Comparative Study On The Biological Functions Of DPBF3,DPBF4 And DPBF5 Members Of Arabidopsis ABI5 Subfamily

The Arabidopsis ABI5 subfamily(AtDPBFs/ABFs/At5G42910)is a class of basic leucine zipper-like(bZIP)transcription factors that are involved in multiple stages of plant growth and development,such as seed germination,timing of flowering,response to hormones such as ABA,and response to adversity such as drought,hypersalinity,and hyperosmolarity.Due to functional redundancy and regulated transcriptional activation activity,knockdown or overexpression of individual genes of the ABI5 subfamily is often not seen as a distinct phenotype.This poses an obstacle to a deep and accurate understanding of the function of individual genes in the ABI5 subfamily.Based on earlier work in our laboratory that CRII of AtDPBF4 has the strongest transcriptional activation activity and that the conserved phosphorylation region has a significant repressive effect on transcriptional activation activity.We directly compared the CRII fragment of AtDPBF4 with AtDPBF3 by removing the transcriptional repression region of each member We constructed the recombinant genes4DIID3 bZIP(recombinant AtDPBF3 gene),4IID4 bZIP(recombinant AtDPBF4 gene),and4IID5 bZIP(recombinant AtDPBF5 gene)by removing the transcriptional repressor region of each member and directly ligating the DNA binding domains of AtDPBF4,AtDPBF4,and AtDPBF5.These recombinant active transcription factor genes were further overexpressed in Arabidopsis as a way to understand the specific roles they play in the plant body.The main findings of this paper are as follows:1.Seed germination of Arabidopsis plants overexpressing recombinant genes 4DIID3 bZIP,4IID4 bZIP,and 4IID5 bZIP was significantly earlier: 59 %,73 %,and 82.5 % for wild type at48 h,54 h,and 72 h were counted.48 %,60 %,and 90 % for plants overexpressing recombinant gene 4IID3 bZIP.48 %,60 %,and 90 % for plants overexpressing recombinant gene 4IID4 bZIP plants were 89 %,92 %,and 92.4 %,respectively.and the germination rates of recombinant gene 4IID5 bZIP plants were 84.2 %,92.4 %,and 94.4 %,respectively.2.The number of rosette leaves in Arabidopsis thaliana plants overexpressing recombinant genes 4DIID3 bZIP,4IID4 bZIP and 4IID5 bZIP increased : the average number of rosette leaves in wild-type plants at 40 days was 10;the overexpression recombinant gene 4IID3 bZIP plants were 12;the overexpression recombinant gene 4IID4 bZIP plants were 14;the overexpression recombinant gene 4IID5 bZIP plant was 14.The rosette leaves of Arabidopsis plants overexpressing recombinant genes 4DIID3 bZIP,4IID4 bZIP,and 4IID5 bZIP were enlarged.The length and width of rosette leaves of wild-type and transgenic plants at 33 d of age were as follows: 0.95 cm long and 0.6 cm wide for wild-type rosettes.1.05 cm long and 0.74 cm wide for plants overexpressing the recombinant gene 4IID3 bZIP.1.1 cm long and 0.8 cm wide for plants overexpressing the recombinant gene 4IID4 bZIP.1.16 cm long and 0.91 cm wide for plants overexpressing the recombinant gene 4IID5 bZIP 0.97 cm wide.3.Arabidopsis plants overexpressing recombinant genes 4DIID3 bZIP,4IID4 bZIP,and4IID5 bZIP delayed threshing: the percentage of wild-type Arabidopsis thaliana plants overexpressing the recombinant gene 4DIID3 bZIP was 66.7 % at 27 days of age.the percentage of Arabidopsis thaliana plants overexpressing the recombinant gene 4DIID4 bZIP was 12 %.the percentage of Arabidopsis thaliana plants overexpressing the recombinant gene 4DIID4 bZIP was 25 %.the percentage of Arabidopsis thaliana plants overexpressing the recombinant gene4DIID5 bZIP was 8 %.Increased number of shoots in Arabidopsis plants overexpressing recombinant genes 4DIID3 bZIP,4IID4 bZIP and 4IID5 bZIP: the number of shoots in 40-dayold wild-type Arabidopsis plants ranged from 1-2 shoots;the number of shoots in overexpression recombinant 4IID3 bZIP plants ranged from 1-5 shoots,the number of shoots in overexpression recombinant 4IID4 bZIP plants ranged from 1-6 shoots.the number of shoots in overexpression recombinant 4IID5 bZIP plants ranged from 1-6 shoots.4.Increase in plant height and angular fruit number of Arabidopsis plants overexpressing recombinant gene 4IID4 bZIP and 4IID5 bZIP: the average plant height was 25 cm for eightweek-old wild-type plants,30 cm for recombinant 4IID4 bZIP plants and 27.8 cm for recombinant 4IID5 bZIP plants.the average number of horn fruits per plant was 10 for eightweek-old wild-type plants,17 for recombinant 4IID4 bZIP plants,and 17 for recombinant4IID5 bZIP plants.The average number of horn fruits per plant was 10 for the eight-week-old wild-type plants,17 for the overexpression of recombinant gene 4IID4 bZIP plants and 12 for the overexpression of recombinant gene 4IID5 bZIP plants.5.Arabidopsis plants overexpressing recombinant genes 4DIID3 bZIP,4IID4 bZIP and4IID5 bZIP were more sensitive to sucrose: the root length of wild-type plants was inhibited by51.6 %.the root length of plants overexpressing the recombinant gene 4IID3 bZIP was inhibited by 62.7 %.the root length of plants overexpressing the recombinant gene 4IID4 bZIP was inhibited by 58.9 %.the root length of plants overexpressing the recombinant gene 4IID5 bZIP was inhibited by 61.67 %.The inhibitory effect of ABA on root length development of Arabidopsis plants overexpressing recombinant genes 4DIID3 bZIP,4IID4 bZIP,and4IID5 bZIP was reduced: 97.45 % of root length was inhibited in wild-type plants by 2.5 μM ABA stress for 14 d;91.5 % of root length was inhibited in plants overexpressing recombinant gene 4IID3 bZIP;90 % of root length was inhibited in plants overexpressing recombinant gene4IID4 bZIP;and 90 % of root length was inhibited in plants overexpressing recombinant gene4IID5 bZIP.4IID4 bZIP plants were suppressed by 93.7 %;overexpression of recombinant gene4IID5 bZIP plants were suppressed by 90 %.6.The drought resistance of Arabidopsis plants overexpressing recombinant genes4DIID3 bZIP,4IID4 bZIP and 4IID5 bZIP was significantly enhanced: the leaves of wild-type and overexpression Arabidopsis seedlings were dried up after 12 days of drought treatment,and the leaves of Arabidopsis plants overexpressing recombinant genes 4DIID3 bZIP,4IID4 bZIP,and 4IID5 bZIP were green and wilted.After 5 days of rehydration,the leaves of wild-type seedlings dried up and died,and the overexpression lines resumed normal growth.Arabidopsis plants overexpressing recombinant genes 4IID3 bZIP,4IID4 bZIP,and 4IID5 bZIP had significantly increased salt tolerance:When three-week-old wild-type and transgenic plants were stressed with 50 m L of 200 m M Na Cl solution for 14 days,the survival rate was 16 % for wild-type plants,90.6 % for overexpression of recombinant 4IID3 bZIP plants,93 % for overexpression of recombinant 4IID4 bZIP plants,and 97.5 % for overexpression of recombinant 4IID5 bZIP plants.7.The m RNA content of 9 genes in ABI5 subfamily of wild type and transgenic plants A.thaliana D3,A.thaliana D4,A.thaliana D5 under different concentrations of Na Cl,sorbitol,sucrose and ABA stress : The m RNA content of 8 members of ABI5 subfamily except ABI5 in A.thaliana D3 was significantly different under four stress treatments.The m RNA content of 9members of ABI5 subfamily in A.thaliana D4 was significantly different under four stress treatments.The m RNA content of 8 members of ABI5 subfamily except ABF4 was significantly different under four stress treatments.8.Transcriptomic analysis of wild-type and transgenic plants A.thaliana D3,A.thaliana D4 and A.thaliana D5: A total of 917 differential genes were screened in the comparison between wild-type plants and transgenic plants A.thaliana D3,of which 577 were up-regulated and 340 were down-regulated.a total of 290 differential genes were screened in the comparison between wild-type plants and transgenic plants A.thaliana D4,of which 112 were up-regulated and 178 were down-regulated.wild-type plants and transgenic plants A.thaliana D5 were screened in the comparison between wild-type plants and transgenic plants A.thaliana D5.A total of 854 differential genes were screened in the comparison between wild-type and transgenic A.thaliana D5,of which 272 were up-regulated and 582 were down-regulated.