This ppt is based on how modification of three Ggamma protein help to design the grain in rice and to increase the yield.

1. Heterotrimeric G protein: Can it help to feed
the world?
Upasana Mohapatra
PALB 6290
Dept. of Plant Biotechnology
UAS, GKVK, Bengaluru

2. CONTENTS
Summary and future prospectives
Case study
G protein signaling in plant stress responses
G protein signaling in plant physiological functions
G protein subunits
Structure and activation of G protein
History
April 13,2018 2Dept. of Plant Biotechnology,UAS(B)

3. HISTORY OF PLANT G-PROTEIN SCIENCE
Urano et al., 2013
April 13,2018 3Dept. of Plant Biotechnology,UAS(B)

4. Structure of G protein
• Heterotrimeric G protein signaling
regulates a wide range of growth
and developmental processes in
animals and plants.
• The membrane bound
heterotrimeric G protein complex
is composed of three subunits:
alpha (Gα), beta (Gβ), and
Gamma (Gγ) , G-protein-coupled
receptors (GPCRs), and regulators
of G-protein signaling proteins
(RGSs).
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( Liu et al.,2017).

5. Activation of G protein
• Activation is mediated by
binding of an extracellular
ligand to a 7-pass trans-
membrane (7TM) G protein-
coupled receptor (GPCR).
• GPCRs are guanine-nucleotide
exchange factors; they cause Gα
to exchange GDP for GTP,
leading to the dissociation of the
complex into Gα -GTP and Gβγ
dimers.
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( Liu et al.,2017).

6. The ‘G’ cycle of animals versus Arabidopsis
Urano et al., 2013
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7. Crystal structure and activation mechanisms of G protein
Urano et al., 2013
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8. Domain structures of Arabidopsis G protein-related proteins
Urano et al., 2013
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9. Domain structures of Arabidopsis G protein-related proteins
Urano et al., 2013April 13,2018 9Dept. of Plant Biotechnology,UAS(B)

10. Structural characteristics of G protein signaling related proteins
Stateczny et al., 2016April 13,2018 10Dept. of Plant Biotechnology,UAS(B)

11. Structural characteristics of G protein signaling related proteins
Stateczny et al., 2016
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12. Model for an intracellular kinase-dependent G
protein cycle in Arabidopsis
Stateczny et al., 2016
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13. Hypothetic model for a membrane associated
kinase-dependent G protein mechanism in
Arabidopsis:
Stateczny et al., 2016
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14. G protein subunits
In Plant cell
• In Arabidopsis-
one canonical Gα (AtGPA1)
one Gβ (AGβ1)
three Gγ(AGγ 1, AGγ 2,AGγ3)
• In rice-one canonical Gα , one
Gβ and 5 Gγ –subunits
• γ -subunits are four times the
average mammalian size.
• Many plant γ -subunits do not
contain an isoprenylation motif
at their C-terminus.
In Human beings
• 23 Gα, five Gβ, and 12 Gγ
subunits
• γ -subunits are very small
proteins (less than 100 amino
acids)
• An obligate requisite in all
animal γ -subunits and
essential for membrane
anchoring.
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Stateczny et al., 2016

15. Physiological Functions of G Protein
Cell proliferation
Root development
Leaf and fruit development
Chloroplast development
Hormonal regulation of seed germination
Auxin and sugar signalling
ABA, Ca2+ and ROS signalling in guard cells.
Stomatal opening and closure
Phytochrome and cryptochrome mediated responses
April 13,2018 15Dept. of Plant Biotechnology,UAS(B) Stateczny et al., 2016

16. 1. Arabidopsis T-DNA insertion lines for Gα (gpa1–4), Gβ (agb1–2) or RGS1 (rgs1–2)
and wild-type Col-0 were grown for 37 days in a short day chamber (8 L : 16 D
cycle, 100 µmol m-2 s-1) at 230C.
2. Nipponbare (wild-type), the Gα knockout (d1,) or Gβ knockdown lines of rice were
grown in a short day chamber (8 L : 16 D cycle, 34 0C during day per 28 0C during
night time, 320 µmol m-2 s-1) for 47 days.
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17. Physiological Function of G Protein
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18. April 13,2018 18Dept. of Plant Biotechnology,UAS(B)

19. The α subunit encoded by a single gene (GPA1) in Arabidopsis, is a
modulator of plant cell proliferation.
1) gpa1 mutants have reduced cell division.
2) Inducible over-expression of GPA1 in Arabidopsis confers
inducible ectopic cell division which causes premature advance of the
nuclear cycle and the premature appearance of a division wall.
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20. •Cell production rates were determined for the RAM and lateral root
formation in gpa1 and agb1 single and double mutants, and in transgenic
lines over expressing GPA1 or AGB1.
•In the RAM that the heterotrimeric complex acts as an attenuator of cell
proliferation, whereas Gα-subunit’s role is a positive modulator.
•For the formation of lateral roots, the Gβγ-dimer acts independently of
the Gα -subunit to attenuate cell division.
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21. April 13,2018 21Dept. of Plant Biotechnology,UAS(B)

22. G Protein in Stress Response
Oxidative stress and hypoxia responses
Pathogen responses
Drought stress
Salinity stress
April 13,2018 22Dept. of Plant Biotechnology,UAS(B)

23. 1. TheAGB1 mutants (agb1-2-1 and agb1-3-2) were more sensitive to drought than
the wild-type.
2. The overexpression of mulberry (Morus alba L.) G-protein b subunit in transgenic
tobacco (Nicotiana tabacum L.) significantly enhanced the plants' drought
tolerance.
3. The transgenic tobacco plants had higher proline contents and peroxidase activities,
and lower malonaldehyde and hydrogen peroxide contents and superoxide free
radical accumulations under drought conditions and antioxidative genes, NtSOD
and NtCAT, are increased in drought-stressed transgenic tobacco plants
April 13,2018 23Dept. of Plant Biotechnology,UAS(B)

24. Overexpression of
mulberry (Morus alba
L.) G-protein β subunit
in transgenic tobacco
(Nicotiana tabacum L.)
TheAGB1 mutants (agb1-2-
1 and agb1-3-2) were more
sensitive
April 13,2018 24Dept. of Plant Biotechnology,UAS(B)

25. •The knockout mutant of the Arabidopsis heterotrimeric G-protein Gβ
subunit, agb1, is hypersensitive to salt, exhibiting a leaf bleaching
phenotype.
•AGB1 is mainly involved in the ionic toxicity component of salinity
stress and plays roles in multiple processes of Na+ homeostasis.
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26. The knockout mutant
of the Arabidopsis
heterotrimeric G-
protein Gβ subunit,
agb1, is hypersensitive
to salt, exhibiting a leaf
bleaching phenotype.
Three-week-old
hydroponically grown
plants treated with 100
mm NaCl for 10 d.
Survival rate is less in
agb-1 mutant plants.
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27. PAMP-triggered immunity
Cell death and defence responses
Signalling against bacterial pathogens
Elicitor-induced stomata closure
Resistance against necrotrophic fungi
Non-host resistance
R protein mediated disease resistance
Heterotrimeric G proteins in plant defence mechanisms
Nitta et al., 2015
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Gβ
Gβ
Gα
Gβ,Gγ
Gα,Gβ
Gα,Gβ,Gγ
Gβ,Gγ

28. Defence-related phenotypes observed in heterotrimeric
G protein mutants
Nitta et al,2015
April 13,2018 28Dept. of Plant Biotechnology,UAS(B)

29. The Gα subunit is not involved in
the Gβ-mediated defense
pathway.
i) chorosis, and necrosis were
less developed in agb1-2
ii) inflorescence growth was also
inhibited in gpa1-4 plants,
although to a lesser extent than in
agb1-2; and
iii) endogenous SA did not affect
resistance to CMV and TuMV
and did not interfere with AGB1-
mediated responses.
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30. Khatri et al,2012
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31. Case study
1. The Gβ protein is essential for plant survival and growth.
2. Gα provides a foundation for grain size expansion.
3. Three Gγ proteins, DEP1, GGC2 and GS3, antagonistically regulate
grain size. DEP1 and GGC2, individually or in combination,
increase grain length when in complex with Gβ.
April 13,2018 31Dept. of Plant Biotechnology,UAS(B)

32. Grain size is a trait for grain quality focused by rice
breeders, as long and slender grains are preferred by rice
consumers in many countries
Recent advances in rice functional genomics facilitated
the cloning of a series of loci controlling grain size,
including genes
1) Grain length -GS3, GL3. An-1, GLW7, GS2 genes
2) Grain width -GW2, GW5, GS5, GW8 , GW7
3) Grain weight-GIF1, GE, TGW6, GW6a, BG1,XIAO
Introduction
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33. Types of Gγ
Type A
• Small in size
• Has C-terminal
CaaX
isoprenylation
motif
• eg-RGG1-
regulation of
abiotic stress
Type B
• Small in size
• Lack CaaX motif
• eg-RGG2-
regulation of
abiotic stress
Type C
• 2 well defined
region-
• 1)an N-terminal
domain with
high similarity to
g subunit
• 2) A C terminal
cystein rich
subunit
• eg- DEP1,GS3-
QTL for grain
size.
April 13,2018 33Dept. of Plant Biotechnology,UAS(B)

34. Methods
1
• Field planting and trait measurement.
• Vector construction and plant transformation -Constructs of GS3-1OE,
GS3-4OE, DEP1OE, dep1OE and GGC2OE , under an ubiquitin gene
promoter.
2
• Generation of knock-out mutants
• Single mutant, double mutant and triple mutant of GS3,DEP1, GGC2
3
• RNA extraction and qRT-PCR.
• Western blot.
4
• Statistical analysis-The two-tailed t test was used for comparing agronomic
traits of each transgenic line with the control
April 13,2018 34Dept. of Plant Biotechnology,UAS(B)

35. •Elevated DEP1 accumulation
increased the grain length by
6.85–9.58% with a normal
plant stature.
•Plants over expressing dep1
showed very similar
phenotype to DEP1Ri plants,
both of which reduced grain
length by ~4.5%, together
with dwarf stature and erect
panicles.
•The expression level of endogenous
DEP1 in dep1OE was not reduced.
• Thus dep1 showed a dominant-negative
effect over DEP1 in regulating grain size
rather than co-suppression of the two
genes.April 13,2018 35Dept. of Plant Biotechnology,UAS(B)

36. •GS3-1OE showed an
average 9.07% reduction in
grain length together with
reduced plant stature.
•GS3-4OE produced even
smaller plants and grain size
with an average 19.10%
reduction of grain length
.
•GS3-1Ri resulted in an
average 5.78% increase of
grain length.
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37. April 13,2018 Dept. of Plant Biotechnology,UAS(B) 37
GS3-1Ri X dep1OE
F1 Reduced grain length, similar to
dep1OE transgenic plants.
F2
Also showed the dep1OE phenotype with
respect to grain size, plant height and
panicle length
Interaction between DEP1 and GS3 in grain size regulation

38. GS3-1ko mutant-increased grain size,
DEP1ko mutant - reduced grain size.
GS3-1koDEP1ko double mutant- intermediate
between that of the single mutants of GS3-1ko
and DEP1ko
GS3-1koDEP1ko double mutant X ZH11
F2
Increased grain length was observed when
over expressing DEP1 in GS3-1Ri
background,
whereas the DEP1OE/GS3-1OE hybrid
showed the GS3-1OE phenotype of short
grain
April 13,2018 38Dept. of Plant Biotechnology,UAS(B)

39. An atypical Gγ subunit GGC2 functions additively with DEP1
Over expression of GGC2 (GGC2OE) in ZH11
increased grain length significantly,
knock-out mutant of GGC2ko in ZH11 reduced the
grain length.
Knocking out DEP1ko GGC2ko resulted in much smaller
grains than either of the single knock-out mutants,
DEP1 and GGC2 worked additively in positive regulation of
grain length.
The DEP1koGGC2koGS3-1ko triple mutant
produced similar phenotype to that of
DEP1koGGC2ko, thus GS3-1ko mutant could not
increase grain length when both DEP1 and
GGC2 were knocked-out
DEP1 and GGC2 functioned positively in regulating grain size in an additive
manner, while the role of GS3 in grain size regulation was to repress the effects
of DEP1 and GGC2 on increasing grain size.
April 13,2018 39Dept. of Plant Biotechnology,UAS(B)

40. Functional dependence of the Gγ subunits on
RGB1 and RGA1.
The transgenic plants with suppressed expression of RGB1 (Knocked down RGB1 by RNAi
(RGB1Ri) )showed reduced grain size and plant height.
GS3-1Ri X RGB1Ri DEP1OE X RGB1Ri
Reduced grain length
Reduced grain length
Overexpressed GGC2 in RGB1Ri.
Overexpressed DEP1 in RGB1Ri.
Reduced grain length
The effects of grain
length increase by
DEP1OE, GGC2OE and
GS3-1Ri are dependent
on RGB1 and RGA1
April 13,2018 40Dept. of Plant Biotechnology,UAS(B)

41. Schematic representation of the functions of the G proteins in grain size regulation
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42. Conclusion
• DEP1 and GGC2, when coupled with RGB1, promote
grain size by tail-mediated signaling.
• GS3,reduces grain size by blocking the interaction of
DEP1 and GGC2 with RGB1.
• The increased grain size is at the cost of fitness. Thus
from fitness viewpoint medium grain is the optimal for
rice reproduction.
• Thus, while DEP1 and GGC2 function to promote grain
size,which is essential for yield increase in breeding,
GS3 plays a role to keep balance for maintaining fitness
in response to natural selection.
April 13,2018 42Dept. of Plant Biotechnology,UAS(B)

43. 1) The interactions of three Gγ proteins can be used for a predictable design of
grain size in rice, by manipulating these genes, individually or in combination, to
improve rice grain yield and quality.
2) As the Gγ proteins are highly conserved in a very wide range of plants,
manipulating these proteins may provide a general strategy for modifying organ
size and yield in crop breeding.
3) G signalling is at the heart of many plant physiologies of agronomic
importance, such as disease resistance, abiotic stress tolerance and harvest index.
Translational work on G protein signalling will certainly improve agriculture by
providing new targets and strategies for increasing yield.
April 13,2018 43Dept. of Plant Biotechnology,UAS(B)

44. Future Prospectives
• What are the receptors upstream of G-proteins, what lies
downstream of G-proteins, and how the proteins connect to the
established modules of hormone, defense or stress-related
signaling.
• Although the G proteins have been shown to have key roles in
regulating plant growth, stress tolerance and grain yield
potential, the molecular mechanisms underlying the
regulation of the heterotrimeric G protein cycles of activation
and deactivation and downstream effectors still remain
unclear.
April 13,2018 44Dept. of Plant Biotechnology,UAS(B)

45. April 13,2018 Dept. of Plant Biotechnology,UAS(B) 45
THANK YOU
Science of G protein is awaiting your
research efforts, to be explored…….

46. REFERENCES
BOTELLA, J.R., 2012, Can heterotrimeric G proteins help to feed the world?. Trends in
plant science, 17(10) : 563-568.
CHEN, J.G., GAO, Y. AND JONES, A.M., 2006, Differential roles of Arabidopsis
heterotrimeric G-protein subunits in modulating cell division in
roots. Plant Physiology, 141(3), pp.887-897.
LIU, C., XU, Y., LONG, D., CAO, B., HOU, J., XIANG, Z. AND ZHAO, A., 2017,
Plant G-protein β subunits positively regulate drought tolerance by
elevating detoxification of ROS. Biochemical and biophysical research
communications, 491(4): 897-902.
STATECZNY, D., OPPENHEIMER, J. AND BOMMERT, P., 2016, G protein signaling
in plants: minus times minus equals plus .Current opinion in plant
biology, 34 :127-135.
SUN, S., WANG, L., MAO, H., SHAO, L., LI, X., XIAO, J., OUYANG, Y. AND
ZHANG, Q., 2018, A G-protein pathway determines grain size in
rice. Nature communications, 9(1): 851.
URANO, D., CHEN, J.G., BOTELLA, J.R. AND JONES, A.M., 2013, Heterotrimeric
G protein signalling in the plant kingdom. Open biology, 3(3): 120186.
XU, Q., ZHAO, M., WU, K., FU, X. AND LIU, Q., 2016, Emerging insights into
heterotrimeric G protein signaling in plants. Journal of Genetics and
Genomics, 43(8): 495-502.
April 13,2018 46Dept. of Plant Biotechnology,UAS(B)