Transcriptome analysis of grain development in hexaploid wheat

Abstract

Background: Hexaploid wheat is one of the most important cereal crops for human nutrition. Molecular understanding of the biology of the developing grain will assist the improvement of yield and quality traits for different environments. High quality transcriptomics is a powerful method to increase this understanding.

Results: The transcriptome of developing caryopses from hexaploid wheat (Triticum aestivum, cv. Hereward) was determined using Affymetrix wheat GeneChip oligonucleotide arrays which have probes for 55,052 transcripts. Of these, 14,550 showed significant differential regulation in the period between 6 and 42 days after anthesis (daa). Large changes in transcript abundance were observed which were categorised into distinct phases of differentiation (6-10 daa), grain fill (12-21 daa) and desiccation/maturation (28-42 daa) and were associated with specific tissues and processes. A similar experiment on developing caryopses grown with dry and/or hot environmental treatments was also analysed, using the profiles established in the first experiment to show that most environmental treatment effects on transcription were due to acceleration of development, but that a few transcripts were specifically affected. Transcript abundance profiles in both experiments for nine selected known and putative wheat transcription factors were independently confirmed by real time RT-PCR. These expression profiles confirm or extend our knowledge of the roles of the known transcription factors and suggest roles for the unknown ones.

Conclusion: This transcriptome data will provide a valuable resource for molecular studies on wheat grain. It has been demonstrated how it can be used to distinguish general developmental shifts from specific effects of treatments on gene expression and to diagnose the probable tissue specificity and role of transcription factors.

Figure 1

Data from two biological replicate samples of developing wheat caryopses. Upper panel: grain fresh weight and dry weight (error bars are least significant difference at P < 0.05 from 1-way analysis of variance). Lower panel: transcriptome of samples measured on wheat Affymetrix arrays. Each line depicts average signal from two replicate samples for a probeset coloured according to expression at 6 days after anthesis (daa). Gene set is those showing significant (P < 0.05; Benjamini-Hochberg false discovery rate multiple-testing correction) differences in expression between stages (14,550 probesets).

Figure 2

Hierarchical clustering of samples by gene expression. Gene set is same as that in Figure 1. Co-expression measure was Pearson correlation. All replicates cluster as pairs, for which the stage is shown as days after anthesis.

Figure 3

Day of anthesis versus normalised expression of probesets grouped into clusters according to their expression profiles by Self-Organising Map algorithm. Each graph represents a different cluster and at the top of the graph the cluster name (which is the position in the map) is shown along with the number of probesets it contains in parentheses. Pie chart classifications for biological process and tissue category were inferred from various sources (see Methods). Unclassified probesets were omitted from the pie charts: the number that were classified are shown in parentheses next to the chart.

Figure 4

Geometric average of expression for all probesets likely to be predominantly expressed in embryo, endosperm or pericarp tissues for both developmental series (left panel) and controlled environment experiment with control (c), drought (d), heat (h) and heat & drought (h&d) treatments (right panels). Embryo, endosperm and pericarp sets contain 104, 496 and 296 probesets, respectively.

Figure 5

Comparison of expression estimates from wheat Affymetrix array (lines) and quantitative reverse transcription PCR (qRT-PCR; points) for nine transcription factors. The left hand panel displays data from the developmental series which are the average of two replicates. The other panels are the unreplicated controlled-environment data for control, dry, hot and hot & dry conditions. Correlation coefficients (r) between the two expression measures are indicated.

Figure 6

Comparison between expression of transcription factors (red lines) and their known targets (green lines). A: Transcripts of the TaSPA, TaPBF and TaGAmyb transcription factors and LMW glutenins. B: Transcripts of TaEmBP and TaVP1 transcription factors and Em proteins. C: Transcripts of HvMYB3-like transcription factor and BTI-CMe trypsin inhibitor protein.

Figure 7

Expression profiles for probesets matching putative NAC (A), YABBY (B) and ARF (C) transcription factors.