Identification of GPAT acyltransferases in cork oak

Background Acyltransferases are enzymes with an important role in the synthesis of both cutin and suberin which are part of the lipophilic barriers, such as epidermis and periderm that protect terrestrial plants against water loss and other external aggressions. During secondary growth in woody plants such as cork oak (Quercus suber L.), the epidermis is replaced by a suberized periderm that includes the phellem (cork), phellogen (cork cambium) and phelloderm tissues. In Q. suber the successive formation of phellem following removal at periodic intervals (every 9 years) allows for exploitation of cork oak on a sustainable basis. The main component of cork (45-50%) is suberin, a complex polymer comprising both aliphatic and aromatic domains and associated waxes [1,2]. Despite the physiological importance of suberin, its biosynthetic pathway as well as its deposition remains largely unknown. Since cork oak is a unique species among terrestrial plants due to its remarkable capacity for cork production, it is expected that suberin biosynthesis and deposition are tightly controlled mechanisms. As a first step to start unraveling these control mechanisms we intend to identify and characterize genes coding for the acyltransferases of the GPAT (glycerol-3-phosphate acyltransferase) family, involved in suberin and cutin synthesis in cork oak. Two ESTs highly similar to GPAT5 (EE 743864 and EE 743865) and one EST (EE743668) highly similar to GPAT4 shown to be strongly up-regulated in the suberin-rich phellem of cork oak tree (Q. suber) were first identified by Soler et al. [3]. Material and methods In this work, phellem tissues from small branches with increasing age (1 to 7 years old) were harvested from cork oak and holm oak (a related but cork non-producing species)at the Instituto Superior de Agronomia (Portugal). Tissues collected during different growth periods were also used for analysis: samples collected during a period of high phellogen activity (April – June, 2009 and 2010) and samples collected during the inactive growth period (January, 2010). Total RNA was successfully extracted from these tissues using a protocol described by Reid et al. [4], with minor modifications. cDNA was synthesized using standard procedures and 5’and 3’-RACE are being performed in order to determine the full-length of putative GPAT coding sequences from Q. suber transcriptome. The expression level of GPAT4 and GPAT5 genes was assessed by quantitative RT_PCR in two different seasonal stages (April and June) in periderm cells from 3 year old branches of Q. suber. The Cp values were converted into relative quantities, using the formula, Q=E, where E (the efficiency of the gene amplification for each primer pair) was calculated using the Real-time PCR Miner algorithm.