Arabidopsis AREB1 and its modified form AREB1-M8 and AREB1-∆QT can activate in rice plant cell
To see whether Arabidopsis AREB1 can also be functionally in rice plant cell, triensient transactivation assays using rice mesophyll protoplast. The effector plasmids carrying full AREB1 or its modified forms AREB1-M8 or AREB1--∆QT were cotransfected into rice protoplasts, with a reporter plasmid, RD29B-GUS, carrying a GUS reporter gene fused to five tandem copies of a 77-bp fragment of the RD29B promoter containing two ABRE motifs (Fig. 1A, B). The experiment was performed in the presence or absence of ABA. In the absence of ABA, GUS activity of full length AREB1 slightly increased higher compared with control (empty plasmid).
However, in the presence of 50 µM ABA, GUS activity of AREB1 greatly enhanced (Fig.1C), suggesting AREB1 expression is dependent on ABA in rice. However, the GUS activity of AREB1-M8 and AREB1-∆QT increased without ABA. Furthermore, addition of ABA did not significantly change the GUS activity in both constructs (Fig.1C) indicating that AREB1-M8 and AREB-∆QT can express independently of ABA. Together, these results suggest that AREB1 acts as transcriptional activator [8] and that Arabidopsis AREB1 and its derivatives can be functionally in rice plant cell.
Selection of transgenic rice plants overexpressing AREB1, AREB1-M8, or AREB1-∆QT Since AREB1 and its modified forms can activate in rice plant cell, transgenic plants were generated by transforming the respective construct into rice tissue under the control by ubiquitous promoter. The primary screening by northern blot revealed that there are 2 lines for each construct that strong expression and the six lines were selected and assigned as Ubi: AREB1#13 and Ubi:AREB1#14 for AREB1; Ubi:AREB1-M8#10 and Ubi:AREB1-M8#15 for AREB1-M8, and AREB1--∆QT#2 and AREB1--∆QT#3 for AREB1-∆QT constructs (Fig.2A). At two-week old seedling stage, transgenic plants overexpressing AREB1 showed similar morphology with compared with control wild type (WT) whereas transgenic plants carrying AREB1-M8 or AREB1-∆QT exhibited smaller than WT in size (Fig.2B). At two-month old, transgenic plants overexpressing AREB1 or AREB1-M8 showed similar plant height. In contrast, plants overexpressing AREB1-∆QT produced very few tiller numbers and therefore they grow slower than control WT (Fig.2C).
Transgenic plants overexpressing AREB1, AREB1-M8 or AREB1-∆QT displayed enhanced drought tolerance at both seedling and mature stages
To evaluate drought stress tolerance in the selective transgenic plants, two independent lines of homozygous lines of T3 generation were used for experiment. At seedling stage, the overexpression lines showed increased and decreased drought resistance, respectively, compared with WT (Fig. 3). When 2 week old seedlings dried for 3.5 hrs in room temperature, the surviving rate of all transgenic lines was over 60% compared with WT just about 20% (Fig.3A,B). Further treatment of dry condition from 3.5 to 4 hrs reduced the surviving rate of transgenic plants whereas no surviving plant was observed in WT plant (Fig.3C,D). All three types of transgenic plants were also resistant to drought at the mature stage (data not shown). These results suggest that Arabidopsis AREB1 and its derivatives function in drought stress tolerance in rice.
0 10 20 30 40 50 60
_ABA ABA
Relative activity (GUS/LUC)
C
Figure 1 Arabidopsis AREB1, AREB1-M8 and AREB1-∆QT can activate in rice protoplast
(A) Scheme of the effector and reporter constructs used in the transactivation analysis with AREB1, AREB1-M8, or AREB1-∆QT. The effector constructs contain the CaMV 35S promoter and TMV V sequence fused to AREB1, AREB1-M8, or AREB1-∆QT cDNA fragments. The reporter construct, RD29B-GUS, contains 77-bp fragments of the RD29B promoter connected tandemly five times. The promoters were fused to the -51 RD29B minimal TATA promoter–GUS construct. Nos-T, nopaline synthase terminator.
(B) Schematic diagram of the effector and reporter constructs used in the transactivation analysis. S D indicates Serine substituted with Aspartic acid.
(C) Transactivation analysis of AREB1, AREB1-M8, and AREB1-∆QT. Protoplasts were cotransfected with the RD29B-GUS reporter. To normalize for transfection efficiency, the pBI35SV-LUC reporter was cotransfected as a control in each experiment. Bars indicate standard deviation of three replicates. ‘‘Relative activity’’ indicates the multiples of expression compared with the value obtained with the pBI221-35SV vector control.
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Discussion
Arabidopsis AREB1 and its derivatives are functional in rice plants. Transient gene expression in rice mesophyll protoplast revealed that AREB1 expression is ABA-dependent. This supports the notion that expression of the intact AREB1 gene alone is insufficient to upregulate its downstream genes under normal growth conditions [8]. Furthermore, AREB1 is required to activate expression of ABRE-dependent downstream genes [7] and AREB1 expression is induced by drought and high salt[8]. On the basis of recent reports that AREB1and its homologs are phosphorylated in vitro or in vivo [7]; [23];[24], phosphorylation of AREB1 may be involved in the modification.
Rice plants expressing AREB1, AREB1-M8 or AREB1--∆QT led to enhanced drought tolerance at both seedling and mature stages . The increased adaptation to dehydration condition may be explained by some reasons, for example, AREB1 synthesizes ABA contents in rice plants which leads to a rapid closure of stomata and therefore reducing water loss during stress occurring.
Another reason to explain the drought tolerance of transgenic plants carrying AREB1 is that AREB1can activate downstream target genes such as RD29B by binding to the promoter region which contains ABRE motif [7].
Rice plant overexpressing AREB1-M8 showed drought tolerance. In Arabidopsis, transgenic plants carrying this gene has less electrolyte leakage compared with normal plant [9]. Electrolyte-leakage analysis is a sensitive measure of loss-of-membrane integrity and is commonly used to assay osmotic injury [25]. On the other hand, transgenic plants overexpressing AREB1-M8 express many ABA-inducible genes such as RD29B without ABA treatment [9].
A B
C
Figure 2 Selection of transgenic rice plants
(A) RNA was extracted from 2 week-old seedlings grown in water culture solution. Nine microgram RNA was used for northern blotting analysis.
(B) Ten-d-old seedlings of selected transgenic plants.
(C) Two month-old seedlings of transgenic plants grown in WAGNER’s pot.
Overexpression of AREB1-M8 or AREB1-∆QT in rice plants also enhanced drought tolerance. The more adaptation of transgenic plants in this case may not involved in altered stomata closure. In Arabidopsis, plants overexpressing AREB1-∆QT showed no remarkable difference in the status of the stomatal opening between the 35S: AREB1-∆QT and wild-type plants grown on soil or on plates for 30 min after excision of the leaves [8].These data together suggest that the enhanced tolerance of the 35S:AREB1-∆QT plants can be attributed to the AREB1-∆QT dependent overexpression of the downstream genes, including LEA class genes, rather than to ABA-mediated stomata closure [8].
Drought is one of the leading factors responsible for the reduction in crop yield worldwide. Due to climate change, in future, more areas are going to be affected by drought and for prolonged periods. Therefore, understanding the mechanisms underlying the drought response is one of the major scientific concerns for improving crop yield.
ABA regulates the expression of most of the target genes through ABA-responsive element (ABRE) binding protein/ABRE binding factor (AREB/ABF) transcription factors. Genes regulated by AREB/ABFs constitute a regulon termed as AREB/ABF regulon. In addition to this, drought responsive genes are also regulated by ABA-independent mechanisms [26].
Figure 3 Transgenic plants overexpressing AREB1, AREB1-M8 or AREB1-∆QT displayed drought tolerance
(A and B) Two week-old seedlings were dried at 28ºC for 3.5 hrs and then transplanted into small pot. The surviving plants were scored after 2 weeks with daily watering (A), and quantitative data showing the surviving rate of transgenic plants and control WT in the dry treatment for 3.5 hrs (B).
(C and D) Two week-old seedlings were dried at 28ºC for 4.0 hrs and then transplanted into small pot. The surviving plants were scored after 2 weeks with daily watering (C), and quantitative data showing the surviving rate of transgenic plants and control WT in the dry treatment for 4.0 hrs (D). Data in (B) and (D) are averages (±) of at least 45 seedlings from three independent experiments.
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Conclusion
In the present study, transgenic rice plants including Ubi:AREB1#13, Ubi:AREB1#14, Ubi:AREB1-M8#10, Ubi:AREB1-M8#15, Ubi:AREB1-∆QT #2 , and Ubi:AREB1-∆QT #2 were generated and showed enhanced drought tolerance. These plants will play a significant role in fight against climate change for food security as well as other purposes for agriculture.
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