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.     

Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis

Flowering is the primary trait affected by ambient temperature changes. Plant microRNAs (miRNAs) are small non-coding RNAs playing an important regulatory role in plant development. In this study, to elucidate the mechanism of flowering-time regulation by small RNAs, we identified six ambient temperature-responsive miRNAs (miR156, miR163, miR169, miR172, miR398 and miR399) in Arabidopsis via miRNA microarray and northern hybridization analyses. We also determined the expression profile of 120 unique miRNA loci in response to ambient temperature changes by miRNA northern hybridization analysis. The expression of the ambient temperature-responsive miRNAs and their target genes was largely anticorrelated at two different temperatures (16 and 23°C). Interestingly, a lesion in short vegetative phase (SVP), a key regulator within the thermosensory pathway, caused alteration in the expression of miR172 and a subset of its target genes, providing a link between a thermosensory pathway gene and miR172. The miR172-overexpressing plants showed a temperature-independent early flowering phenotype, suggesting that modulation of miR172 expression leads to temperature insensitivity. Taken together, our results suggest a genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs under non-stress temperature conditions.     

miR172 signals are incorporated into the miR156 signaling pathway at the SPL3/4/5 genes in Arabidopsis developmental transitions

In plants, developmental timing is coordinately regulated by a complex signaling network that integrates diverse intrinsic and extrinsic signals. miR172 promotes photoperiodic flowering. It also regulates adult development along with miR156, although the molecular mechanisms underlying this regulation are not fully understood. Here, we demonstrate that miR172 modulates the developmental transitions by regulating the expression of a subset of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which are also regulated by miR156. The SPL3/4/5 genes were upregulated in the miR172-overproducing plants (35S:172) and its target gene mutants that exhibit early flowering. In contrast, expression of other SPL genes was not altered to a discernible level. Kinetic measurements of miR172 abundance in the transgenic plants expressing the MIR156a gene driven by a β-estradiol-inducible promoter revealed that expressions of miR172 and miR156 are not directly interrelated. Instead, the 2 miRNA signals are integrated at the SPL3/4/5 genes. Notably, analysis of developmental patterns in the 156?×?172 plants overproducing both miR172 and miR156 showed that whereas vegetative phase change was delayed as observed in the miR156-overproducing plants (35S:156), flowering initiation was accelerated as observed in the 35S:172 transgenic plants. Together, these observations indicate that although miR172 and miR156 play distinct roles in the timing of developmental phase transitions, there is a signaling crosstalk mediated by the SPL3/4/5 genes.     

The role of miR156 in developmental transitions in Nicotiana tabacum

Plants undergo a series of developmental transitions during their life cycle. After seed germination, plants pass through two distinct phases: the vegetative phase in which leaves are produced and the reproductive phase in which flowering occurs. Based on the reproductive competence and morphological changes, the vegetative phase can be further divided into juvenile and adult phases. Here, we demonstrate that the difference between juvenile and adult phase of Nicotiana tabacum is characterized by the changes in leaf size, leaf shape as well as the number of leaf epidermal hairs(trichomes). We further show that miR156, an age-regulated microR NA, regulates juvenile-to-adult phase transition in N. tabacum. Overexpression of miR156 results in delayed juvenile-to-adult transition and flowering. Together, our results support an evolutionarily conserved role of miR156 in plant developmental transitions.     

miRNA control of vegetative phase change in trees

After germination, plants enter juvenile vegetative phase and then transition to an adult vegetative phase before producing reproductive structures. The character and timing of the juvenile-to-adult transition vary widely between species. In annual plants, this transition occurs soon after germination and usually involves relatively minor morphological changes, whereas in trees and other perennial woody plants it occurs after months or years and can involve major changes in shoot architecture. Whether this transition is controlled by the same mechanism in annual and perennial plants is unknown. In the annual forb Arabidopsis thaliana and in maize (Zea mays), vegetative phase change is controlled by the sequential activity of microRNAs miR156 and miR172. miR156 is highly abundant in seedlings and decreases during the juvenile-to-adult transition, while miR172 has an opposite expression pattern. We observed similar changes in the expression of these genes in woody species with highly differentiated, well-characterized juvenile and adult phases (Acacia confusa, Acacia colei, Eucalyptus globulus, Hedera helix, Quercus acutissima), as well as in the tree Populus x canadensis, where vegetative phase change is marked by relatively minor changes in leaf morphology and internode length. Overexpression of miR156 in transgenic P. x canadensis reduced the expression of miR156-targeted SPL genes and miR172, and it drastically prolonged the juvenile phase. Our results indicate that miR156 is an evolutionarily conserved regulator of vegetative phase change in both annual herbaceous plants and perennial trees.     

Identification and expression analysis of miRNAs in germination and seedling growth of Tibetan hulless barley

Germination and seedling growth are crucial for plant development and agricultural production. While, the regulatory mechanisms during these processes in Tibetan hulless barley (Hordeum vulgare L. var. nudum) are not well understood. Given the regulatory roles of microRNAs (miRNAs) in crop plants and the irreplaceability of barley in the highland area of China, we herein presented a genome-wide survey of miRNAs to reveal a potential regulatory network in the early developmental stages of two Tibetan hulless barleys, from which a total of 156 miRNAs was identified including 35 known and 121 novel ones. Six of the identified novel miRNAs were further experimentally validated. According to the evolutionary analysis, miR156, miR166, miR168, and miR171 were conserved across Tibetan hulless barleys and eight other seed plants. Expression profiles of ten known miRNAs showed that they were involved in phytohormone signaling, carbohydrate and lipid metabolism, as well as juvenile-adult transition during barley development. Moreover, a total of 1280 genes targeted by 101 miRNAs were predicted from both barley libraries. Three genes (PLN03212, MATE eukaryotic, and GRAS) were validated via the RNA ligase-mediated 5′-rapid amplification of cDNA ends (RLM-5' RACE) to be the targets of hvu-miR159a, hvu-miR166a, and hvu-miR171-3p, respectively. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of putative targets, the most abundant pathways were related to “metabolism”.These results revealed that miRNA-target pairs participating in the regulation of multigene expression and the embryonic development of Tibetan hulless barleys were controlled by complex mechanisms involving the concordant expression of different miRNAs and feedback loops among miRNAs as well as their targets. The study provides insight into the regulatory network of barley miRNAs for better understanding of miRNA functions during germination and seedling growth.     

The COP1 ortholog PPS regulates the juvenile-adult and vegetative-reproductive phase changes in rice

Because plant reproductive development occurs only in adult plants, the juvenile-to-adult phase change is an indispensable part of the plant life cycle. We identified two allelic mutants, peter pan syndrome-1 (pps-1) and pps-2, that prolong the juvenile phase in rice (Oryza sativa) and showed that rice PPS is an ortholog of Arabidopsis thaliana CONSTITUTIVE PHOTOMORPHOGENIC1. The pps-1 mutant exhibits delayed expression of miR156 and miR172 and the suppression of GA biosynthetic genes, reducing the GA(3) content in this mutant. In spite of its prolonged juvenile phase, the pps-1 mutant flowers early, and this is associated with derepression of RAP1B expression in pps-1 plants independently of the Hd1-Hd3a/RFT1 photoperiodic pathway. PPS is strongly expressed in the fourth and fifth leaves, suggesting that it regulates the onset of the adult phase downstream of MORI1 and upstream of miR156 and miR172. Its ability to regulate the vegetative phase change and the time of flowering suggests that rice PPS acquired novel functions during the evolution of rice/monocots.     

Genome-wide identification of vegetative phase transition-associated microRNAs and target predictions using degradome sequencing in Malus hupehensis

Background

A long juvenile period between germination and flowering is a common characteristic among fruit trees, including Malus hupehensis (Pamp.) Rehd., which is an apple rootstock widely used in China. microRNAs (miRNAs) play an important role in the regulation of phase transition and reproductive growth processes.

Results

M. hupehensis RNA libraries, one adult and one juvenile phase, were constructed using tree leaves and underwent high-throughput sequencing. We identified 42 known miRNA families and 172 novel miRNAs. We also identified 127 targets for 25 known miRNA families and 168 targets for 35 unique novel miRNAs using degradome sequencing. The identified miRNA targets were categorized into 58 biological processes, and the 123 targets of known miRNAs were associated with phase transition processes. The KEGG analysis revealed that these targets were involved in starch and sucrose metabolism, and plant hormone signal transduction. Expression profiling of miRNAs and their targets indicated multiple regulatory functions in the phase transition. The higher expression level of mdm-miR156 and lower expression level of mdm-miR172 in the juvenile phase leaves implied that these two small miRNAs regulated the phase transition. mdm-miR160 and miRNA393, which regulate genes involved in auxin signal transduction, could also be involved in controlling this process. The identification of known and novel miRNAs and their targets provides new information on this regulatory process in M. hupehensis, which will contribute to the understanding of miRNA functions during growth, phase transition and reproduction in woody fruit trees.

Conclusions

The combination of sRNA and degradome sequencing can be used to better illustrate the profiling of hormone-regulated miRNAs and miRNA targets involving complex regulatory networks, which will contribute to the understanding of miRNA functions during growth, phase transition and reproductive growth in perennial woody fruit trees.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1125) contains supplementary material, which is available to authorized users.     

Reproductive competence from an annual and a perennial perspective

Plants at early stages of development undergo a juvenile phase during which they are not competent to flower in response to environmental stimuli. The length of this phase varies among species and is extended in perennial plants particularly. In annuals, temporal changes in expression of microR156 (miR156), miR172, and their targets are correlated with the transition from the juvenile to the adult phase and flowering. This developmental transition in perennials is probably more complex than in other plants and the molecular mechanisms are less well understood. In addition, once perennials become adult and capable of reproduction they still keep some meristems in the vegetative state that contribute to their polycarpic growth habit. Juvenility and polycarpy, although considered as two different processes in perennials, might be related.     

Redundant and specific roles of individual MIR172 genes in plant development

Evolutionarily conserved microRNAs (miRNAs) usually have high copy numbers in the genome. The redundant and specific roles of each member of a multimember miRNA gene family are poorly understood. Previous studies have shown that the miR156-SPL-miR172 axis constitutes a signaling cascade in regulating plant developmental transitions. Here, we report the feasibility and utility of CRISPR-Cas9 technology to investigate the functions of all 5 MIR172 family members in Arabidopsis. We show that an Arabidopsis plant devoid of miR172 is viable, although it displays pleiotropic morphological defects. MIR172 family members exhibit distinct expression pattern and exert functional specificity in regulating meristem size, trichome initiation, stem elongation, shoot branching, and floral competence. In particular, we find that the miR156-SPL-miR172 cascade is bifurcated into specific flowering responses by matching pairs of coexpressed SPL and MIR172 genes in different tissues. Our results thus highlight the spatiotemporal changes in gene expression that underlie evolutionary novelties of a miRNA gene family in nature. The expansion of MIR172 genes in the Arabidopsis genome provides molecular substrates for the integration of diverse floral inductive cues, which ensures that plants flower at the optimal time to maximize seed yields.

This study uses CRISPR-Cas9 technology to investigate the functions of all five miR172 genes in Arabidopsis, finding that miRNA172 family members exhibit distinct expression pattern and exert functional specificity in regulating meristem size, trichome initiation, stem elongation, shoot branching and floral competence.     

Identification and validation of Asteraceae miRNAs by the expressed sequence tag analysis

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a vital role in the regulation of gene expression. Despite their identification in hundreds of plant species, few miRNAs have been identified in the Asteraceae, a large family that comprises approximately one tenth of all flowering plants. In this study, we used the expressed sequence tag (EST) analysis to identify potential conserved miRNAs and their putative target genes in the Asteraceae. We applied quantitative Real-Time PCR (qRT-PCR) to confirm the expression of eight potential miRNAs in Carthamus tinctorius and Helianthus annuus. We also performed qRT-PCR analysis to investigate the differential expression pattern of five newly identified miRNAs during five different cotyledon growth stages in safflower. Using these methods, we successfully identified and characterized 151 potentially conserved miRNAs, belonging to 26 miRNA families, in 11 genus of Asteraceae. EST analysis predicted that the newly identified conserved Asteraceae miRNAs target 130 total protein-coding ESTs in sunflower and safflower, as well as 433 additional target genes in other plant species. We experimentally confirmed the existence of seven predicted miRNAs, (miR156, miR159, miR160, miR162, miR166, miR396, and miR398) in safflower and sunflower seedlings. We also observed that five out of eight miRNAs are differentially expressed during cotyledon development. Our results indicate that miRNAs may be involved in the regulation of gene expression during seed germination and the formation of the cotyledons in the Asteraceae. The findings of this study might ultimately help in the understanding of miRNA-mediated gene regulation in important crop species.