1) Seven aquaporin genes were cloned from pearl millet using homology-based cloning. The genes were evolutionarily closer to maize than rice. 2) Pearl millet genotypes with different transpiration responses to high vapor pressure deficit were identified. The aquaporin genes showed increased expression in low transpiring genotypes under low and high vapor pressure deficit conditions. 3) Tobacco plants were genetically transformed with the PgPIP2;6 pearl millet aquaporin gene. Transgenic tobacco plants showed improved performance under drought stress, salinity stress, and heat stress compared to wild type plants, demonstrating the potential of aquaporin genes to improve abiotic stress tolerance in crops.

1. Feb
2017
Role of the pearl millet Aquaporin genes
in abiotic stress tolerance
Reddy PS*, Divya K, Sivasakthi K, Tharanya M, Lale A, Bhatnagar-Mathur P, Kholova J, Sharma KK, Vadez V
*E-mail: p.sudhakarreddy@cgiar.org
Introduction
• Pearl millet (Pennisetum glaucum L.) is an important cereal crop of semi-arid
tropics, which is grown across India and Sub-Saharan Africa. It is well adapted to
grow in regions of very low rainfall and it can withstand long periods of drought.
• Aquaporins are members of Major Intrinsic Proteins (MIP) family known to be
present in the cell membranes and are involved in the conductance of water.
• Aquaporin proteins are of special interest for water deficit adaptations as
changes in AQPs have been linked to improved abiotic stress response.
Objective
Harnessing the role of Aquaporins in water conservation and abiotic stress
tolerance improvement in Pearl Millet.
Results
1. Seven PgAQP genes were cloned using homology based gene cloning approach
and evolutionarily closer to maize than rice.
0
2
4
6
8
10
12
14
Leaf Root Leaf Root Leaf Root Leaf Root
ICMR 1122 ICMR 1086 ICMR 1152 ICMR 1078
Relativeexpression
Genotypes
PIP1.1 PIP1.2 PIP2.1 PIP2.3
PIP2.6 TIP1.1 TIP2.2
Figure 2. Transpiration rate (Tr) response
to increasing VPD in High Resolution
Cross (HRC) genotypes.
Figure 3. Relative expression of PgAQP genes under
high VPD conditions using qPCR.
3. PgPIP2;6 transgenic tobacco plants were generated and functional
characterization was done in homozygous T2 generation plants.
LB
35s pro npt Poly A 35s pro PgPIP2;6 NosT
RBa. pMDC100 comprises AQP cassette
b. Steps involved in tobacco transformation
c. PCR
confirmation of
putative transgenic
tobacco plants
Figure 4. Genetic transformation of tobacco with 35s-PgPIP2;6-NosT construct using the Leaf
disc method.
Conclusion
PgPIP2;6 can be deployed to engineer stress tolerant transgenic crops for
sustained growth and productivity under unfavorable conditions.
Acknowledgements
Science and Engineering Research Board (SERB), DST, Govt. of India for the
research grant through Young Scientist Scheme SB/YS/LS-12/2013. This work has
been undertaken as part of the CGIAR Research Program on Dryland Cereals.
2. Pearl millet genotypes showing contrasting transpiration response to high
vapour pressure deficit (VPD) were identified. PgAQPs exhibited increased
expression in VPD-insensitive genotypes under low and high VPD conditions.
4. PgPIP2;6 transgenic plants showed a better performance under drought stress
and salinity compared to wild type plants.
e. Transpiration reduction of heat stressed
transgenic and wild type plants in the
presence of aquaporin inhibitor AgCl2
.
f. Relative water content measurement of
drought stressed plants transgenic treated with
aquaporin inhibitor AgCl2
.
� �
a. Visual difference in wild type and transgenic leaf
discs after 7 days of 300 mM salt stress.
b. Depicting leaf disc assay under salt
stress.
c. The relative total chlorophyll levels of salt
stressed leaf discs from transgenic as well as
wild type plants.
d. Lipid peroxidation levels in salt stressed
plants.
Figure 1. Cloning and sequence analysis of pearl millet AQP genes.
Cloning
ARGAGGTSGAYG
TCCSGAARGART
Degenerate primers from related species
Phylogenetic analysis
Motif 1 Motif 2 Motif 3
PgPIP1;1 1 MEGKEEDVRLGANKYSERQPIGTAAQGGDDKDYKEPPPAPLFEPGELKSWSFYRAGIAEFVATFLFLYITILTVMG--------VSKSNTKCATVGIQGIA 93
PgPIP1;2 1 MEGKEEDVRLGANKYSERQPIGTAAQGSDDKDYKEPPPAPLFEPGELKSWSFYRAGIAEFVATFLFLYISILTVMG--------VSKSQSKCATVGIQGIA 93
PgPIP2;1 1 MGKDDVIES-----------GAGGGEFAAKDYTDPPPAPLIDAAELGSWSLYRAVIAEFIATLLFLYITVLTVIGYKHQTDPTASGADAACGGVGILGIA 89
PgPIP2;6 1 MGKEVDVSA-----------LEAGG---VRDYADPPPAPLVDIDELGKWSLYRAVIAEFVATMLFLYITVATVIGYKHQTDASASGPDAACGGVGILGIA 86
PgTIP1;1 1 MAAAGIRLCRSESIAVGSHQEVYHPGALKAAFAEFISTLIFVFAGQGSGMAFS------KLSPGG-SSPAGLISAA 69
PgTIP2;2 1 MSGNIAFGRFGDSFSAASLRAYVAEFISSLVFVFAGVGSAIAYR------KLSDGAPLDATGLVAVA 61
. .* ***... .*.. . . *. *
Motif 4 Motif 5
PgPIP1;1 94 WSFGGMIFALVYCTAGISGGHINPAVTFGLFLARKLSLTRAIFYIIMQCLGAICGAGVVKGFQQGLYMGNGGGANTVASGYTKGSGLGAEIIGTFVLVYTV 194
PgPIP1;2 94 WSFGGMIFALVYCTAGISGGHINPAVTFGLFLARKLSLTRAVFYMIMQCLGAICGAGVVKGFQEGLYMGNGGGANMVASGYTKGDGLGAEIVGTFVLVYTV 194
PgPIP2;1 90 WAFGGMIFVLVYCTAGISGGHINPAVTFGLFLARKVSLVRALLYIVAQCLGAICGVGLVKAFQSAYFDRYGGGANSLASGYSRGTGLGAEIIGTFVLVYTV 190
PgPIP2;6 87 WAFGGMIFILVYCTAGISGGHINPAVTFGLFLARKVSLVRAILYMAAQCLGAICGVALVKGFQSGFYARYGGGANEVSAGYSTGTGLAAEIVGTFVLVYTV 187
PgTIP1;1 70 IAHAFALFVAVSVGANISGGHVNPAVTFGAFVGGNITLFRGILYWIAQLLGSTVACFLLRFSTGGLPTGTFG-----LTGISVWEALVLEIVMTFGLVYTV 165
PgTIP2;2 62 VCHGFALFVAVAIGANISGGHVNPAVTFGLVLGGQITMLTGLFYWIAQLVGAIVGAVLVQFSTG-VATPTHG-----LSGIGTLEGVVMEIIVTFGLVYIV 156
. .* * *.*****.******* . ... . * * .*. ... * .* . **. ** *** *
H2
Motif 6 Motif 5
PgPIP1;1 195 FSATDAKRNARDSHVPILAPLPIGFAVFLVHLATIPITGTGINPARSLGAAIIYNRDHAWSDHWIFWVGPFIGAALAAIYHQVIIRA------LPFKSRD 288
PgPIP1;2 195 FSATDAKRNARDSHVPILAPLPIGFAVFLVHLATIPITGTGINPARSLGAAIIYNRSQAWDDHWIFWVGPFIGAALAAIYHQVIISA------IPFKSRS 288
PgPIP2;1 191 FSATDPKRNARDSHVPALAPLPIGFAVFMVHLATIPVTGTGINPARSLGAAVIYNKDKPWDDHWIFWVGPFVGAAIAAFYHQYILRAGAIKALGSFRSNA 290
PgPIP2;6 188 FSATDPKRNARDSHIPVLAPLPIGFAVFMVHLPTIPITGTGINPARSLGAAVVYNNSKAWSDQWIFWVGPFIGAAIAALYHQIVLRA-SARGYGSFRSNA 286
PgTIP1;1 166 YATAVD---PKKGSLGTIAPIAIGFIVGANILVGGAFDGASMNPAVSFGPALVS---WSWGYQWVYWVGPLIGGGLAGIIYEVLFIS---HTHEQLPSTDY 257
PgTIP2;2 157 YATAAD---PKKGSLGTIAPIAIGFIVGANILVAGPFSGGSMNPARSFGPAVAS---GDFTNIWIYWVGPLVGGGLAGIVYRYIYMC---GDHAPVASSEF 248
.... . . .**. *** * * * .*** * * *. *..**** .* .* . . *
H5 LE1 LE2/P2 P3 P4P5
Sequence analysis for PgAQPs
Figure 5. Functional characterization of PgPIP2;6 transgenic tobacco plants under salt, drought
and heat stress treatments.
a. pMDC100 comprises PgPIP2;6 cassette
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