1. EXPLOITING DUCKWEED AS A SOURCE OF BIO-ENERGY Yiheng Yan, Hai Shi, Jorg Schwender and John Shanklin Brookhaven Natl Lab Evan Ernst, Rob Martienssen Cold Spring Harbor Lab Alex Krichevsky, Vitaly Citovsky SUNY Stony Brook Picture by Wenqin Wang

2. G OAL S TATEMENT Develop aquatic flowering plants (Lemnaceae) as alternatives to algae for biofuel feedstocks Determine the genome sequence of several duckweed species, and screen for lipid content Develop genetic tools to manipulate oil content in fast growing duckweed species

3. Q UAD C HART O VERVIEW Start date: 9/3/2010 End date: 9/2/2012 Percent completion: 20% Barriers addressed Ft-C Feedstock genetics and development Total project funding –DOE share $2,750,000 –Contractor share $692,568 Funding received in FY09 $0 Funding for FY10 $2,750,000 ARRA Funding $0 Timeline Budget Barriers Co-PIs R. Martienssen (CSHL), John Shanklin (BNL), Jorg Schwender (BNL) Vitaly Citovsky (SUNY Stony Brook) Project management: R.Martienssen (CSHL) Partners

4. P ROJECT O VERVIEW Lemnaceae (duckweed) are the world’s smallest, but fastest growing aquatic flowering plants Used for basic research, environmental monitoring and waste water remediation Very high rates of biomass accumulation make them an attractive target for engineering biofuel feedstocks

5. Prospect Park, New York DUCKWEED IS A CLONAL AQUATIC PLANT

6. BUT CAN ALSO PRODUCE FLOWERS…

7. Duckweed “resting fronds” or turions make more starch than corn kernels, and may be persuaded to make oil

8. 1- APPROACH Determine gene catalog of various species of duckweed selected for oil content Identify candidate genes that are known to regulate the components of the oil biosynthetic pathways Develop tools for gene manipulation Establish an efficient transformation protocol to enable rapid testing and screening of genes of interest

9. 2 - T ECHNICAL A CCOMPLISHMENTS / P ROGRESS /R ESULTS Key milestones addressed so far A1 - Establish database schema and website for duckweed research A2 - Collect paired-end sequence read data from >10 species of duckweed A3 - Assemble first Lemna genome sequence and annotate B4 -Screen growth conditions for factors that increase oil accumulation B6 - Establish and characterize transient expression of genes in duckweed B7 - Use determined genome sequence to identify gene targets for manipulation of expression levels in duckweed B8 - Identify potential genes for heterologous expression in duckweed C - Development of Stable Genetic Transformation System for Lemna

10. A1. We are constructing a web portal for duckweed research. We held an international meeting to discuss this in October 2009, and obtained agreements from A. Stomp and E. Lam to provide literature and strain information from the international stock center (Rutgers). Sequence access will also be provided through this portal. A2, A3. We have obtained a draft sequence of Lemna gibba genome (450Mb), transcriptome and small RNAs, and we have identified 9 candidate genes whose orthologs regulate fatty acid production in vegetative tissues of Arabidopsis A. Lemna gibba genome sequencing

11. 11 Lemna gibba genome sequencing: Lemna gibba G3 DWC131 data collection LibraryMean est. (bp)Std. dev. (bp)Reads (M)GAxII Lanes 300 bp fragment26123.5593 x 101 bp PE 400 bp fragment37324.21023 x 101 bp PE 500 bp fragment49036.21503 x 101 bp PE 5 Kb mate pairTBD 251 x 76 bp PE Total sequenced: 33 Bbp Raw coverage: ~74x # >200nt# >100KntN50 (bp)NG50 (bp)Longest (bp) Size (Mbp) Contigs471,436-1,8761,47633,244401 Scaffolds140,4995416,08518,907270,981507 Lemna gibba G3 DWC131 Assembly (450 Mbp)

12. Lemna gibba transcriptome sequencing: Lemna gibba G3 poly A+ RNA-seq # >152ntN50 (bp)L50 #Longest (bp)Size (Mbp) PE Contigs31,30563866606,19514.8 Lemna gibba G3 poly A+ transcript assembly

13. PICKLE: A REGULATORY GENE FOR OIL BIOSYNTHESIS

14. Aims B and C: efficient transformation and transient expression systems The lack of facile genetic transformation is a barrier to progress in realizing duckweed’s bioenergy potential Transformation through callus has been reported, but takes 3-4 months and is expensive and easily lost to contamination We have made progress in establishing an efficient transformation protocol to enable rapid testing and screening of genes of interest This is the critical enabling technology on which the entire program depends and was therefore assigned highest priority Cordula Kruse et al. Aquatic Botany 72(2002)175-181, Vunsh, R; Li, JH; Hanania, U, et al, Plant Cell Reports 26(2007)1511-1519 Yamato YT et al. IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT37(2001) 349-353 J.Li et al. Plant Cell Rep(2004) 22:457- 464

15. CURRENT APPROACH Duckweed proliferate vegetatively We hypothesize that by directly transforming the meristem cells we will be able to obtain stably transformed daughter fronds Current protocol utilizes direct infiltration of Agrobacterium solution into the meristem region of mature fronds Picture by Wenqin Wang

16. INFILTRATION OF DUCKWEED FROND USING 5 ML SYRINGE

17. SPIRODELA POLYRHIZA EXHIBIT STRONG SILENCING MECHANISM 100 individual Spirodela polyrhiza fronds were transformed using the direct infiltration method with an construct containing the GFP reporter gene Number of fronds expressing GFP drops continuously over time and none of the fronds initially transformed retains GFP expression after 23 days Percent of fronds of Spirodela polyrhiza transformed with 35S:GFP

18. INTRODUCING P19 PROTEIN Originally discovered in Cymbidium ringspot virus Has been shown to be an RNA-silencing suppressor P19 suppresses RNA silencing by sequestering siRNAs Would this protein extend the time of GFP expression in duckweed? Lorant Lakatos et al, The EMBO Journal(2004) 23, 876-884

19. EXPRESSION OF P19 PROTEIN STABILIZES TRANSGENE EXPRESSION Fronds transformed with construct containing both GFP and P19 retain GFP expression for the life of the frond + P19 - P19 Percent of fronds of Spirodela polyrhiza transformed with 35S:GFP +/- P19

20. STABLE TRANSFORMANTS ARE RAPIDLY ACHIEVED AND OCCUR AT HIGH FREQUENCY 33% of fronds transformed with GFP+P19 produce lines that retain GFP expression across >4 generations Entire transformation process including selection takes 4 weeks

21. 3 - R ELEVANCE Our goal is to increase oil yield in aquatic flowering plants, whose biomass and growth characteristics are uniquely suited as biofuel feedstocks Lemna spp. are already in widespread commercial use for animal feed and bioremediation of waste water Increased oil yield will therefore impact the economics of oil production from biofuels in the near term The genome sequences, and genetic tools for manipulation that we develop will be applied to improving aquatic plants as biofuels long after the project is complete

22. 4- CRITICAL SUCCESS FACTORS We have established a facile and efficient transformation protocol that does not rely on transformation of callus tissues and regeneration with high transient transformation efficiency (60%+) and significant stable transformation rate (33%) in ~4 weeks We have determined the sequence of Lemna gibba and identified genes, promoters and miRNA for gene knockdown We are now in a position to rapidly test and screen for genes we hypothesize will increase oil accumulation in vegetative tissues

23. REMAINING OBSTACLES AND FUTURE RESEARCH Though GFP expression is retained through >4 generations, expression of the transgene is biased towards expression in vascular tissues This likely results from enhanced vascular tissue- expression of the 35S promoter in monocots We will switch to using native duckweed promoters to achieve more uniform transgene expression

24. S UMMARY We have completed the first genome and the first transcriptome sequence of Lemna gibba, and we can now compare this with other species being sequenced here and elsewhere ( Spirodela polyrhiza, at JGI) We have used the sequence to identify candidate genes that regulate fatty acid biosynthesis, as well as tools (microRNA) with which they can be manipulated We have achieved stable transformation of duckweed allowing these genes to be manipulated and tested Future work will profile lipids and genes from a variety of species to optimize oil production Our work will form the basis for a database of sequence and network model information to maximize biofuel potential

25. A DDITIONAL S LIDES

26. Total Lipid Total fatty acids are about 3.5ug/mg FW, or about 0.35%. Per fresh weight. Assuming the tissue is 90% water, then this increases to about 3.5% on a DW basis

27. At leaf 5 At Seeds DWC 202DWC 204DWC 206DWC 207DWC 208DWC 211 origin TAG A neutral lipid TLC experiment indicated the presence of TAG in Duckweed cultures The effects of stress were most pronounced in one line. While the increase in TAG is 3-4 fold, it only raises the TAG percent (relative to fresh weight) from 0.0025% to 0.01%. Assuming 90% of the fresh weight is water this would be more like an increase from 0.025% to 0.1% DW compared to dry Arabidopsis seeds which contain around 40%. Spirodela polyrrhiza (Giant Duckweed) Contains TAG and Stores More In Response To Nutrient and Cold Stress

28. P19 ENHANCES EXPRESSION LEVEL OF GFP Addition of P19 not only prolongs GFP expression of transformed fronds, but also increases the expression level of the transgene -P19 +P19

29. Result (i.e. object number surface area, etc…) MONITORING LEMNACEAE GROWTH BY LEAF AREA MEASUREMENTS

30. LEMNACEAE GROWTH CURVES UNDER DIFFERENT GROWTH CONDITIONS Lemna gibbaSpirodela polyrrhiza +G -G +G -G Legend: +G supplemented with glucose -G not supplemented with glucose Next steps: Analyze fatty acid content and lipid composition Construct metabolic network model Perform metabolic flux analysis

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