Dynamic Regulation of Novel and Conserved miRNAs Across Various Tissues of Diverse Cucurbit Species

MicroRNA genes (miRNAs) encoding small non-coding RNAs are abundant in plant genomes and play a key role in regulating several biological mechanisms. Five conserved miRNAs, miR156, miR168-1, miR168-2, miR164, and miR166 were selected for analysis from the 21 known plant miRNA families that were recovered from deep sequencing data of small RNA libraries of pumpkin and squash. A total of six novel miRNAs that were not reported before were found to have precursors with reliable fold-back structures and hence considered novel and were designated as cuc_nov_miRNAs. A set of five conserved, six novel miRNAs, and five uncharacterized small RNAs from the deep sequencing data were profiled for their dynamic regulation using qPCR. The miRNAs were evaluated for differential regulation across the tissues among four diverse cucurbit species, including pumpkin and squash (Cucurbita moschata Duch. Ex Poir. and Cucurbita pepo L.), bitter melon (Momordica charantia L.), and Luffa (Loofah) (Luffa acutangula Roxb.). Expression analysis revealed differential regulation of various miRNAs in leaf, stem, and fruit tissues. Importantly, differences in the expression levels were also found in the leaves and fruits of closely related C. moschata and C. pepo. Comparative miRNA profiling and expression analysis in four cucurbits led to identification of conserved miRNAs in cucurbits. Predicted targets for two of the conserved miRNAs suggested miRNAs are involved in regulating similar biological mechanisms in various species of cucurbits.     

MicroRNAs in the shoot apical meristem of soybean

Plant microRNAs (miRNAs) play crucial regulatory roles in various developmental processes. In this study, we characterize the miRNA profile of the shoot apical meristem (SAM) of an important legume crop, soybean, by integrating high-throughput sequencing data with miRNA microarray analysis. A total of 8423 non-redundant sRNAs were obtained from two libraries derived from micro-dissected SAM or mature leaf tissue. Sequence analysis allowed the identification of 32 conserved miRNA families as well as 8 putative novel miRNAs. Subsequent miRNA profiling with microarrays verified the expression of the majority of these conserved and novel miRNAs. It is noteworthy that several miRNAs* were expressed at a level similar to or higher than their corresponding mature miRNAs in SAM or mature leaf, suggesting a possible biological function for the star species. In situ hybridization analysis revealed a distinct spatial localization pattern for a conserved miRNA, miR166, and its star speciessuggesting that they serve different roles in regulating leaf development. Furthermore, localization studies showed that a novel soybean miRNA, miR4422a, was nuclear-localized. This study also indicated a novel expression pattern of miR390 in soybean. Our approach identified potential key regulators and provided vital spatial information towards understanding the regulatory circuits in the SAM of soybean during shoot development.     

Deep Sequencing of Maize Small RNAs Reveals a Diverse Set of MicroRNA in Dry and Imbibed Seeds

Seed germination plays a pivotal role during the life cycle of plants. As dry seeds imbibe water, the resumption of energy metabolism and cellular repair occur and miRNA-mediated gene expression regulation is involved in the reactivation events. This research was aimed at understanding the role of miRNA in the molecular control during seed imbibition process. Small RNA libraries constructed from dry and imbibed maize seed embryos were sequenced using the Illumina platform. Twenty-four conserved miRNA families were identified in both libraries. Sixteen of them showed significant expression differences between dry and imbibed seeds. Twelve miRNA families, miR156, miR159, miR164, miR166, miR167, miR168, miR169, miR172, miR319, miR393, miR394 and miR397, were significantly down-regulated; while four families, miR398, miR408, miR528 and miR529, were significantly up-regulated in imbibed seeds compared to that in dry seeds. Furthermore, putative novel maize miRNAs and their target genes were predicted. Target gene GO analysis was performed for novel miRNAs that were sequenced more than 50 times in the normalized libraries. The result showed that carbohydrate catabolic related genes were specifically enriched in the dry seed, while in imbibed seed target gene enrichment covered a broad range of functional categories including genes in amino acid biosynthesis, isomerase activity, ligase activity and others. The sequencing results were partially validated by quantitative RT-PCR for both conserved and novel miRNAs and the predicted target genes. Our data suggested that diverse and complex miRNAs are involved in the seed imbibition process. That miRNA are involved in plant hormone regulation may play important roles during the dry-imbibed seed transition.     

MicroRNA Expression Profile during Aphid Feeding in Chrysanthemum (Chrysanthemum morifolium)

MicroRNAs (miRNAs) are important regulators of gene expression, affecting many biological processes. As yet, their roles in the response of chrysanthemum to aphid feeding have not been explored. Here, the identity and abundance of miRNAs induced by aphid infestation have been obtained using high-throughput Illumina sequencing platform. Three leaf small RNA libraries were generated, one from plants infested with the aphid Macrosiphoniella sanbourni (library A), one from plants with mock puncture treatment (library M), and the third from untreated control plants (library CK). A total of 7,944,797, 7,605,251 and 9,244,002 clean unique reads, ranging from 18 to 30 nucleotides (nt) in length, were obtained from library CK, A and M, respectively. As a result, 303 conserved miRNAs belonging to 276 miRNAs families and 234 potential novel miRNAs were detected in chrysanthemum leaf, out of which 80, 100 and 79 significantly differentially expressed miRNAs were identified in the comparison of CK-VS-A, CK-VS-M and M-VS-A, respectively. Several of the differentially abundant miRNAs (in particular miR159a, miR160a, miR393a) may be associated with the plant''s response to aphid infestation.     

microRNA profiling of root tissues and root forming explant cultures in Medicago truncatula

Plant root architecture is regulated by the initiation and modulation of cell division in regions containing pluripotent stem cells known as meristems. In roots, meristems are formed early in embryogenesis, in the case of the root apical meristem (RAM), and during organogenesis at the site of lateral root or, in legumes, nodule formation. Root meristems can also be generated in vitro from leaf explants cultures supplemented with auxin. microRNAs (miRNAs) have emerged as regulators of many key biological functions in plants including root development. To identify key miRNAs involved in root meristem formation in Medicago truncatula, we used deep sequencing to compare miRNA populations. Comparisons were made between: (1) the root tip (RT), containing the RAM and the elongation zone (EZ) tissue and (2) root forming callus (RFC) and non-root forming callus (NRFC). We identified 83 previously reported miRNAs, 24 new to M. truncatula, in 44 families. For the first time in M. truncatula, members of conserved miRNA families miR165, miR181 and miR397 were found. Bioinformatic analysis identified 38 potential novel miRNAs. Selected miRNAs and targets were validated using Taqman miRNA assays and 5′ RACE. Many miRNAs were differentially expressed between tissues, particularly RFC and NRFC. Target prediction revealed a number of miRNAs to target genes previously shown to be differentially expressed between RT and EZ or RFC and NRFC and important in root development. Additionally, we predict the miRNA/target relationships for miR397 and miR160 to be conserved in M. truncatula. Amongst the predictions, were AUXIN RESPONSE FACTOR 10, targeted by miR160 and a LACCASE-like gene, targeted by miR397, both are miRNA/target pairings conserved in other species.     

Genome-Wide Identification and Analysis of MicroRNAs Involved in Witches’-Broom Phytoplasma Response in Ziziphus jujuba

MicroRNAs (miRNAs) play an important role in responding to biotic and abiotic stresses in plants. Jujube witches’-broom a phytoplasma disease of Ziziphus jujuba is prevalent in China and is a serious problem to the industry. However, the molecular mechanism of the disease is poorly understood. In this study, genome-wide identification and analysis of microRNAs in response to witches’-broom was performed. A total of 85 conserved miRNA unique sequences belonging to 32 miRNA families and 24 novel miRNA unique sequences, including their complementary miRNA* strands were identified from small RNA libraries derived from a uninfected and witches’-broom infected Z. jujuba plant. Differentially expressed miRNAs associated with Jujube witches’-broom disease were investigated between the two libraries, and 12 up-regulated miRNAs and 10 down- regulated miRNAs identified with more than 2 fold changes. Additionally, 40 target genes of 85 conserved miRNAs and 49 target genes of 24 novel miRNAs were predicted and their putative functions assigned. Using the modified 5’-RACE method, we confirmed that SPL and MYB were cleaved by miR156 and miR159, respectively. Our results provide insight into the molecular mechanisms of witches’-broom disease in Z. jujuba.     

Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing

Hickory (Carya cathayensis Sarg.) is an economically important woody plant in China, but its long juvenile phase delays yield. MicroRNAs (miRNAs) are critical regulators of genes and important for normal plant development and physiology, including flower development. We used Solexa technology to sequence two small RNA libraries from two floral differentiation stages in hickory to identify miRNAs related to flower development. We identified 39 conserved miRNA sequences from 114 loci belonging to 23 families as well as two novel and ten potential novel miRNAs belonging to nine families. Moreover, 35 conserved miRNA*s and two novel miRNA*s were detected. Twenty miRNA sequences from 49 loci belonging to 11 families were differentially expressed; all were up-regulated at the later stage of flower development in hickory. Quantitative real-time PCR of 12 conserved miRNA sequences, five novel miRNA families, and two novel miRNA*s validated that all were expressed during hickory flower development, and the expression patterns were similar to those detected with Solexa sequencing. Finally, a total of 146 targets of the novel and conserved miRNAs were predicted. This study identified a diverse set of miRNAs that were closely related to hickory flower development and that could help in plant floral induction.     

Cloning and characterization of maize miRNAs involved in responses to nitrogen deficiency

Although recent studies indicated that miRNAs regulate plant adaptive responses to nutrient deprivation, the functional significance of miRNAs in adaptive responses to nitrogen (N) limitation remains to be explored. To elucidate the molecular biology underlying N sensing/signaling in maize, we constructed four small RNA libraries and one degradome from maize seedlings exposed to N deficiency. We discovered a total of 99 absolutely new loci belonging to 47 miRNA families by small RNA deep sequencing and degradome sequencing, as well as 9 new loci were the paralogs of previously reported miR169, miR171, and miR398, significantly expanding the reported 150 high confidence genes within 26 miRNA families in maize. Bioinformatic and subsequent small RNA northern blot analysis identified eight miRNA families (five conserved and three newly identified) differentially expressed under the N-deficient condition. Predicted and degradome-validated targets of the newly identified miRNAs suggest their involvement in a broad range of cellular responses and metabolic processes. Because maize is not only an important crop but is also a genetic model for basic biological research, our research contributes to the understanding of the regulatory roles of miRNAs in plant adaption to N-deficiency stress.     

Cloning and characterization of microRNAs from Brassica napus

A library containing approximately 40,000 small RNA sequences was constructed for Brassica napus. Analysis of 3025 sequences obtained from this library resulted in the identification of 11 conserved miRNA families, which were validated by secondary structure prediction using surrounding sequences in the Brassica genome. Two 21 nt small RNA sequences reside within the arm of a pre-miRNA like stem-loop structure, making them likely candidates for novel non-conserved miRNAs in B. napus. Most of the conserved miRNAs were expressed at similar levels in a F1 hybrid B. napus line and its four double haploid progeny that showed marked variations in phenotypes, but many were differentially expressed between B. napus and Arabidopsis. The miR169 family was expressed at high levels in young leaves and stems, but was undetectable in roots and mature leaves, suggesting that miR169 expression is developmentally regulated in B. napus.