Overexpression of an ethylene-forming ACC oxidase (ACO) gene precedes the Minute Hilum seed coat phenotype in Glycine max

Abstract

Background: To elucidate features of seed development, we investigated the transcriptome of a soybean isoline from the germplasm collection that contained an introgressed allele known as minute hilum (mi) which confers a smaller hilum region where the seed attaches to the pod and also results in seed coat cracking surrounding the hilum region.

Results: RNAs were extracted from immature seed from an extended hilum region (i.e., the hilum and a small ring of tissue surrounding the hilum in which the cracks form) at three different developmental stages:10-25, 25-50 and 50-100 mg seed fresh weight in two independent replicates for each stage. The transcriptomes of these samples from both the Clark isoline containing the mi allele (PI 547628, UC413, iⁱ R t mi G), and its recurrent Clark 63 parent isoline (PI 548532, UC7, iⁱ R T Mi g), which was used for six generations of backcrossing, were compared for differential expression of 88,648 Glyma models of the soybean genome Wm82.a2. The RNA sequence data obtained from the 12 cDNA libraries were subjected to padj value < 0.05 and at least two-fold expression differences to select with confidence genes differentially expressed in the hilum-containing tissue of the seed coat between the two lines. Glyma.09G008400 annotated as encoding an ethylene forming enzyme, ACC oxidase (ACO), was found to be highly overexpressed in the mi hilum region at 165 RPKMs (reads per kilobase per million mapped reads) compared to the standard line at just 0.03 RPKMs. Evidence of changes in expression of genes downstream of the ethylene pathway included those involved in auxin and gibberellin hormone action and extensive differences in expression of cell wall protein genes. These changes are postulated to determine the restricted hilum size and cracking phenotypes.

Conclusions: We present transcriptome and phenotypic evidence that substantially higher expression of an ethylene-forming ACO gene likely shifts hormone balance and sets in motion downstream changes resulting in a smaller hilum phenotype and the cracks observed in the minute hilum (mi) isoline as compared to its recurrent parent.

Keywords: ACC oxidase; Defective seed coat; Ethylene forming enzyme; Expression profiling; Glycine max; Minute hilum; RNA-Seq; Seed development; Soybean.

Fig. 1

Hilum phenotypic differences between seeds of Clark isolines UC7 (iⁱ R T Mi g) and UC413 (iⁱ R t mi G) at three late developmental stages. Seeds in the left hand panels have normal size hilum (UC7) while the ones in the right (UC413) have the minute hilum phenotype and associated cracks in the seed coat. Seed coat cracks from the hilum and beyond are formed at a late stage of seed maturation and clearly seen in UC413 dry seed in the bottom right panel. The three stages of development shown to the right of the image are: full seed (R7), mid-desiccation (R7-R8) and dry seed (R8). The white oval lines around the hilum in the upper panels represent the area of the hilum and immediate seed coat region, designated the EH (extended hilum), dissected from seeds at the early maturation stages of EH10 (10–25 mg seed), EH25 (25–50 mg), and EH50 (50–100 mg) seed fresh weight developmental stages (not shown) . All genotypes are homozygous in the inbred isolines

Fig. 2

Percentages of genes by annotation categories overexpressed in the UC7 (iⁱ R T Mi g) (blue) or minute hilum UC413 (iⁱ R t mi G) (red) isolines at three seed developmental stages. a EH10: 10–25 mg; b EH25: 25–50 mg; c EH50:50–100 mg. The absolute number of genes in the 23 categories is shown in Table 2. The rDNA gene category has been omitted for calculation of percentages (total within a category per isoline/total overexpressed genes in the isoline)

Fig. 3

Overexpression of genes in the ethylene biosynthesis and signaling pathways in the minute hilum Clark isoline. The overexpressed genes in the minute hilum region of UC413 are framed and highlighted in a color background. ACC (l-aminocyclopropane-l-carboxylate); ACS (ACC Synthase); ACO (ACC Oxidase) catalyzes the last step in the synthesis of ethylene; ETO protein inhibitor of ACC Synthase; ETR2 and EIN4, ethylene receptors; RAN1 a cupper transporter; CTR1 a Raf-like kinase; EIN2 positive regulator of ethylene pathway; EIN3 transcription factor activated by EIN2; ERFs, transcription factors that turn on target genes that activate ethylene responses

Fig. 4

Comparison of numbers of overexpressed cell wall genes by category in the standard UC7 (iⁱ R T Mi g) and minute hilum UC413 (iⁱ R t mi G) Clark isolines. From top to bottom are the three seed maturation developmental stages measured EH10, EH25 and EH50 for the extended hilum region of the seed coats. The numbers of genes are in parentheses

Fig. 5

A model for altered hormone responses leading to the minute hilum trait based on global expression changes between the minute hilum UC413 (iⁱ R t mi G) and standard UC7 (iⁱ R T Mi g) isolines. Thick red arrows indicate upregulation and thick blue arrows indicate downregulation in the mi isoline UC413 of the indicated transcripts (boxed) or hormones (circled)

Fig. 6

Comparative expression of ACO transcripts. (a) Illustration of differential expression of the two most closely related ACO Glyma models (red: Glyma.09G008400.1 and blue: Glyma.15G112700.1) in the RNA-Seq of the extended hilum regions (EH) or whole seed coat samples (WS) during the indicated developmental stage of either the recurrent parent isoline UC7 (Mi) or the minute hilum isoline UC413 (mi). The average of the RPKMs for two replicates is graphed for the EH10, EH25, and EH50 data. The full data set is shown in Additional file 8a. (b) Graph of RPKMs values of each of the two ACO Glymas from a set of 39 total RNA-Seq libraries representing a range of developmental stages and tissues from the Williams cultivar. See Additional files 8 b,c for the full data of 11 ACO genes and more description of the libraries

Fig. 7

A representation of the promoter region of Glyma.09G008400.1 and the differences in amplicons from three different soybean lines. The 1505 bp sequence shown is that of the Williams 82 reference genome. Bases in bold italics at the ends are the primers used. Green highlighting indicates the 5’UTR region in the gene model, blue highlighting denotes the first two exons. The box and yellow highlight mark the five different WAAAG elements in the promoter regions of both strands. The 50 bp in red font indicate the repetitive AT region in the promoter. Within this region are the observed differences of the three sequenced amplicons as clearly shown in the alignments of Additional file 10. Both UC7 (iⁱ R T Mi g) and UC413 (iⁱ R t mi G) are identical and both lack 26 nucleotides of the AT region that are in red small letters. Amplicon sequencing of the cultivar Williams, the recurrent parent of the reference genome Williams 82 lacks 4 of the bases in the AT rich region which are also underlined in red. The bases in blue font mark the site of indel variants called by the VCF file of the whole genome sequence (Additional file 9), but they do not indicate the exact size of the deletions found in the AT-rich region as determined definitively by amplicon sequencing (Additional files 10 and 11)