1. DREB genes play an important role in improving crop tolerance to stresses like drought, salt, and cold. DREB transcription factors regulate stress-responsive genes allowing plants to adapt. 2. Case studies showed overexpression of DREB genes enhanced stress tolerance in crops like rice and sugarcane. Co-transformation with DREB and other stress genes improved tolerance more than single DREB genes. 3. DREB genes respond differently to various stresses. DREB1 genes respond mainly to cold while DREB2 genes respond to dehydration and heat. Proper expression analysis of DREB genes is important for abiotic stress tolerance in transgenic crops.

1. 2
IInd Seminar on
Wel-come
Vijay kumar S.
2015A28D
“role of dreb -genes in crop improvement”

2. Conclusions
Case studies
Biological role of DREB transcripstion factor.
Mechanism of DREB transcription factor.
What is DREB gene …??
Introduction
2

3. Some facts about stresses…
 The extremes of the major environmental conditions: soil moisture, salt
concentration and temperature, reduce potential crop yields by as much as
70%.
 Every year significant losses occur due to sudden frost and unusual freezing
temperatures in winter and late cold spring.
 Globally, approximately 22% of the agricultural land is saline and more than
50% of all arable lands expected by the year 2050.
 Drought is a widespread phenomenon in many regions and expected to
increase further.
 In general, crops are lost to the tune of 25% due to insects, due to diseases
including that of nematodes up to 20%
(Akthar et al., 2012)

4. (Agarwal et al., 2006)

5. Transcription factors/regulatory protein
• TFs are protein complexes that responses
to specific signals and in accordance
regulate expression of protein coding
gens by binding to the control region of
DNA
• These regulatory proteins are involved in
the control of primary and secondary
metabolism, growth and developmental
programs, as well as responses to
environmental stimuli.
5

6. 6
Important transcription factors responses to stresses:
1. Ap2/ERF
2. Basic-domain
leucine zipper
(bzip)
3. MYC
4. MYB
5. WRKY

7. what is DREB..?
• Dehydration Responsive Element Binding
• DREB transcription factors are one of the largest sub-families
of transcriptional regulators in plants
• DREB proteins are involved in responses to abiotic stresses
• DREB exhibit extensive auto-regulation and cross-regulation
that facilitates transcriptional reprogramming in plant
• The DREB gene with 9bp conserved core sequence was first
identified in Arabidopsis in 1993
• The first isolated cDNAs encoding DRED protein in 1997
7

8. Structural characteristics of DREB TFs
• All TFs have a DNA binding domain containing a short peptide region called
the DNA-binding motif.
• Proteins belongs to DREB have highly conserved DNA binding domain called
AP2 /ERF domain consisting of ~60 amino acids.
• DNA-binding domain that directly interact with GCC box or dehydration
responsive element(DRE) or C-repeat element (CRT) cis-acting element at the
promoter of downstream target gene.
• The amino acids Valine (at position 14) and glutamic acid (19) in the AP2/ERF
domain are quite conserved and play a central role in recognition and binding
specificity of Cis elements.
8

9. • The entry of DREB proteins is mediated by one or two nuclear localization signal
(NLS).
• AP2 domain play a central role in recognition and binding specificity of cis
element. 30 aa sequence rich in basic and hydrophobic in nature.
• All the DREB/CBF genes posses carboxyl terminal acidic region. This region is
supposed to be a transcriptional activator motif.
• DSAW and LWSY motif are the signature sequences are specifically found in
A1 sub group.
• The DREB/CBF-type proteins have a basic-amino acid-rich stretch having
consensus sequence PKRPAGRTKFRETRHP as NLS.
9

10. Structure of the DREB domain
10

11. Classification of DREB subfamily and their induction
11

12. The newly discovered non-plant protein bearing an AP2 domain are predicted to be
HNH endonucleases
Horizontal transfer of an HNH-AP2 endonuclease from bacteria or virus in to
plants may lead to the origin of the AP2/ERF FAMILY
origin and evolution
12
(Magnani et al., 2004)

13. Stress signal perception pathways of DREB
13
(Lata and Prasad 2011)

14. Expression analysis of DREB
In Arabidopsis, out of six DREB1 genes DREB1A, DREB1B and DREB1C were
expressed at high levels under cold stress but not under drought or high-salinity
stress.
It is well known that expressions of the A-1 group of genes is induced by low
temperature, but not by drought or high-salt stress,
while A-2 group genes are regulated by salt and drought, but not by cold.
The different abiotic stress signalling pathways are assumed to interact and share
some common elements that formed as potential ‘node’ for crosstalk.
14

15. DREB1/CBF (A-1) subgroup — major regulator of
cold-stress responses
15

16. DREB2 subgroup — dual functions in dehydration
and heat shock responses
16

17. Case studies
17

18. Material:
1. Samba Mahsuri cultivar
2. TF- AtDREB1A
18

19. Expression cassette PCR analysis - TO
Southern analysis of To Copy no detection
RT-PCR of TO with DREB primers RT-PCR of TO with actin primers
19

21. 21

22. 22

23. 23

24. After withdrawal of water for
28 days
After 2 days of re-watering
Vandana Rasi BPT-control T5 transgenic lines
Evaluation
and
Screening
24

25. Conclusion for case study-1
1. Drought tolerance at both vegetative and reproductive stages.
2. DREB1A functioned in stress tolerance pathway.
3. Samba Mahsuri - a premium variety.
4. Selected transgenic lines would be valuable resource.
5. They can be used as a direct variety.
25

26. Material:
1. wild lotus
2. SN1301 with a CaMV 35S
3. NaCL, ABA, mannitol, low tem.
26

27. NnDREB2 expression patterns in response to various treatments
and in different organs.
27

28. Screen of transgenic plants by RT-PCR method and
GUS staining
28

29. p
29

30. Expression profiling analysis of 22 stress-related genes
30

31. Obj: Assessing the expression of the abiotic stress tolerance in the single-gene and
double-gene transformants
Material:
i. DREB2 gene from E. arundinaceus
ii. Co 86032 - popular sugarcane variety
iii. PDH45 gene driven by Port Ubi 2.3 promoter
31

32. V0 stage at 25 % soil moisture V1 stage at 8.1 and 25 % soil moisture stress
V0 stage at 25 % soil moisture V1 stage at 8.1 and 25 % soil moisture stress
E
a
D
R
E
B
2
C
o
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t
r
a
n
s
f
o
r
m
e
d
32

33. 33
EaDREB2 transgenics
Co-transformed transgenics

34. 34

35. A. EaDREB2 transgenics
B. Co-transformed events
35

36. Assay for salinity tolerance EaDREB2 36

37. Assay for salinity tolerance Co-transformed 37

38. Bud germination %
EaDREB2 transgenic plants Co-transformed transgenic plants
38

39. Conclusion for case study-3
Transformation of sugarcane with the EaDREB2 gene under the control of the Port
Ubi 2.3 promoter enhanced the tolerance to water deficit and salinity stress
through improved physiological adaptation .
Improved salinity tolerance was observed in the co-transformed events compared
to that of EaDREB2 and enhanced stress-related gene expression.
Thus, for combining drought and salinity tolerance, pyramiding of these two genes
seems to be a better approach.
Further replicated field studies are needed to confirm their tolerance to drought
and salinity, along with the yield potential and water use efficiency.
39

40. 40
(Plants with 7-8 leaves were
exposed to 450 C for 36h and then
returned to 220C for 3 weeks)

41. 41
Effect of heat stress on electrolyte leakage in transgenic
chrysanthemum
Expression of the AtDREB1A gene analyzed by RT-PCR
under heat conditions in transgenic plants exposed to 45C

42. Effects of heat stress on the photosynthetic capacity of
transgenic plants
42

43. 43
Conclusion :- AtDREB1A TF enhanced the CO2 assimilation rates and
Rubisco activity under heat stress condition in Chrysanthemum

44. Aim: To establish protocols for the development of genome-specific and allele specific
markers in common wheat using the Dreb1 genes.
Material:
1. Opata 85 T. aestivum L., AABBDD, 2n=6x=42)
2. DS107 T. durum (AABB, 2n=4x=28),
3. UR203 T. urartu (AA, 2n=2x=14),
4. 2046 Aegilops. speltoides (BB, 2n=2x=14),
5. Y2009 Ae. tauschii (DD, 2n=2x=14).

45. Results:
 Development of the genome specific primers and chromosome assignments of
the Dreb1 genes
 the five primer pairs designed in the study will be useful as FMs to trace each
locus during MAS in search of more drought-resistant wheat varieties revealed.
Mapping of Dreb-B1

46. 46
Response of DREB genes to various stresses. ( Lata and Prasad, 2011)

47. 47
Stress response of overexpressing DREBs in transgenic plants. ( Lata and Prasad, 2011)

48. 48Conclusions
Further research is needed to uncover the regulatory mechanism of drought
response and tolerance under field conditions.
Considerable progress can be made in improving crop tolerance by
incorporating AP2/ERF genes.
Optimizing DREB technology through reducing growth abnormalities and
enhancing the introduced trait to the level of commercial value.
DREB as a candidate gene and developing proper functional marker used
for MAS and allele-mining in breeding programmes

49. 49
Crop
improvement DREB