The transcriptome of the developing grain: a resource for understanding seed development and the molecular control of the functional and nutritional properties of wheat

Human survival has relied heavily upon the food contained in the seeds of the three major cereals, rice, wheat and maize since the beginnings of agriculture [1]. Wheat (Triticum aestivum L.) feeds nearly 30% of the world population [2]. Bread wheat has a unique genetic makeup, being an allohexaploid (2n = 6× = 42) with three sub-genomes, A, B, D, and a novel seed composition due especially to the presence of the gluten proteins with characteristic features that enable the production of diverse food products [3]. The wheat caryopsis (dry seed) is comprised of three key tissues, the pericarp, embryo, and endosperm [4]. Anatomically, the largest part of the mature seed, the endosperm, consist mainly of starchy endosperm cells with an outer layer specialized layer known as the aleurone [5]. As the wheat grain matures, the viability of the starchy endosperm cells is lost while that of the aleurone cells is maintained [6]. The starchy endosperm cells contain 60-70% starch and 10-15% storage proteins and form the main food source in the seed [7].

The development of the wheat caryopsis begins following anthesis (flower development) and pollination and proceeds until a physiologically mature seed is formed. Based on morphological characteristics the process can be divided into distinct developmental stages [8] that have been classified, based upon the presence of distinguishable botanical structures, into five distinct stages, tissues undifferentiated (0-7 – days post anthesis (dpa)), embryo differentiation (7-14dpa), lateral root primordia initiation (14-21dpa), caps on lateral roots with appearance of primary leaves (21-31dpa), and fully differentiated (31-50dpa) [9]. Based on growth phases these five botanical stages have been placed into three distinct phases, grain enlargement (0-14dpa, Zadok’s scale: Z69-Z75), grain filling (15-35dpa, Z75-Z87), and physiological maturity (36-50dpa, Z87-Z92) [10, 11]. However this categorization may vary with genotype, growth environment, temperature, and other factors [12]. Structural and anatomical features of the maturing wheat caryopsis like the development of transfer cells [13], endosperm [4, 5, 7, 14–20] and aleurone [21] have been well studied. Accumulation of starch and storage protein in the starchy endosperm contributes 70–85% of the total grain yield [7] and has been intensely studied [4, 5, 15, 16, 18, 20, 22].

Although the anatomy and composition of the wheat caryopsis has been well studied there are few reports on analysis of the role of gene expression in controlling grain formation. There are studies of the transcriptome using microarray [23–25] and NGS based methods [26–28]; but each study investigated only a single cultivar. In a continuation of our earlier work identifying differentially expressed genes that underlie aleurone and starchy endosperm differentiation [29]; we now report a study of genes expressed during seed development across wheat cultivars.

We report here, transcriptome profiling of the wheat caryopsis studied at two stages, at the boundary of grain enlargement and grain filing (14dpa, representing early grain filling) and at physiological maturity (30dpa, representing late grain filling) using RNA-Seq for 35 diverse cultivars (Fig. 1) [30]. The differences in expression and functional roles of these genes were characterized [31, 32] and their metabolic roles analysed [33]. In addition to explaining the role of gene expression in seed development, this study provides a platform for analysis of genetics of grain traits associated with grain yield, functional performance in food products and nutritional value in human and animal diets.

Early analysis of differentially expressed genes in this data set facilitated the discovery of a gene associated with bread making quality (wbm) of wheat [35] that led us to study the global gene expression pattern across all genotypes of this study between the 14dpa and 30dpa stages. More recently, pathway mapping of this data through KEGG helped us discover a C₄ pathway of photosynthesis without Kranz anatomy in wheat grains [36]. A compilation of the differentially regulated genes between 14dpa and 30dpa stages is now presented in this article (Additional file 14; Table 4). Based on the metabolism in which the pathways were involved, the 73 pathways were grouped into nine broad categories, nucleotide metabolism; amino acid metabolism; carbohydrate metabolism; respiratory pathways; photosynthesis; lipid metabolism; hormone biosynthesis; vitamin metabolism; and specialized metabolism (Table 3; Additional file 14). Although some metabolic pathways are highly conserved across taxa [37] like carbohydrate metabolism, respiratory pathways and nucleotide metabolism, others are highly diversified and specialized in some taxa [38].

Carbohydrate metabolic processes and photosynthesis are the dominant biological process ontologies identified at the 14dpa stage suggesting the likely importance of ear photosynthesis in determining grain yield [39, 40]. Phosphohexokinase and hexokinase type IV glucokinase are the key regulatory genes involved in carbohydrate metabolism differing between early and late grain filling stages. Transaminase that converts serine to hydroxy-pyruvate leading to glycerate formation (glyoxylate and dicarboxylate metabolism) is the key differentially expressed gene during early-grain filling that is linked with the photorespiration process (Table 4; Additional files 14 and 19). This data from carbohydrate metabolism also correlates well with photosynthesis and photorespiration being tightly linked and differentially regulated during the early-grain filling stage (14dpa). Key genes that are expressed differentially in carbohydrate metabolism, phosphohexokinase and hexokinase type IV glucokinase are also involved in respiratory metabolism (Additional file 14).

Photosynthesis is one of the crucial metabolic process that directly impacts on yield of the wheat grain. Genes involving porphyrin and chlorophyll metabolism are differentially expressed during early grain filling stage (Table 4). Genes involving C₄ photosynthesis are differentially expressed between early and late grain filling stages leading us to discover the C₄ photosynthetic pathway in the pericarp tissues of wheat grain [36]. The anatomy with which non-Kranz C₄ photosynthetic pathway being accomplished were termed as Bose anatomy [41] in honour of his discovery on the role of malic acid (4C) in photosynthesis in Hydrilla sp. that was much later known to be single-cell C4 type. The RuBisCO small subunit was also differentially expressed during different developmental stages (Table 4; Additional file 14).

Amino-acyl tRNA biosynthesis with its ligases and synthase (glutamine-hydrolysing) are the key differentially expressed genes between early and late grain filling in nucleotide metabolism (Table 4 and Additional file 19). Transaminase is the key gene being differentially regulated for various types of amino acid metabolism (Table 4 and Additional file 14). It is also the best example of a transcripts that is being differentially expressed in different metabolic pathways of amino acid metabolism (Additional files 14 and 19). Lactoperoxidase of phenylalanine metabolism under amino acid metabolism is the best example for the transcript IDs that are differentially expressed across the early and late grain filling stages (Additional files 14 and 19). Tetrahydrofolate (THF) dependent aminomethyltransferase (EC2.1.2.10) of glycine, serine and threonine metabolism is a key differentially expressed gene in D14 and D30 stages (Table 4) and involved in photorespiration process of glycine decarboxylase system (GDS) by being the T-subunit component [42]. Similarly, P-subunit of GDS, dehydrogenase (aminomethyl-transferring; EC1.4.4.2) from glycine, serine and threonine metabolism is the key differentially expressed gene only during the D14 stage and is involved in the photorespiration process (Table 4).

Phospholipase A2 is the key differentially expressed gene that is involved in multiple pathways of lipid metabolism, glycerophospholipid metabolism, ether lipid metabolism, arachidonic acid metabolism, linoleic acid metabolism, and alpha-linolenic acid metabolism (Table 4). Except for ether lipid metabolism which is differentially expressed only during 14dpa stage, the others are differentially expressed both at 14dpa and 30dpa stages with different transcript IDs during different developmental stages (Additional file 14). Different hydrolase genes that are pathway specific and involved in fatty acid biosynthesis and fatty acid elongation pathways are differentially expressed at 14dpa stage; while ligase genes are differentially expressed at 14dpa stage and involved in fatty acid biosynthesis and degradation pathways (Table 4).

The monooxygenase gene is the key differentially expressed transcript at 14dpa stage involved in the steroid biosynthetic pathway and categorized under hormone biosynthesis (Table 4; Additional file 14). There are other monooxygenase genes that are differentially expressed and involved in amino acid metabolism, lipid metabolism and specialized metabolism and hence caution is required while manipulating this gene. Reductase is the key differentially expressed gene and rate limiting step during late-grain filling for one carbon pool by folate pathway being categorized under Vitamin metabolism (Table 4; Additional file 14). It is involved in conversion of folate to dihydrofolate and tetrahydrofolate (THF) – involved in photorespiration – involved in glycine, serine and threonine metabolism. This suggests that silencing of reductase (EC1.5.1.3) in plants might downregulate THF formation and in turn possibly minimize photorespiration. However, THF is known to be involved as a co-factor in various other one carbon metabolic processes like purine biosynthesis, pantothenate biosynthesis, organellar protein biosynthesis, and methionine biosynthesis [43]. So, how far the downregulation of reductase could be used in selectively restricting photorespiration during the late-grain filling stages without affecting the normal physiology of the plant is unclear. Ammonia-lyase and geranyl-diphosphate synthase respectively for phenylpropanoid and terpenoid backbone metabolic pathways involved in specialized metabolism are key genes that are differentially expressed at the 14dpa and 30dpa stages (Table 4; Additional files 14 and 19).

This study provides an atlas of transcripts expressed during early (14dpa) and late (30dpa) grain filling in wheat. Among those, the key transcripts involving various metabolic pathways that are significantly differentially expressed at grain filling stages during wheat grain development were compiled (Additional file 14). Next generation sequencing (NGS) based transcriptome analysis (RNA-Seq) in combination with functional annotation has proved to be a very robust tool helpful in discovering novel genes such as the wheat bread making gene [35] and genes controlling flour yield and entire metabolic pathways (grain specific C₄ photosynthesis) [36], improving our understanding on the complex metabolic networks at different developmental stages. The present resource will facilitate breeding selection for key genes in a metabolic pathway or a key metabolic pathway in a network involving specific developmental stages. Selection of genes expressed during grain filling identified in this dataset might be used for improving yield and grain quality. For example, a focus on silencing the genes that are involved in photorespiration, H-subunit (protein component with no independent catalytic activity) or P-subunit (Aminomethyl-transferring dehydrogenase) or T-subunit (THF dependent aminomethyltransferase) might be helpful in improving grain yield through reduced photorespiration [44]. Reducing photorespiration might be a significant contribution to achieve the goal of 20:20 Wheat® [45] - to achieve the productivity of 20 t per hectare in 20 years - through increased photosynthetic efficiency.