Regulation of gene expression by miRNAs has been comprehensively investigated in animals and plants [3
]. In the case of higher plants, Arabidopsis
and rice miRNA targets have been widely studied by high throughput sequencing [13
]. Soybean is a polyploid crop plant having a complex and large genome compared to Arabidopsis
and rice. The number of identified miRNAs and their potential targets in soybean is limited. To date, degradome sequencing has been reported for only one soybean tissue, namely the very young whole seed extracted 15
days after flowering from the cultivar Heinong44 [21
]. In order to study the regulation of gene expression during soybean seed development, we constructed and sequenced five distinct degradome libraries using cotyledons and seed coats as a tissue source from different seed developmental stages of the cultivar Williams. The seed coat is primarily maternal tissue while the cotyledons represent the embryo of the next generation. Both tissues have the same genotype in inbred soybean lines.
After computational analysis using the Cleaveland pipeline [24
], we identified a total of 183 potential targets of 53 miRNA families in five different seed developmental stage specific degradome libraries. Subsequent analysis and identification of cotyledon and seed coat specific miRNAs and their targets give us a better understanding of the regulation of gene expression in a spatial manner during soybean seed development at later stages of seed development in a widely used Maturity Group III cultivar “Williams”. The soybean genome sequence and predicted gene models used in this study are derived from a closely related isoline Williams82 [15
]. Validation of four Auxin Response Factor (ARF) genes as targets of gma-miR160 indicates degradome sequencing as an efficient strategy to identify miRNA targets in plants.
Comparison of the data in our report with the first soybean degradome data reported by Song et al. [21
] from a very early stage of soybean seed development in the cultivar Heinong44 revealed a number of miRNA targets in common among the data sets including many transcription factors such as ARF, MYB, TCP, NF-Y, Growth Regulatory Factor, HD-ZIP, PPR, SBP and NAC family protein. In contrast, we found some other miRNA targets such as Permease family protein, LRR domain containing protein, transmembrane protein 14
C, Serine-Threonine kinase, BRE expressed protein and transcription factor TFIID only in our degradome libraries that represented later stages of seed development of the cultivar Williams. Overall there were 65 Glyma model targets in common between both of the data sets, 80 found only in the Heinong44 data set, and 118 found only in the Williams data sets.
The 183 identified targets from our degradome analyses belonged to 57 different annotation groupings ( Additional file 1
). Comparing those to the Song et al. paper, 11 annotation groups are in common, 18 are unique to the Heinong44 very young seed, and 46 are unique to the five libraries of the Williams data sets (Table
). Many of the identified soybean miRNA targets belong to diverse gene families of transcription factors such as ARFs, MYBs, TCPs, NACs, HD-ZIPs and NF-Y subunits (Tables
and and Additional file 1
). Many of these transcription factors are known to regulate diverse aspects of plant growth and development. Patterning and outgrowth of lateral organs in plants depend on the expression of HD-ZIP transcription factors that specify adaxial/upper cell fate [35
]. We identified a number of HD-ZIP transcription factors as targets for gma-miR166 in our degradome libraries. MYB family members in rice, which are targeted by miR159, appear to play an important role in response to the presence of abscisic acid (ABA) during plant embryonic development, suggesting their roles in seed development [37
]. In this study, we detected a number of MYB family transcription factors regulated by gma-miR159 (Additional file 1
. The gma-miRNA156 family members target sites in numerous proteins containing the Squamosa Promoter Binding (SBP) domain. SBP and SBP-LIKE (SPLs) proteins play multiple roles in plant development [39
]. In Arabidopsis
, rice and in some other plants, miR156 regulates leaf development by targeting Squamosa-Promoter Binding protein-like (SBP) transcription factors [31
]. The identification of SBP as a target of gma-miR160 may indicate the additional level of regulation for SBP during soybean seed development.
Comparison of miRNA target annotations between the Williams and Heinong44 data sets
Our analyses of the early, mid and late maturation developmental stages of soybean show a number of targets similar to those found by Song et al., [21
] in the very young seeds of the cultivar Heinong44 including the SPB transcription factors. One notable difference was the absence in our degradome data of miRNA172 targets which include members of the AP2 transcription factor family. From inspection of sequenced small RNA populations from the 50–75
mg seed coats and cotyledons of Williams [27
], we find only a few occurrences of the miR172 family (less than 30 occurrences per million reads) while some family members of the miR156 family are highly abundant (99,000 per million) in the cotyledons. We speculate that miR172 and/or its targets may be more abundant in the very young seed used by the Song et al. group [21
] and not prevalent in the mid-maturation seed that we have examined. In Arabidopsis
, miR172 has been reported to be involved in the regulation of flowering time and floral development [40
]. Alternatively, the AP2 factors may not be detected as targets in the degradome data if translational repression by miR172 is operative as has been shown in Arabidopsis
flower development [41
]. Nuclear Factor Y (NF-Y) was shown to control a variety of agronomically important traits, including drought tolerance, flowering time, and seed development [42
]. We detected seven NF transcription factor YA subunit mRNAs specifically in seed coats that are targets of miR169 family members that occur in both seed coats and cotyledons ( Additional file 1
). These targets may indicate some specific regulation of NF-YA transcription factors during soybean seed development.
To obtain a deeper understanding of soybean seed development, we investigated tissue specific miRNA target identification in the cotyledons and seed coats at different seed developmental stages. Based on the degradome data, we identified some miRNAs that may act differentially in the cotyledons versus the seed coats to degrade their targets (Tables
and ). F-box proteins involved in auxin-stimulated protein degradation (TIR1-like) were among the identified targets specifically found in soybean cotyledon (Table
). The LRR kinases have been reported to play important roles in plant development and brassinosteroid and ABA signaling [43
]. We identified several LRR domain containing proteins as targets for gma-miR393, gma-miR1523 and gma-miR2109 (Tables
and ). The presence of these miRNA targets implies their regulation during soybean seed development. As a storage organ, the soybean seed contains significant amounts of lipid and protein. Thus the regulation of energy metabolism is very important during seed development. We identified a number of targets in the soybean cotyledon such as NADP/FAD oxidoreductase, ribose-5-phosphate isomerase, GTPase activating proteins and ferredoxin related proteins which are related to energy metabolism. Both in the soybean cotyledon and seed coat, we found pentatricopeptide repeat (PPR) proteins as targets of miR1520 (Table
and Additional file 1
) which regulates gene expression in the mitochondria and chloroplasts [44
]. Since we constructed our degradome libraries using cotyledons and seed coats from different seed developmental stages, we identified targets of miRNAs during a broad range of soybean seed development.
Auxin is an important phytohormone in higher plants. It acts as a key player in plant development [46
]. As the transducer of auxin signaling, ARFs play vital roles in plant development, including shoot, root and flower formation [47
]. In Arabidopsis
, miR160 and miR167 are involved in auxin signaling via regulation of ARF genes [1
]. In rice, a number of ARF encoding genes have been identified which are regulated by osa-miR160 and osa-miR167, respectively [33
]. In our study, we identified a large number of ARF genes as targets for different miRNAs such as gma-miR160 and miR167. In the cotyledon (100–200
mg) degradome library ( Additional file 1
), we identified five targets annotated as Auxin Response Factors (ARFs) for gma-miR160, and four of these, Glyma12g08110.1Glyma12g29720.1Glyma14g33730.1
were validated by RLM-5’RACE showing precise cleavage as expected (Figure
). These results suggested that gma-miR160 could participate in auxin signaling via down-regulation of ARFs during soybean seed developmental stages. The cleaved mRNAs captured by the degradome procedure indicate that the levels of the ARF mRNA targets are likely to be decreased, but qRTPCR or RNA sequencing data would be needed to directly confirm the effect on mRNA levels for a particular ARF target gene.
In our degradome libraries, miRNA targets are involved in major transitions between each stage of seed development and transcription factors account for approximately half of these targets. Of 183 identified targets in our soybean degradome libraries, GO analysis for biological function indicates that these genes are mainly involved in developmental and metabolic processes (Figure
and Additional file 2
). Enrichment of developmentally related genes as target miRNAs suggests the high level of regulation of gene expression during soybean seed development. The larger number of targets found in the 300–400
mg desiccating, yellow cotyledons of late maturation implies that post-transcriptional regulation by miRNAs may aid in shifting the developmental program of the immature soybean cotyledons from biosynthesis of storage reserves to a catabolic role in utilization of those reserves during seed germination and growth. The miRNA targets verified by degradome sequencing will provide useful information for understanding and revealing significant roles of miRNAs during soybean seed development.