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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.

ORGANISM(S): Glycine max

PROVIDER: GSE146287 | GEO | 2020/10/16

REPOSITORIES: GEO

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Project description:Molecular characterization of leaf development has not been well studied in soybean. Studies have shown that genomic regions controlling multifoliate leaf morphology in Glycine max also regulates important agronomic characters including yield, seed weight, seed number, shattering, plant growth, and flowering. Two soybean isolines that differed in leaf phenotype were profiled by high throughput RNA and small RNA (sRNA) sequencing. A Clark isoline, homozygous for a dominant mutant allele, Lf1, that specifies a five-foliate compound leaf was compared to wild type Clark that is homozygous for the standard allele that produces trifoliate leaves. Although Lf1 is dominant, it presents variable expressivity as the young plantlets with the Lf1Lf1 genotype initially have trifoliate leaves in the first few weeks, after which they transition to five-foliate leaves. At later developmental stages, they begin to produce four-foliate or trifoliate leaves. In RNA-Seq experiments, a total of 91 and 95 million reads were generated in each lane of Illumina sequencing for the shoot tip of wild type Clark standard (CS) and mutant Clark five-foliate (CF) libraries, respectively. Of these, ~70% million reads aligned to the 78,743 target Glyma models from the reference soybean genome (cv. Williams 82) with maximum of 3 mismatches and up to 25 alignments. Where as in vegetative bud, 56 (CS) to 59 (CF) million reads were produced and of these ~80% aligned to the soybean reference genome. The comparative studies of the transcript profiles of the wild-type versus mutant line revealed a number of differentially expressed genes. A total of 1,296 and 2,083 genes were up-regulated in the shoot tip of CS and CF, respectively that showed ≥2-fold expression difference. On the contrary in the vegetative bud, much smaller number of genes 14 (CS) and 94 (CF) showed increased transcript abundance. In sRNA analysis, a collection of 200,447 and 268,508 unique sRNA sequences isolated from shoot tip tissue of CS and CF were aligned to the soybean reference genome and their target glyma models were predicted using bioinformatics. This sRNA analysis at genome level reveals differences in size distribution of classes in the CS and CF. This study provides insight into the initial understanding of leaf development in soybean by revealing a number of genes and sRNAs differentially expressed between the CS and CF.

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

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