Bimal Paudel Mike Tran Jai Rohila Jose Gonzalez Arvid Boe Gautam Sarath Paul Rushton South Dakota State University Brookings SD PCGSD Differences for biomass production and level of senescence between the PCGSD and PCGND populations during late September 2013 ID: 745650
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Slide1
Gene discovery for delayed senescence in bioenergy crops to improve total biomass production
Bimal Paudel, Mike TranJai Rohila, Jose Gonzalez, Arvid Boe, Gautam Sarath, Paul RushtonSouth Dakota State University, Brookings, SDSlide2
PCG-SD
Differences for biomass production and level of senescence between the PCG-SD and PCG-ND populations during late September 2013.PCG-NDBiomassSenescence rateSlide3
Bottleneck for the genetic improvement programs:
What is the molecular difference between the late senescence germplasm and the early senescence ones?Understand the fundamental molecular basis of senescence in perennial grasses.Situation:Untimely senescence in perennial grasses causes low biomass harvestResearch Question:Little knowledge of molecular markers or gene functionsSlide4
Experimental Design
Spring Pre-SenescencePost-SenescenceWinterBefore 3rd week of AugustLast week of September
Chlorophyll Data
Pre-Senescence
Post-Senescence
Post-Senescence
Pre-SenescenceSlide5
Tissue
TreatmentGroupSample#
1
Switchgrass
Clone # 5
(Early Senescence)
1. Before Senescence
A
Sample# 1
Sample# 2
Sample# 3
2. After Senescence
B
Sample# 4
Sample# 5
Sample# 6
2
Switchgrass
Clone # 4
(Late Senescence)
1. Before Senescence
C
Sample# 7
Sample# 8
Sample# 9
2. After Senescence
D
Sample# 10
Sample# 11
Sample# 12
3
Prairie
Cordgrass
-ND
(Early Senescence)
1. Before Senescence
E
Sample# 13
Sample# 14
Sample# 15
2. After SenescenceFSample# 16Sample# 17Sample# 184Prairie Cordgrass-SD (Late Senescence)1. Before SenescenceGSample# 19Sample# 20Sample# 212. After SenescenceHSample# 22Sample# 23Sample# 24
Samples for the Proteomics Experiment Slide6
Early Senescence
Late SenescenceProteomics WorkflowAnalysis by Typhoon TRIOQuantification by DeCyderGel staining by Sypro-RubySpot
picked
and
digested by trypsin
Protein ID by MALDI-TOF MS and
NCBI data
base
search, and GO annotation
Cy3
Cy5
Pre-
Sene
Post-
sene
Pre-
Sene
Post-
seneSlide7
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59
37
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81
120
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123
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127
88
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Gel-1
276
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A1
/
C7
Protein samples of
SG, and PCG leaves,
c
ontrol and treatment samples,
were labeled with Cy3 (green) and Cy5 (red), respectively and mixed in equal ratios; proteins were separated by two-dimensional PAGE in the first dimension on a
14
cm IPG strip, pH
3.0
-
10.0
and in the second dimension on a 12.5% acrylamide SDS-gel. The Isoelectric points (
pI
) and molecular mass (in
kDa
) are noted. Color coding: green spots indicates protein abundance is high in
Cy3,
red spots indicates protein abundance is high in
Cy5,
yellow spots
indicates
where protein abundance is similar in both the cases.Slide8
Highest percentage of proteins
that are differentially expressed were involved known to be involved in Photosynthesis, ATP synthesis, and Carbohydrate metabolism.What we have foundDifferential expression of different categories of proteins during senescence processSlide9
18910Number of differentially expressed proteins in PCGNumber of differentially expressed proteins in SGUP: 4Down: 5UP: 5Down: 13UP: 5Down: 5Number of differentially expressed proteins in PCG and SG during senescence when the ratio of proteins was observed after/before senescence
19
proteins are differentially expressed in PCG, whereas 28 proteins are differentially expressed in SG. Among those proteins 10 proteins are common in both PCG and
SG
PCG
SGSlide10
2
1) Putative aconitate hydratase, cytoplasmic (ACOC_ORYSJ); 2) Ribulose bisphosphate carboxylase large chain (RBL_SETIT); 3) Ribulose bisphosphate carboxylase large chain (RBL_SETIT); 4) Ribulose bisphosphate carboxylase large chain (RBL_AVESA); 5) unknown (gi|223974857); 6) Oxygen-evolving enhancer protein 1, chloroplastic (PSBO_HELAN); 7) glutathione S-transferase (gi|46276327); 8) Ribulose bisphosphate carboxylase large chain (RBL_LIQST); 9) hypothetical protein (gi|413933720); 10) hypothetical protein (gi|147843505);Proteins differentially expressed with same pattern during senescence in all four cultivars of PCG and SG B/A for Early SG, D/C for Late SG, F/E for Early PCG,
and H/G
for late
PCG. Slide11
β
-Ketoadepyl CoA thiolaseAconitase hydrataseSuccinate dehydrogenaseCysteine proteaseSucrose phosphate synthaseUp-regulation in conversion of starch, lipids, and proteins to hexoses and towards sucroseSignal for source to sink translocation, early floral development, and early senescence
TCA cycle
Glyoxylate
cycle &Gluconeogenesis
β
-oxidation
Proteolysis
A new hypothesis being developed for early/delayed senescence in perennial grasses
Proposed model
during
the
senescence, which
signals for early floral development, translocation of sugars from source to
sink.
We
found
up-regulation
of
five
proteins,
during the process of senescence whereas 3-proteins: β-
Ketoadepyl
CoA
thiolase
, Cysteine protease, and Sucrose phosphate synthase were constitutively
down-regulated
in late
senescing.Slide12
Constitutively overexpressed photosynthesis machinery in “late senescing” cultivar of PCG compared to “early senescing”Slide13
Number
Protein Name1Transketolase, chloroplastic OS=Zea mays PE=1 SV=12 Sedoheptulose-1,7-bisphosphatase, chloroplastic OS=Triticum aestivum PE=2 SV=1 3 Fructose-bisphosphate aldolase, chloroplastic OS=Oryza sativa subsp. japonica GN=Os11g0171300 PE=1 4 glutathione S-transferase GSTF14 [Oryza sativa Japonica Group] 5 S-adenosylmethionine synthase 1 OS=Brassica juncea GN=SAMS1 PE=2 SV=1
6
Ras
-related protein RABB1b OS=Arabidopsis thaliana GN=RABB1B PE=2 SV=1
7
14-3-3-like protein OS=
Pisum
sativum
PE=2 SV=1
8
Oxysterol
-binding protein-related protein 1D OS=Arabidopsis thaliana GN=ORP1D PE=2 SV=1
9
Probable sucrose-phosphate synthase 1 OS=
Craterostigma
plantagineum
GN=SPS1 PE=2 SV=1
10
beta-
ketoadipyl
CoA
thiolase
[
Leptothrix
cholodnii
SP-6]
11
cysteine protease 1 precursor [
Zea
mays]
12
Putative cytochrome c oxidase subunit II PS17 (Fragments) OS=
Pinus
strobus
PE=1 SV=113ATP synthase subunit alpha, chloroplastic OS=Saccharum hybrid GN=atpA PE=2 SV=214ATP synthase subunit beta, chloroplastic OS=Sorghum bicolor GN=atpB PE=3 SV=115Oxygen-evolving enhancer protein 1, chloroplastic OS=Solanum lycopersicum GN=PSBO PE=2 SV=2Ratio of ExpressionSlide14
WRKY Genes and Sencescence
Functional genomic studies of individual WRKY transcription factors has provided clear evidence that specific WRKY proteins are regulators of senescenceHere, we identify the members of the WRKY gene family that are present in Version 1.1 of the genome sequence of switchgrass. We identified 191 full length WRKY genes and named them PviWRKY1-PviWRKY191 using TOBFAC pipeline that we used to find the WRKY gene family in Brachypodium distachyon (Tripathi et al., 2012)In addition, we found an additional 49 WRKY-containing sequences that did not encode a full length gene. The incomplete WRKY genes were named PartialWRKY1-Partial WRKY49 and will be added to the list of complete genes or pseudogenes when additional sequence data become available.A combined phylogenetic tree of all switchgrass and Arbabidopsis WRKY domains and several other senescence-inducible genes from other plants. The senescence-associated Eigengene Set 13 switchgrass genes are indicated in red.Slide15
Senescence associated WRKY genes from switchgrass
Senescence associated WRKY genes from switchgrass and other plants.SwitchgrassArabidopsis*Other plants*The other plants include rice, banana, and Medicago truncatulaSlide16
Conclusion
Determining the molecular genes/proteins associated with the senescence in perennial grasses.2. We have identified 10 genes through proteomics approach that could serve as functional markers in screening SG and PCG germplasm for delayed senescence.3. We are in the process of elucidating the senescence molecular pathway in perennial grasses.Slide17
Acknowledgements
Xijin Ge, Bioinformatics expert and collaboratorMoustafa Eldakak, Manali Shirke