Diterpenes & Transcriptional Profile of CYPs Genes during Coffea arabica fruit  Development

Date: Sunday, January 12, 2014
Time: 5:20 PM
Room: Pacific Salon 4-5 (2nd Floor)
Luiz F. P. Pereira , Embrapa Café, Londrina, Brazil
Douglas S. Domingues , Instituto Agronômico do Paraná, Londrina, Brazil
Gonçalo Amarante Guimarães Pereira , Genomics and Expression Laboratory - University of Campinas (UNICAMP), Campinas, Brazil
Marcelo Falsarella Carazzolle , Genomics and Expression Laboratory - University of Campinas (UNICAMP), Campinas, Brazil
Osvaldo Reis Junior , Genomics and Expression Laboratory- UNICAMP, Campinas, Brazil
Luiz G. E. Vieira , IAPAR (LBI-AMG), Londrina, Brazil
Maria Brigida dos Santos Scholz , Instituto Agronômico do Paraná, Londrina, Brazil
Cíntia Sorane Good-Kitzberger , Instituto Agronômico do Paraná, Londrina, Brazil
Priscila Mary Yuyama , UFRGS, Montpellier, France
Suzana Tiemi Ivamoto , Instituto Agronômico do Paraná, Londrina, Brazil
Coffee oil is rich in kaurane family diterpenes, mainly cafestol (CAF) and kahweol (KAH), which are related with plant defense mechanisms, nutraceutical and sensorial beverage characteristics. In plants, the cytochrome P450s gene family (CYPs) is usually involved in the synthesis and interconversion of most plant secondary metabolites – which probably includes the diterpenes biosynthesis. Therefore, the aim of this study was to elucidate the CAF and KAH final biosynthesis steps combining biochemical and transcriptional analyses. We measured CAF and KAH by HPLC in flowers as well as fruit perisperm in several stages (30 to 210 days after flowering – DAF) to have the pattern of CAF and KAH tissue accumulation during fruit/grain development. CAF levels were detected mainly in flowers as well as in the perisperm decreasing after 120 DAF. On the other hand, KAH concentration increased with perisperm development reaching a peak at 120 DAF. Based on this HPLC analysis of diterpenes, 12 RNA-Seq libraries were obtained for Coffea arabica cv. IAPAR59: leaves, flowers and perisperm tissue from fruits along development.  A total of 41.881.572 sequences were generated using Illumina, HiSeq2000. After clusterization, 127.600 contigs were formed with an average size of 1264bp.  From those, 65480 were considered unique splicing variants (unigenes). With BLAST analysis we detected more than 250 CYPs  which eight were used for further transcriptional analysis by qPCR in leaves, flowers and fruits in three developmental stages (90, 120 and 150 DAF). For five genes we observed a similar pattern between gene transcription and diterpenes concentration levels. Three CYPs (CaCYP76F2_1, CaCYP82C4, CaCYP74A1) had transcriptional patterns similar to CAF accumulation (most accumulated in flowers). On the other hand, two CYPs (CaCYP71A4_1 and CaCYP701A3) were related with KAH accumulation, lower in leaf and flower, but with increasing detection during fruit development. These five CYPs warrant further investigation as potential candidate genes involved in the final stages of CAF and KAH biosynthetic pathway providing us important clues and valuable information for future analysis of coffee diterpene synthesis. This is the first work with Illumina sequencing of coffee fruit tissue, which is providing important information on key genes related to  enzymes and metabolites  involved in fruit ripening and cup quality.