these results indicate that mutant lines had a lower accumulation of ROS when treated with ABA and drought while higher accumulation when treated with heat stress. These results corresponded well with the respective drought- and heat-tolerance phenotypes of these mutants.