1. Gene discovery for delayed senescence in bioenergy crops to improve total biomass production
Bimal Paudel, Mike Tran
Jai Rohila, Jose Gonzalez, Arvid Boe, Gautam Sarath, Paul Rushton
South Dakota State University, Brookings, SD

2. Biomass Senescence rate PCG-SD PCG-ND
Differences for biomass production and level of senescence between the PCG-SD and PCG-ND populations during late September 2013.
Biomass
Senescence rate
PCG-SD
PCG-ND

3. Bottleneck for the genetic improvement programs:
Situation:
Untimely senescence in perennial grasses causes low biomass harvest
Bottleneck for the genetic improvement programs:
Little knowledge of molecular markers or gene functions
Research Question:
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.

4. Before 3rd week of August
Experimental Design
Before 3rd week of August
Pre-Senescence
Post-Senescence
Winter
Last week of September
Spring
Pre-Senescence
Post-Senescence
Pre-Senescence
Post-Senescence
Chlorophyll Data

5. Samples for the Proteomics Experiment
Tissue
Treatment
Group
Sample# 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) C
Sample# 7
Sample# 8
Sample# 9 D
Sample# 10
Sample# 11
Sample# 12 3
Prairie Cordgrass-ND E
Sample# 13
Sample# 14
Sample# 15 F
Sample# 16
Sample# 17
Sample# 18 4
Prairie Cordgrass-SD G
Sample# 19
Sample# 20
Sample# 21 H
Sample# 22
Sample# 23
Sample# 24

6. Proteomics Workflow Early Senescence Late Senescence Pre-Sene Pre-Sene
Cy3
Cy5
Pre-Sene
Post-sene
Pre-Sene
Post-sene
Analysis by Typhoon TRIO
Quantification by DeCyder
Gel staining by Sypro-Ruby
Spot picked and digested by trypsin
Protein ID by MALDI-TOF MS and NCBI data base search, and GO annotation

7. 1 3 4
Gel-1 6 2 17
263 5 7 8 9 10
276 12 13
A1 / C7 20 21 15 16
277 19 18 11 14 22 24
264
265 28 30 26 29 23 31
278 33 34 25 32 35 36 44 45 46 47
279 48
280
281
282 27 37 39 43 54 49 50 51 57 58
284
285 59 60
283 38 40 41
Protein samples of SG, and PCG leaves, control 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 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. 52
266 56
286 53 62 42 55 63 70 61
267
269 65 81 64 69 71 83 66 73 76 95 84 75 74 82 85 68 72
100 67
268 97
289
288 86 87 77 79
107 96 90
287
104
108 98 92 91 89 88 80
101 78
290
109
270 93 94 99
102
103
105
292
291
294
293
117
121
110
111
113
106
112
126
127
119
120
114
122
115
116
271
118
123
124
125
129
131
128
296
297
138
295
143
130
140
146
147
132
139
144
134
133
137
298
141
142
149
151
145
156
150
152
153
155
136
154
135
158
148
184
299
160
175
181
185
159
163
162
161
157
172
170
174
176
177
182
183
166
168
173
179
180
167
169
300
171
196
187
164
194
197
178
186
189
209
165
193
198
201
204
188
191
192
195
199
200
206
301
190
205
211
272
202
212
203
302
207
208
210
215
216
217
218
213
214
303
226
219
221
223
224
227
229
232
220
305
225
222
273
274
228
304
240
241
230
233
275
239
235
234
237
306
238
231
249
250
253
248
247
236
245
244
242
243
307
251
246
252
309
308
262
254
255
259
260
256
258
261
312
310
257
313
311 7

8. What we have found
Differential expression of different categories of proteins during senescence process
Highest percentage of proteins that are differentially expressed were involved known to be involved in Photosynthesis, ATP synthesis, and Carbohydrate metabolism.

9. Number of differentially expressed proteins in PCG and SG during senescence when the ratio of proteins was observed after/before senescence 18 9 10
Number of differentially expressed proteins in PCG
Number of differentially expressed proteins in SG
UP: 4
Down: 5
UP: 5
Down: 13
PCG SG
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

10. Proteins differentially expressed with same pattern during senescence in all four cultivars of PCG and SG 2
B/A for Early SG, D/C for Late SG, F/E for Early PCG, and H/G for late PCG.
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| ); 6) Oxygen-evolving enhancer protein 1, chloroplastic (PSBO_HELAN); 7) glutathione S-transferase (gi| ); 8) Ribulose bisphosphate carboxylase large chain (RBL_LIQST); 9) hypothetical protein (gi| ); 10) hypothetical protein (gi| );

11. A new hypothesis being developed for early/delayed senescence in perennial grasses
β-Ketoadepyl CoA thiolase
Cysteine protease
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.
β-oxidation
Proteolysis
Aconitase hydratase
Succinate dehydrogenase
TCA cycle
Glyoxylate cycle &Gluconeogenesis
Sucrose phosphate synthase
Up-regulation in conversion of starch, lipids, and proteins to hexoses and towards sucrose
Signal for source to sink translocation, early floral development, and early senescence

12. Constitutively overexpressed photosynthesis machinery in “late senescing” cultivar of PCG compared to “early senescing”

13. Ratio of Expression Number Protein Name 1
Transketolase, chloroplastic OS=Zea mays PE=1 SV=1 2
Sedoheptulose-1,7-bisphosphatase, chloroplastic OS=Triticum aestivum PE=2 SV=1 3
Fructose-bisphosphate aldolase, chloroplastic OS=Oryza sativa subsp. japonica GN=Os11g 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
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=1 13
ATP synthase subunit alpha, chloroplastic OS=Saccharum hybrid GN=atpA PE=2 SV=2 14
ATP synthase subunit beta, chloroplastic OS=Sorghum bicolor GN=atpB PE=3 SV=1 15
Oxygen-evolving enhancer protein 1, chloroplastic OS=Solanum lycopersicum GN=PSBO PE=2 SV=2
Ratio of Expression

14. WRKY Genes and Sencescence
Functional genomic studies of individual WRKY transcription factors has provided clear evidence that specific WRKY proteins are regulators of senescence
Here, 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.
Figure 6. Senescence associated WRKY genes from switchgrass.
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.
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.

15. Senescence associated WRKY genes from switchgrass
Senescence associated WRKY genes from switchgrass and other plants.
Switchgrass
Arabidopsis
*Other plants
*The other plants include rice, banana, and Medicago truncatula
Figure 7. Senescence associated WRKY genes from switchgrass and other plants.
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, senescence-associated WRKY genes from Arabidopsis (blue), switchgrass (red) and other plants (green) are shown with inset close ups of clusters of senescence associated WRKY gene clusters. The other plants include rice, banana, and Medicago truncatula. Higher plants groups of WRKY genes are shown. The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

16. 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.

17. Acknowledgements Xijin Ge, Bioinformatics expert and collaborator
Moustafa Eldakak, Manali Shirke