Comprehensive research in various plants shows that the metabolic pathway of anthocyanin biosynthesis is affected by environmental factors and regulated by microRNAs through post-transcriptional regulation. In seedlings of Brassica rapa Tsuda, the accumulation of anthocyanin is induced by light. However, the roles of BrmiR828 in the light-induced synthesis of anthocyanin in Brassica rapa remain to be explored. Here, a primary transcript of BrmiR828 was identified to be located on the chromosomes of the A03 sub-genome. Five candidate MYB family genes were predicted as targets of BrmiR828 in the database of Brassica rapa (BRAD, V1.1) by using psRNATarget. The transcript abundance of mature BrmiR828 was reduced in seedlings of Brassica rapa Tsuda under blue light irradiation comparing with dark treatment. However, Real-time PCR showed the transcript level of the five candidate targets, Bra004162, Bra022602, Bra001917, Bra029113, and Bra039763 was up-regulated when the seedlings exposed to blue or UV-A light. Trans-acting siRNA gene 4 (BrTAS4) was also identified to have a higher transcript level under blue and UV-A light irradiation than that in dark treatment. RNA ligase mediated 5′amplification of cDNA ends (RLM-5′ RACE) showed that BrmiR828 can splice the mRNA of Bra039763, Bra022602, and BrTAS4 on binding sites. Phylogenetic analysis of candidate BrMYBs targets along with MYBs from Arabidopsis thaliana showed that Bra039763, Bra004162, Bra001917, Bra029113, and Bra022602 are classified to the same group with AtMYB75, AtMYB114, AtMYB90, AtMYB113, and AtMYB82 which are involved in the anthocyanin biosynthetic pathway. As a result, light-induced down-regulation of BrmiR828 can target BrTAS4, BrPAP1 (Bra039763), MYB82 (Bra022602) to negatively regulate their transcript levels leading to the accumulation of MYB transcription factors that positively regulate anthocyanin biosynthesis in light-exposed seedlings of Brassica rapa.

Growth, development, and pigment synthesis in Brassica rapa subsp. rapa cv. Tsuda, a popular vegetable crop, are influenced by light. Although microRNAs (miRNAs) have vital roles in the metabolic processes and abiotic stress responses of plants, whether miRNAs play a role in anthocyanin biosynthesis and development of Tsuda seedlings exposed to light is unknown. Seventeen conserved and 226 novel miRNAs differed at least 2-fold in response to blue and UV-A light compared with levels after a dark treatment. Real time PCR showed that BrmiR159, BrmiRC0191, BrmiRC0460, BrmiRC0323, BrmiRC0418, BrmiRC0005 were blue light-induced and northern blot revealed that the transcription level of BrmiR167 did not differ significantly among seedlings treated with dark, blue or UV-light. BrmiR156 and BrmiR157 were present in the greatest amount (number of reads) and among their 8 putative targets in the SPL gene family, only SPL9 (Bra004674) and SPL15 (Bra003305) increased in expression after blue or UV-A exposure. In addition, miR157-guided cleavage of target SPL9 mRNAs (Bra004674, Bra016891) and SPL15 mRNAs (Bra003305, Bra014599) took place 10 or 11 bases from the 5′ ends of the binding region in the miR157 sequence. A set of miRNAs and their targets involved in the regulation of the light-induced photomorphogenic phenotype in seedlings of Brassica rapa was identified, providing new insights into blue and UV-A light-responsive miRNAs in seedlings of Tsuda and evidence of multiple targets for the miRNAs and their diverse roles in plant development.

Background: Cold tolerance is important for plants’ geographical distribution and survival in extreme seasonal variations of climate. However, Populus simonii × P. nigra shows wide adaptability and strong cold resistance. Transcriptional and post-transcriptional regulation of cold-responsive genes is crucial for cold tolerance in plants. To understand the roles of regulatory RNAs under cold induction in Populus simonii × P. nigra, we constructed cDNA and small RNA libraries from leaf buds treated or not with −4 °C for 8 h for analysis. Results: Through high-throughput sequencing and differential expression analysis, 61 miRNAs and 1229 DEGs were identified under cold induction condition in Populus simonii × P. nigra. The result showed that miR167a, miR1450, miR319a, miR395b, miR393a-5p, miR408-5p, and miR168a-5p were downregulated, whereas transcription level of miR172a increased under the cold treatment. Thirty-one phased-siRNA were also obtained (reads ≥ 4) and some of them proceeded from TAS3 loci. Analysis of the differentially expressed genes (DEGs) showed that transcription factor genes such as Cluster-15451.2 (putative MYB), Cluster-16493.29872 (putative bZIP), Cluster-16493.29175 (putative SBP), and Cluster-1378.1 (putative ARF) were differentially expressed in cold treated and untreated plantlets of Populus simonii × P. nigra. Integrated analysis of miRNAs and transcriptome showed miR319, miR159, miR167, miR395, miR390, and miR172 and their target genes, including MYB, SBP, bZIP, ARF, LHW, and ATL, were predicted to be involved in ARF pathway, SPL pathway, DnaJ related photosystem II, and LRR receptor kinase, and many of them are known to resist chilling injury. Particularly, a sophisticated regulatory model including miRNAs, phasiRNAs, and targets of them was set up. Conclusions: Integrated analysis of miRNAs and transcriptome uncovered the complicated regulation of the tolerance of cold in Populus simonii × P. nigra. MiRNAs, phasiRNAs, and gene-encoded transcription factors were characterized at a whole genome level and their expression patterns were proved to be complementary. This work lays a foundation for further research of the pathway of sRNAs and regulatory factors involved in cold tolerance.