Wathugala G.D. Lakmini1, Heather Knight and Marc R. Knight · Wathugala G.D. Lakmini1, Heather...
1
Wathugala G.D. Lakmini 1 , Heather Knight 2 and Marc R. Knight 2 1 Department of Crop Science, Faculty of Agriculture, University of Ruhuna, Mapalana, Kamburupitiya, Sri Lanka, 2 School of Biological and Biomedical Sciences, Durham of University, South Road, Durham, United Kingdom. DH1 3LE The current rate of climate change predicts that plants will become subject to increasing extremes of environmental stress. Rapid population increases in developing countries also demand higher yield from crop production, often from sub-optimal agricultural areas. Genetic engineering can help meet these needs through the development of crops with greater stress tolerance. Mediator is transcriptional co-activators which convey DNA bound transcriptional regulators and enhancers to the general RNA polymerase II transcription machinery and mediator genes are recently identified in plants (Fig. 1). So far it has showed their great involvement in regulation of plant stress tolerance by controlling transcription of stress genes (Fig. 2). SFR6 (SENSITIVE TO FREEZING6) is one of plant mediator protein which has identified first with its involvement to tolerance against freezing in Arabidopsis. The freezing sensitivity of sfr6 mutant is lack of expression of downstream genes in CBF cold response pathway (Fig. 3). Apart from that there was preliminary evidence that sfr6 mutant is sensitive for other biotic and abiotic stresses. Therefore, the objective of this research was to screen the involvement of SFR6 to regulate other environmental stresses. Activation Repression Med16/SFR6 Figure 1: Topological organization of yeast mediator (Guglielmi et al., 2004) Figure 2: Function of mediator in transcriptional activation and repression (Bjorklund and Gustafsson, 2005) Figure 3: Involvement of SFR6 upon low temperature induced gene expression To examine the role of Med 16/SFR6, At4g04920 was over-expressed in wild type Arabidopsis and sfr6-1 mutant. Then freezing sensitivity and KIN2 expression were measured in transgenic plants. To examine the sensitivity of sfr6 mutant to different environmental stresses, sfr6 mutants were subjected to range of environmental stresses along with wild type Arabidopsis. Homologue was cloned from rice and its orthology was tested transferring OsSFR6 to sfr6-1 mutant. Complementation of sfr6‐1 mutant by wild type AtSFR6 0 0.5 1 1.5 2 2.5 Col-0 sfr6-1 L#1 L#2 L#5 KIN2/βTUB4 0 10 20 30 40 50 60 Col-0 sfr6-1 sfr6-2 sfr6-3 MCA8Expression (RQ) Untreated Treated Sensitivity of sfr6 mutants to UV radiation and biotic stresses 0 500 1000 1500 2000 2500 3000 Col-0 sfr6-1 sfr6-2 sfr6-3 PR1Expression (RQ) Untreated Treated 0 20 40 60 80 100 120 0 5 10 15 20 % Seedlings survived UV‐C (KJ/m2) Col-0 sfr6‐1 sfr6‐2 sfr6‐3 Figure 6: Sensitivity of sfr6 mutants to UV Irradiance. (A) Number of seedlings survived 10 days after UV irradiance. (B,C) Real time PCR of PR1 and MC8 transcripts in 7 day old seedlings of sfr6 alleles subjected to 5KJ/m 2 UV irradiance. Error bars show ±RQ. A B A B C Figure 4: (A) Transformation of wild type AtSFR6 in to the sfr6‐1 mutant restores its freezing tolerance. Line 3, 4, 5 and 6 are in sfr6‐1 background. (B) Real time PCR of KIN2 transcripts in 7 day old seedlings of sfr6‐1 overexpressing 35S::AtSFR6, subjected to 4 o C for 6 h. Error bars show ±RQ. Col 0 sfr6 1 L#1 L#2 L#5 Introducing the wild type SFR6 gene into the sfr6-1 mutant should rescue the mutant phenotype. This assay shows that At4g04920 in the sfr6-1 mutant compliments mutant phenotypes. The transgene rescues seedling colour from yellow green to dark green, the plants regain freezing tolerance (Fig.4A), and express KIN2 gene to wild type levels (Fig.4B). 0 2000 4000 6000 8000 10000 12000 Col-0 sfr6-1 sfr6-2 sfr6-3 PR1Expression (RQ) Untreated Treated 3 4 5 6 7 8 9 Day 0 Day 1 Day 2 Day 3 Log cfu/cm 2 Days after inoculation Col-0 eds‐1 sfr6‐1 AtSFR6 orthologues from other crop plants Figure 7: Sensitivity of sfr6 mutants to virulent P. syringae infiltration. (A) Comparison of the spread of lesions caused by P. syringae, 5 days after infiltration. (B) The growth of strain by scoring colony forming units (cfu). (C) Real time PCR of PR1 transcripts in 5 weeks old plants of sfr6 alleles inoculated with P. Syringae virulent strain. Error bars show ±RQ. A B C 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Col-0 sfr6-1 L#8 L#10 L#19 KIN2expression (RQ) Figure 5: (A) Transformation of OsSFR6 in to the sfr6‐1 mutant restores its freezing tolerance. Line 3, 4 and 5 are in sfr6‐1 background, (B) Real time PCR of KIN2 transcripts in 7 day old seedlings of sfr6‐1 overexpressing 35S::OsSFR6, subjected to 4 o C for 6 h. Error bars show ±RQ. AtSFR6 homologue from rice was identified (OsSFR6), and its functional l i d b f i O SFR6 f6 1 Sensitivity of sfr6 mutants to range of environmental stresses were tested and found, in addition to known roles of SFR6, SFR6 also has roles in protecting against UV irradiance and pathogen infection in Arabidopsis, by showing reduced level of UV (Fig. 6) and pathogen (Fig. 7) induced gene expression. Results demonstrate the requirement of SFR6/MED16 for the activation of many but not all stress response gene expression, and indicated conserved AtSFR6 function in rice. However, the mechanism of regulation of stress induced gene expression via SFR6/MED16 remains to be further investigated. The future research on specific roles fi di id l b i d fh hl l f l di ill id A B 1. Bjorklund S, Gustafsson CM (2005) The yeast Mediator complex and its regulation. Trends in Biochemical Sciences 30: 240-244 2. Boyce, J. M., H. Knight, et al. (2003). The sfr6 mutant of Arabidopsis is defective in transcriptional activation via CBF/DREB1 and DREB2 and shows sensitivity to osmotic stress. Plant Journal 34(4): 395-406 3. Backstrom S, N. Elfving et al. (2007) Purification of a plant mediator from Arabidopsis thaliana identifies PFT1 as the Med25 subunit. Molecular Cell 26: 717-729 4. Guglielmi B, NL van Berkum et al. (2004) A high resolution protein interaction map of the yeast Mediator complex. Nucleic Acids Research . 32: 5379-5391 5. Knight, H., E. L. Veale, et al. (2004). "The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif . Plant Cell 16(9): 2541-2541. 6. Knight, H., S.G. Mugford, et al. (2009). Identification of SFR6, a key component in cold acclimation acting post translational on CBF function. Plant journal 58: 97-108. complementation was tested by transferring OsSFR6 to sfr6-1 mutant. Transgenic plants complemented all sfr6-1 mutant phenotypes including freezing sensitivity (Fig.5A) and KIN2 expression (Fig.5B). of individual subunits and of the whole complex of plant mediator will widen our knowledge of the transcriptional regulation of gene expression in plant and will create new routes to improve crop tolerance to environmental stresses.
Transcript of Wathugala G.D. Lakmini1, Heather Knight and Marc R. Knight · Wathugala G.D. Lakmini1, Heather...
Wathugala G.D. Lakmini1, Heather Knight2 and Marc R. Knight2
1Department of Crop Science, Faculty of Agriculture, University of Ruhuna, Mapalana, Kamburupitiya, Sri Lanka, 2School of Biological and Biomedical Sciences, Durham of University, South Road, Durham, United Kingdom. DH1 3LE
The current rate of climate change predicts that plants will become subject to increasing extremes of environmental stress. Rapid population increases indeveloping countries also demand higher yield from crop production, often from sub-optimal agricultural areas. Genetic engineering can help meet these needsthrough the development of crops with greater stress tolerance. Mediator is transcriptional co-activators which convey DNA bound transcriptional regulators andenhancers to the general RNA polymerase II transcription machinery and mediator genes are recently identified in plants (Fig. 1). So far it has showed their greatinvolvement in regulation of plant stress tolerance by controlling transcription of stress genes (Fig. 2). SFR6 (SENSITIVE TO FREEZING6) is one of plant mediatorprotein which has identified first with its involvement to tolerance against freezing in Arabidopsis. The freezing sensitivity of sfr6 mutant is lack of expression ofdownstream genes in CBF cold response pathway (Fig. 3). Apart from that there was preliminary evidence that sfr6 mutant is sensitive for other biotic and abioticstresses. Therefore, the objective of this research was to screen the involvement of SFR6 to regulate other environmental stresses.
ActivationRepression
Med16/SFR6
Figure 1: Topological organization of yeast mediator (Guglielmi et al., 2004)Figure 2: Function of mediator in transcriptional activation and repression (Bjorklund and Gustafsson, 2005)
Figure 3: Involvement of SFR6 upon low temperature induced gene expression
To examine the role of Med 16/SFR6, At4g04920 was over-expressed in wild type Arabidopsis and sfr6-1 mutant. Then freezing sensitivity and KIN2 expression weremeasured in transgenic plants. To examine the sensitivity of sfr6 mutant to different environmental stresses, sfr6 mutants were subjected to range of environmentalstresses along with wild type Arabidopsis. Homologue was cloned from rice and its orthology was tested transferring OsSFR6 to sfr6-1 mutant.
Complementation of sfr6‐1 mutant by wild type AtSFR6
0
0.5
1
1.5
2
2.5
Col-0 sfr6-1 L#1 L#2 L#5
KIN2/βTU
B4
0
10
20
30
40
50
60
Col-0 sfr6-1 sfr6-2 sfr6-3
MCA8Expression (RQ)
Untreated
Treated
Sensitivity of sfr6 mutants to UV radiation and biotic stresses
0
500
1000
1500
2000
2500
3000
Col-0 sfr6-1 sfr6-2 sfr6-3
PR1Expression (R
Q)
Untreated
Treated
0
20
40
60
80
100
120
0 5 10 15 20
% Seedlings survived
UV‐C (KJ/m2)
Col-0sfr6‐1sfr6‐2sfr6‐3
Figure 6: Sensitivity of sfr6 mutants to UV Irradiance. (A) Number of seedlings survived 10 days after UV irradiance. (B,C) Real time PCR of PR1 and MC8 transcripts in 7 day old seedlings of sfr6 alleles subjected to 5KJ/m2 UV irradiance. Error bars show ±RQ.
A B
A B C
Figure 4: (A) Transformation of wild type AtSFR6 in to the sfr6‐1 mutant restores its freezing tolerance. Line 3, 4, 5 and 6 are in sfr6‐1 background. (B) Real time PCR of KIN2 transcripts in 7 day old seedlings of sfr6‐1overexpressing 35S::AtSFR6, subjected to 4oC for 6 h. Error bars show ±RQ.
Col 0 sfr6 1 L#1 L#2 L#5
Introducing the wild type SFR6 gene into the sfr6-1 mutant should rescue themutant phenotype. This assay shows that At4g04920 in the sfr6-1 mutantcompliments mutant phenotypes. The transgene rescues seedling colour fromyellow green to dark green, the plants regain freezing tolerance (Fig.4A), andexpress KIN2 gene to wild type levels (Fig.4B).
0
2000
4000
6000
8000
10000
12000
Col-0 sfr6-1 sfr6-2 sfr6-3
PR1 Expression (R
Q)
Untreated
Treated
3
4
5
6
7
8
9
Day 0 Day 1 Day 2 Day 3
Log cfu/cm
2
Days after inoculation
Col-0eds‐1
sfr6‐1
AtSFR6 orthologues from other crop plants
/ Q
Figure 7: Sensitivity of sfr6 mutants to virulent P. syringae infiltration. (A) Comparison of the spread of lesions caused by P. syringae, 5 days after infiltration. (B) The growth of strain by scoring colony forming units (cfu). (C) Real time PCR of PR1 transcripts in 5 weeks old plants of sfr6 alleles inoculated with P. Syringae virulent strain. Error bars show ±RQ.
A B C
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Col-0 sfr6-1 L#8 L#10 L#19
KIN2expression (R
Q)
Figure 5: (A) Transformation of OsSFR6 in to the sfr6‐1 mutant restores its freezing tolerance. Line 3, 4 and 5 are in sfr6‐1 background, (B) Real time PCR of KIN2 transcripts in 7 day old seedlings of sfr6‐1 overexpressing 35S::OsSFR6, subjected to 4oC for 6 h. Error bars show ±RQ.
AtSFR6 homologue from rice was identified (OsSFR6), and its functional l i d b f i O SFR6 f 6 1
Sensitivity of sfr6 mutants to range of environmental stresses were tested and found, in addition to known roles of SFR6, SFR6 also has roles in protecting against UV irradiance and pathogen infection in Arabidopsis, by showing reduced level of UV (Fig. 6) and pathogen (Fig. 7) induced gene expression.
Results demonstrate the requirement of SFR6/MED16 for the activation of many but not all stress response gene expression, and indicated conserved AtSFR6 function in rice. However, the mechanism of regulation of stress induced gene expression via SFR6/MED16 remains to be further investigated. The future research on specific roles
f i di id l b i d f h h l l f l di ill id
A B
1. Bjorklund S, Gustafsson CM (2005) The yeast Mediator complex and its regulation. Trends in Biochemical Sciences 30: 240-2442. Boyce, J. M., H. Knight, et al. (2003). The sfr6 mutant of Arabidopsis is defective in transcriptional activation via CBF/DREB1 and DREB2 and shows sensitivity to osmotic stress. Plant Journal 34(4): 395-4063. Backstrom S, N. Elfving et al. (2007) Purification of a plant mediator from Arabidopsis thaliana identifies PFT1 as the Med25 subunit. Molecular Cell 26: 717-7294. Guglielmi B, NL van Berkum et al. (2004) A high resolution protein interaction map of the yeast Mediator complex. Nucleic Acids Research . 32: 5379-53915. Knight, H., E. L. Veale, et al. (2004). "The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif . Plant Cell 16(9): 2541-2541.6. Knight, H., S.G. Mugford, et al. (2009). Identification of SFR6, a key component in cold acclimation acting post translational on CBF function. Plant journal 58: 97-108.
complementation was tested by transferring OsSFR6 to sfr6-1 mutant. Transgenic plants complemented all sfr6-1 mutant phenotypes including freezing sensitivity (Fig.5A) and KIN2 expression (Fig.5B).
of individual subunits and of the whole complex of plant mediator will widen our knowledge of the transcriptional regulation of gene expression in plant and will create new routes to improve crop tolerance to environmental stresses.
