Feb 2017

Role of the pearl millet Aquaporin genes in abiotic stress toleranceReddy 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.

ObjectiveHarnessing the role of Aquaporins in water conservation and abiotic stress tolerance improvement in Pearl Millet.

Results1. Seven PgAQP genes were cloned using homology based gene cloning approach

and evolutionarily closer to maize than rice.

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Leaf Root Leaf Root Leaf Root Leaf Root

ICMR 1122 ICMR 1086 ICMR 1152 ICMR 1078

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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.

ConclusionPgPIP2;6 can be deployed to engineer stress tolerant transgenic crops for sustained growth and productivity under unfavorable conditions.

AcknowledgementsScience 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.

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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

ARGAGGTSGAYGTCCSGAARGART

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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