Identification of miRNAs and Their Target Genes Using Deep Sequencing and Degradome Analysis in Trifoliate Orange [<Emphasis Type="Italic">Poncirus trifoliate</Emphasis> (L.) Raf]

To identify novel as well as conserved miRNAs in citrus, deep sequencing of small RNA library combined with microarray was performed in precocious trifoliate orange (an early flowering mutant of trifoliate orange, Poncirus trifoliata L. Raf.), resulting in the obtainment of a total of 114 conserved miRNAs belonging to 38 families and 155 novel miRNAs. The miRNA star sequences of 39 conserved miRNAs and 27 novel miRNAs were also discovered among newly identified miRNAs, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 172 and 149 genes were identified as targets of conserved miRNAs and novel miRNAs, respectively. GO and KEGG annotation revealed that high ranked miRNA-target genes were those implicated in biological and metabolic processes. To characterize those miRNAs expressed at the juvenile and adult development stages of citrus, further analysis on the expression profiles of these miRNAs through hybridizing the commercial microarray and real-time PCR was performed. The results revealed that some miRNAs were down-regulated at adult stage compared with juvenile stage. Detailed comparison of the expression patterns of some miRNAs and corresponding target genes revealed the negative correlation between them, while few of them are positively correlated.     

MicroRNA gene regulation cascades during early stages of plant development

MicroRNAs (miRNAs) regulate various developmental programs of plants. This review focuses on miRNA involvement in early events of plant development, such as seed germination, seedling development and the juvenile to adult phase transition. miR159 and miR160 are involved in the regulation of seed germination through their effects on the sensitivity of seeds to ABA. miR156 and miR172 play critical roles in the emergence of vegetative leaves at post-germinative stages, which is important for the transition to autotrophic growth. The phase transition from the juvenile to adult stage in both monocots and dicots is also regulated by miR156 and miR172. In these early developmental processes, there are miRNA gene regulation cascades where the miR156 pathway acts upstream of the miR172 pathway. Moreover, targets of miR156 and miR172 exert positive feedback on the expression of MIR genes that suppress themselves. The early events of plant development appear to be controlled by complex mechanisms involving sequential expression of different miRNA pathways and feedback loops among miRNAs and their target genes.     

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.     

Embryo and endosperm development is disrupted in the female gametophytic capulet mutants of Arabidopsis

The female gametophyte of higher plants gives rise, by double fertilization, to the diploid embryo and triploid endosperm, which develop in concert to produce the mature seed. What roles gametophytic maternal factors play in this process is not clear. The female-gametophytic effects on embryo and endosperm development in the Arabidopsis mea, fis, and fie mutants appear to be due to gametic imprinting that can be suppressed by METHYL TRANSFERASE1 antisense (MET1 a/s) transgene expression or by mutation of the DECREASE IN DNA METHYLATION1 (DDM1) gene. Here we describe two novel gametophytic maternal-effect mutants, capulet1 (cap1) and capulet2 (cap2). In the cap1 mutant, both embryo and endosperm development are arrested at early stages. In the cap2 mutant, endosperm development is blocked at very early stages, whereas embryos can develop to the early heart stage. The cap mutant phenotypes were not rescued by wild-type pollen nor by pollen from tetraploid plants. Furthermore, removal of silencing barriers from the paternal genome by MET1 a/s transgene expression or by the ddm1 mutation also failed to restore seed development in the cap mutants. Neither cap1 nor cap2 displayed autonomous seed development, in contrast to mea, fis, and fie mutants. In addition, cap2 was epistatic to fis1 in both autonomous endosperm and sexual development. Finally, both cap1 and cap2 mutant endosperms, like wild-type endosperms, expressed the paternally inactive endosperm-specific FIS2 promoter GUS fusion transgene only when the transgene was introduced via the embryo sac, indicating that imprinting was not affected. Our results suggest that the CAP genes represent novel maternal functions supplied by the female gametophyte that are required for embryo and endosperm development.     

Identification of the Conserved and Novel miRNAs in Mulberry by High-Throughput Sequencing

miRNAs are a class of non-coding endogenous small RNAs. They play vital roles in plant growth, development, and response to biotic and abiotic stress by negatively regulating genes. Mulberry trees are economically important species with multiple uses. However, to date, little is known about mulberry miRNAs and their target genes. In the present study, three small mulberry RNA libraries were constructed and sequenced using high-throughput sequencing technology. Results showed 85 conserved miRNAs belonging to 31 miRNA families and 262 novel miRNAs at 371 loci. Quantitative real-time PCR (qRT-PCR) analysis confirmed the expression pattern of 9 conserved and 5 novel miRNAs in leaves, bark, and male flowers. A total of 332 potential target genes were predicted to be associated with these 113 novel miRNAs. These results provide a basis for further understanding of mulberry miRNAs and the biological processes in which they are involved.