Expression profiles of genes involved in fatty acid and triacylglycerol synthesis in developing seeds of Jatropha (Jatropha curcas L.)

Abstract

Jatropha (Jatropha curcas L., Euphorbiaceae), a potential biodiesel plant, has created tremendous interest all over the world for the use of its seed oil as a commercial source of biodiesel. Due to the unreliability of oil content in its seeds and low economic returns planting of jatropha in agriculture was restricted. Investigating the molecular basis of storage lipid accumulation during seed development is an immediate need to understand genetic factors regulating storage lipid biosynthesis in jatropha seeds. In this study, we characterized the seed development and lipid accumulation from female flowers pollinated to mature seeds, and investigated temporal expression profiles of 21 lipid genes involved in different steps of the pathways leading to fatty acid and TAG synthesis within jatropha developing seeds using quantitative real-time PCR technology. Concomitantly, 17 genes increased their expression levels in developing seeds compared to their expression in leaf, but showed various temporal expression patterns and different relative-maximum ratio ranging from 2.8 to 1,919,280-fold in developing seeds. Five gene groups with distinct temporal patterns were identified by clustering analysis of expression data. Two gene groups including 15 genes presented up-regulated expression patterns correlated with storage lipid accumulation in developing seeds. This study provided not only the initial information on promoter activity for each gene, but also a first glimpse of the global patterns of gene expression and regulation, which are critical to understand the molecular basis of lipid biosyntheses, identifying the rate-limiting genes during seed development and to create improved varieties by genetic engineering.

Introduction

Jatropha (Jatropha curcas L., Euphorbiaceae) is a great potential biodiesel crop, commonly known as purging nut or physic nut, and native to Mexico and Central America. Due to its wealth seed oil in the form of triacylglycerol (TAG) that serves as biodiesel feedstock and its growth property such as drought hardiness, small gestation period and wide adaptation to soil conditions, jatropha has created tremendous interest all over the world for the use of its seed oil (storage lipids) as a commercial source of biodiesel [1], [2], [3], [4]. Currently, jatropha has been planted as a biodiesel crop in India, China, South America and Africa [5]. However, jatropha is still an undomesticated plant with unreliable seed yield and seed oil content and low economic returns in agriculture. The available germplasm growing in the wild lacks genetic information and has low genetic diversity [5]. There is an immediate need for genetic enhancement and metabolic engineering of jatropha which could create improved varieties to serve as its application in agriculture [6]. Investigating the molecular basis of storage lipid (i.e. TAG) accumulation during seed development is an essential prerequisite to understand genetic factors regulating storage lipid biosynthesis in jatropha seeds and to create improved varieties by genetic engineering.

Although molecular mechanism of storage lipid biosynthesis in higher plants may be variable, in general, the pathway of storage lipid biosynthesis mainly includes two conceptually simplified systems: fatty acid (FA) synthesis and TAG assembly. The step of FA synthesis produced a common and unique system intermediate, cytosolic acyl-CoA, using direct or indirect products of photosynthesis in plasmids. The TAG assembly sequentially consumed the acyl-CoA produced in the front system using substrate Glycerol 3-phosphate (G-3-P) in Endoplasmic reticulum (ER) [7]. The latter step was called as the Kennedy Pathway [8]. In developing seeds, quality and quantitative of the storage lipids synthesized depends on the composition of its constituent TAGs and a number of enzymes which participated in FA synthesis and transfer during photosynthesis in leaf and TAG assembly during the Kennedy Pathway [9], [10], [11]. Such control is exerted at the level of de novo FA synthesis in the plastids, in subsequent modification reactions on these FAs and at the level of microsomal acyltransferases in ER. We had known much about the individual enzymes and of genes coding their activities [11], and useful changes in TAG quality or yield have been made by genetic manipulation [12], [13], [14], [15]. However, little is known about the overall expression and regulation profiles of multiple genes involved in lipid biosynthesis from de novo carbon flux to TAG during seed development [16], [17]. This aspect of storage lipid accumulation is important for attempts to identify the key rate-limiting enzyme genes which regulate storage lipid accumulation during seed development in crops. In particular, raising overall oil yields from oleaginous seeds for providing feedstock for biodiesel production by genetic manipulation technology is a critical issue to facilitate the development of biodiesel in industry. Jatropha is a potential woody biodiesel plant, but the main problem associated with jatropha seeds is its oil content is unreliable [5]. Little is known about its lipid biosynthetic enzymes or how FA and TAG biosynthesis is regulated in jatropha seed [18]. Investigating the molecular basis of storage lipid accumulation during seed development is necessary to understand the physiological reasons for affecting unreliability of oil content in seeds and identify the rate-limiting enzyme genes which regulate storage lipid biosynthesis from de novo carbon flux to TAG during seed development.

Current efforts on genetic enhancement and metabolic engineering of oilseeds have been shifted towards searching for additional genes or general transcription factors that may up-regulate multiple activities or entire pathways leading to oil biosynthesis [13], [19]. Therefore, knowledge of the expression overview of multiple genes and their regulation during Jatropha seed oil biosynthesis is needed to further understand the regulatory mechanisms controlling storage lipid accumulation. In this study, we obtained 21 genes involved in different steps of the pathways leading to fatty acid and TAG synthesis, and characterized their expression profiles during the time-course of seed development. Our results provided not only the initial information on promoter activity for each gene, but also a first glimpse of the global patterns of gene expression and regulation, which are critical to understand the molecular basis of lipid biosyntheses and identify the rate-limiting genes which regulate storage lipid biosyntheses during seed development in jatropha.