Small RNA profiling for identification of microRNAs involved in regulation of seed development and lipid biosynthesis in yellowhorn.

BACKGROUND: Yellowhorn (Xanthoceras sorbifolium), an endemic woody oil-bearing tree, has become economically important and is widely cultivated in northern China for bioactive oil production. However, the regulatory mechanisms of seed development and lipid biosynthesis affecting oil production in yellowhorn are still elusive. MicroRNAs (miRNAs) play crucial roles in diverse aspects of biological and metabolic processes in seeds, especially in seed development and lipid metabolism. It is still unknown how the miRNAs regulate the seed development and lipid biosynthesis in yellowhorn. RESULTS: Here, based on investigations of differences in the seed growth tendency and embryo oil content between high-oil-content and low-oil-content lines, we constructed small RNA libraries from yellowhorn embryos at four seed development stages of the two lines and then profiled small RNA expression using high-throughput sequencing. A total of 249 known miRNAs from 46 families and 88 novel miRNAs were identified. Furthermore, by pairwise comparisons among the four seed development stages in each line, we found that 64 miRNAs (53 known and 11 novel miRNAs) were differentially expressed in the two lines. Across the two lines, 15, 11, 10, and 7 differentially expressed miRNAs were detected at 40, 54, 68, and 81 days after anthesis, respectively. Bioinformatic analysis was used to predict a total of 2654 target genes for 141 differentially expressed miRNAs (120 known and 21 novel miRNAs). Most of these genes were involved in the fatty acid biosynthetic process, regulation of transcription, nucleus, and response to auxin. Using quantitative real-time PCR and an integrated analysis of miRNA and mRNA expression, miRNA-target regulatory modules that may be involved in yellowhorn seed size, weight, and lipid biosynthesis were identified, such as miR172b-ARF2 (auxin response factor 2), miR7760-p3_1-AGL61 (AGAMOUS-LIKE 61), miR319p_1-FAD2-2 (omega-6 fatty acid desaturase 2-2), miR5647-p3_1-DGAT1 (diacylglycerol acyltransferase 1), and miR7760-p5_1-MED15A (Mediator subunit 15a). CONCLUSIONS: This study provides new insights into the important regulatory roles of miRNAs in the seed development and lipid biosynthesis in yellowhorn. Our results will be valuable for dissecting the post-transcriptional and transcriptional regulation of seed development and lipid biosynthesis, as well as improving yellowhorn in northern China.

Comparative RNA-Seq Analysis of High- and Low-Oil Yellow Horn During Embryonic Development.

Yellow horn (Xanthoceras sorbifolium Bunge) is an endemic oil-rich shrub that has been widely cultivated in northern China for bioactive oil production. However, little is known regarding the molecular mechanisms that contribute to oil content in yellow horn. Herein, we measured the oil contents of high- and low-oil yellow horn embryo tissues at four developmental stages and investigated the global gene expression profiles through RNA-seq. The results found that at 40, 54, 68, and 81 days after anthesis, a total of 762, 664, 599, and 124 genes, respectively, were significantly differentially expressed between the high- and low-oil lines. Gene ontology (GO) enrichment analysis revealed some critical GO terms related to oil accumulation, including acyl-[acyl-carrier-protein] desaturase activity, pyruvate kinase activity, acetyl-CoA carboxylase activity, and seed oil body biogenesis. The identified differentially expressed genes also included several transcription factors, such as, AP2-EREBP family members, B3 domain proteins and C2C2-Dof proteins. Several genes involved in fatty acid (FA) biosynthesis, glycolysis/gluconeogenesis, and pyruvate metabolism were also up-regulated in the high-oil line at different developmental stages. Our findings indicate that the higher oil accumulation in high-oil yellow horn could be mostly driven by increased FA biosynthesis and carbon supply, i.e. a source effect.