Developmental changes in Scots pine transcriptome during heartwood formation Kean-Jin Lim, Tanja Paasela, Teemu Teeri University of Helsinki Anni Harju, Martti Venäläinen, Katri Kärkkäinen Luke Punkaharju, June 2016 Heartwood of Scots pine (Pinus sylvestris) is naturally decay resistant Variation in wood extractives correlates with decay resistance — Variation in wood extractives is large and mostly genetic Trees higest in heartwood extractives are most decay resistant. Harju & Venäläinen 2006. Can J. For Res. Heartwood — Does not contain living cells — Reserve materials (e.g. starch) have been converted to ”heartwood substances” — May have different color, lower permeability and increased decay resistance than sapwood — In conifers, heartwood is usually dryer than sapwood Robinia Pinus Picea Heartwood formation Magel 2000 — Type 1 heartwood: Accumulation of (phenolic) extractives takes place in tissue between sapwood and heartwood (transition zone). — Type 2 heartwood: Precursors to phenolics accumulate gradually in ageing sapwood. — Pine heartwood is thought to be of Type 2. Heartwood formation — Much evidence in literature summarizes that heartwood formation takes place during the dormant season. — Pine — From midsummer to autumn (Fukuzawa et al. 1980) — From midsummer to dormant season (Shain and Mackay 1973) — No specific period for heartwood formation (Bergström et al. 1999) Heartwood extractives in Scots pine — Resin acids abietic acid — 50% of heartwood extractives — Stilbenes — 15% of heartwood extractives — Free fatty acids pinosylvin — 35% of heartwood extractives linoleic acid oleic acid Bergström 2003: Forestry 76, 45-53. Induction of stilbene biosynthesis — Developmental during heartwood formation — Wounding — Ultraviolet radiation — Ozone — Pathogens Harju et al. 2009. Tree Physiology 29(1):19-25. Important questions — Do developmental and induced synthesis capacity correlate? — Can we do early selection for durable pine heartwood? — DNA markers — Seedling testing Developmental regulation of pinosylvin biosynthesis — Adult trees sampled by taking increment cores Sampling by year rings 1 2 3 4 Annual rings 5 1 2 3 5 6 7 8 6 4 7 8 Pine increment core harvested on 02 June 2010 STS RT-PCR Tree number: 20 Leppävirta stand June 7, 2011 SW TZ TZ SW L335 L285 H415 H328 STS RT-PCR TZ SW TZ SW TZ SW TZ SW TZ: Transition zone SW: Sapwood STS RT-PCR L335 L285 H415 H328 Ubiquitin ligase RT-PCR TZ SW TZ SW TZ SW TZ SW (reference gene) Ubiquitin ligase RT-PCR RNA-seq transcriptome library pipeline Wet lab Dry lab Raw colourspaceFASTA read files Total RNA isolation ribosomal RNA depletion Transcriptome library preparation RNA-seq sequencing, SOLiD platform RNA-seq transcriptome library pipeline Wet lab Dry lab Raw colourspaceFASTA read files Total RNA isolation Referenced mapping SHRiMP2 ribosomal RNA depletion Mapped reads - SAM --> --> sorted BAM Picard tools - Mark duplicates Transcriptome library preparation Differential expression analysis edgeR RNA-seq sequencing, Downstream analysis SOLiD platform Blast2GO etc. Genes in Scots pine transition zone: Upregulated Downregulated Genes upregulated in Scots pine transition zone Genes downregulated in Scots pine transition zone Biosynthesis of pine stilbenes OMT Stilbene pathway Pinosylvin biosynthesis pathway RefTag FC Annotation logFC logCPM FDR H1TZ H2TZ L1TZ L2TZ H1SW H2SW L1SW L2SW TC154526 13 Phenylalanine Ammonia Lyase 3,714 7,116 1E-46 653 772 977 618 37 72 56 47 TC999999 13 4-Coumarate--Coa Ligase 3,739 3,465 4E-23 72 65 58 35 5 5 1 4 TC154538 13 Pinosylvin Synthase 3,728 7,757 1E-30 1332 1174 1325 955 37 146 45 98 TC999999 5 O-Methyltransferase 2,212 7,118 4E-21 753 894 656 430 122 194 125 98 TC188881 9 S-Adenosylmethionine Synthetase 3,179 4,82 6E-27 152 166 192 85 14 21 12 12 Biosynthesis of diterpene resin acids diTPS Abietadiene synthase CYP720B Abietadienol/abietadienal oxidase, a multisubstrate, multifunctional cytochrome P450 enzyme Zulak KG, Bohlmann J (2010) J. Integr. Plant Biol. 52, 86–97. Biosynthesis of diterpene resin acids Resin acid biosynthesis RefTag Annotation H328TZ H415TZ L285TZ L335TZ H328SW H415SW L285SW L335SW BQ698297 Diterpene synthase 14 3 11 2 13 20 9 2 BX677599 Abietadiene synthase 0 0 0 0 3 0 0 0 CF475293 Diterpene synthase 0 0 0 0 0 0 0 0 CF475347 Diterpene synthase 2 0 1 2 2 1 1 0 CF478561 Levopimaradiene/abietadiene synthase 4 1 5 0 5 6 4 1 CK909868 Diterpene synthase 3 0 6 0 5 9 5 4 CT578084 Abietadiene synthase 5 1 5 0 18 12 14 3 GW772599 Abietadiene synthase 2 0 0 0 3 4 4 2 TC155445 Diterpene synthase 37 1 13 1 32 20 7 3 TC156847 Abietadiene synthase 3 2 9 0 2 3 4 0 TC158139 Diterpene synthase 33 0 6 1 41 30 29 6 TC167434 Levopimaradiene/abietadiene synthase 9 1 18 0 17 2 5 0 TC183034 Levopimaradiene/abietadiene synthase 1 0 0 0 0 0 0 0 TC183398 Levopimaradiene/abietadiene synthase 9 3 3 0 11 10 9 3 TC184285 Diterpene synthase 17 3 11 0 21 14 4 4 TC188188 Levopimaradiene/abietadiene synthase 0 1 1 0 4 1 0 0 TC188572 Diterpene synthase 1 0 0 0 0 1 0 0 TC189520 Diterpene synthase 13 0 1 0 20 14 13 1 TC196667 Diterpene synthase 18 9 13 4 23 26 32 8 TC198006 Diterpene synthase 21 29 25 17 33 30 16 15 Year around sampling, selected genes — ACO - ACC oxidase — AUX - Auxin responsive — BFN - Bifunctional nuclease — 4CL - 4-Coumarate:CoA ligase — MYB - MYB-like TF — STS - Pinosylvin synthase — NAC - NAC domain TF — OMT - O-methyl transferase — SuSy - Sucrose synthase — TPS - Diterpene synthase — DAHPS - DAHP synthase — CYP - Abietadienol oxidase — PAL - Phenylalanine — Histone ammonia lyase — Actin Stilbene pathway Correlation of expression profiles Tree C Tree D Main observations — Heartwood stilbenes are synthesized in the transition zone during the growing season — From the stilbene point of view, Scots pine heartwood is of Type 1 — Heartwood resin acids are synthesized in the sapwood in early spring — From the resin acid point of view, Scots pine heartwood is of Type 2 — Programmed cell death marks heartwood formation — Ethylene may be involved, but not as a sufficient signal Thanks!.