MicroRNAs (miRNAs) are approximately 19 ~ 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs have critical functions in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interaction with specific target mRNAs. Camellia sinensis is one of the most important commercial beverage crops in the world. However, miRNAs associated with cold stress tolerance in C. sinensis remains unexplored. The use of high-throughput sequencing can provide a much deeper understanding of miRNAs. To obtain more insight into the function of miRNAs in cold stress tolerance, Illumina sequencing of C. sinensis sRNA was conducted. Solexa sequencing technology was used for high-throughput sequencing of the small RNA library from the cold treatment of tea leaves. To align the sequencing data with known plant miRNAs, we characterized 106 conserved C. sinensis miRNAs. In addition, 215 potential candidate miRNAs were found, among, which 98 candidates with star sequences were chosen as novel miRNAs. Both congruously and differentially regulated miRNAs were obtained, and cultivar-specific miRNAs were identified by microarray-based hybridization in response to cold stress. The results were also confirmed by quantitative real-time polymerase chain reaction. To confirm the targets of miRNAs, two degradome libraries from two treatments were constructed. According to degradome sequencing, 455 and 591 genes were identified as cleavage targets of miRNAs from cold treatments and control libraries, respectively, and 283 targets were present in both libraries. Functional analysis of these miRNA targets indicated their involvement in important activities, such as development, regulation of transcription, and stress response. We discovered 31 up-regulated miRNAs and 43 down-regulated miRNAs in ‘Yingshuang’, and 46 up-regulated miRNA and 45 down-regulated miRNAs in `Baiye 1’ in response to cold stress, respectively. A total of 763 related target genes were detected by degradome sequencing. The RLM-5′RACE procedure was successfully used to map the cleavage sites in six target genes of C. sinensis. These findings reveal important information about the regulatory mechanism of miRNAs in C. sinensis, and promote the understanding of miRNA functions during the cold response. The miRNA genotype-specific expression model might explain the distinct cold sensitivities between tea lines.

Background: MicroRNAs (miRNAs), involving in various biological and metabolic processes, have been discovered and analyzed in quite a number of plants species, such as Arabidopsis, rice and other plants. However, there have been few reports about grapevine miRNAs in response to gibberelline (GA₃). Results: Solexa technology was used to sequence small RNA libraries constructed from grapevine berries treated with GA₃ and the control. A total of 122 known and 90 novel grapevine miRNAs (Vvi-miRNAs) were identified. Totally, 137 ones were found to be clearly responsive to GA₃, among which 58 were down-regulated, 51 were up-regulated, 21 could only be detected in the control, and seven were only detected in the treatment. Subsequently, we found that 28 of them were differentially regulated by GA₃, with 12 conserved and 16 novel Vvi-miRNAs, based on the analysis of qRT-PCR essays. There existed some consistency in expression levels of GA₃-responsive Vvi-miRNAs between high throughput sequencing and qRT-PCR essays. In addition, 117 target genes for 29 novel miRNAs were predicted. Conclusions: Deep sequencing of short RNAs from grapevine berries treated with GA3 and the control identified 137 GA₃-responsive miRNAs, among which 28 exhibited different expression profiles of response to GA₃ in the diverse developmental stages of grapevine berries. These identified Vvi-miRNAs might be involved in the grapevine berry development and response to environmental stresses.

Grape (Vitis vinifera) is highly sensitive to gibberellin (GA), which effectively induce grape parthenocarpy. Studies showed that miR160s and their target AUXIN RESPONSIVE FACTOR (ARF) responding hormones are indispensable for various aspects of plant growth and development, but their functions in GA-induced grape parthenocarpy remain elusive. In this study, the morphological changes during flower development in response to GA treatments were examined in the ‘Rosario Bianco’ cultivar. The precise sequences of VvmiR160a/b/c/d/e and their VvARF10/16/17 target genes were cloned, sequenced and characterized. The phylogenetic relationship and intron-exon structure of VvARFs and other ARF family members derived from different species were investigated. All VvmiR160s (except VvmiR160b) and VvARF10/16/17 had the common cis-elements responsive to GA, which support their function in GA-mediated grape parthenocarpy. The cleavage role of VvmiR160s-mediated VvARF10/16/17 was verified in grape flowers. Moreover, spatio-temporal expression analysis demonstrated that among VvmiR160 family, VvmiR160a/b/c highly expressed at late stage of flower/berry development, while VvARF10/16/17showed a reverse expression trend. Interestingly, GA exhibited a long-term effect through inducing the expression of VvmiR160a/b/c/e to increase their cleavage product accumulations from 5 to 9 days after treatment, but GA enhanced the expressions of VvARF10/16/17 only at short term. Pearson correlation analysis based on expression data revealed a negative correlation between VvmiR160a/b/c and VvARF10/16/17 in flowers not berries during GA-induced grape parthenocarpy. This work demonstrated that the negative regulation of VvARF10/16/17 expression by VvmiR160a/b/c as key regulatory factors is critical for GA-mediated grape parthenocarpy, and provide significant implications for molecular breeding of high-quality seedless berry.

The effects of ultrasound-enhanced hot air drying on the drying characteristics, microstructure and bioactive profile of germinated highland barley seeds (GHB) were studied. GHB was dried by hot air at 55 °C and 70 °C and ultrasonic intensities of 125.1 W/dm² and 180.2 W/dm², respectively. The results showed that when the drying temperature was 55 °C or 70 °C, the sonicated groups could shorten the drying time by 17.4–26.1% or 18.8–31.3%, respectively. Ultrasound drying at 125.1 W/dm² and 55 °C could mostly increase the content of organic selenium and the rehydration rate, improve the color and maintain the original structure of GHB. Compared with hot air drying alone, the phenolic content did not increase due to ultrasound-enhanced hot air drying. Therefore, drying at an ultrasonic intensity of 125.1 W/dm² and a temperature of 55 °C could effectively shorten the drying time, and enhance the quality of GHB.

In this study, an ultrasound-assisted aqueous two-phase (ATP) extraction method was used for the extraction and purification of phenolic compounds from grape pomace. The effect of acoustic energy densities (AED, 41.1, 63.5, 96.1, 111.2 W/L) and temperatures (20, 30, 40°C) on the yield of phenolics was investigated. An artificial neural network (ANN) was successfully used to correlate the extraction parameters with phenolic yield. Then, a diffusion model based on Fick's second law was used to model the mass transfer process during ultrasound-assisted ATP extraction and evaluate the effective diffusion coefficient of phenolics. The results revealed the increase in AED, and the temperature increased the effective diffusivity of phenolics. The HPLC analysis of anthocyanins and flavonols showed that ultrasound significantly increased the extraction yield of anthocyanins compared with the traditional method. High amounts of rutin and myricetin were recovered using the ATPS systems. Sugars were mainly distributed in the bottom phase, whereas phenolics were located in the top phase. Conclusively, ultrasound-assisted aqueous two-phase (ATP) extraction can be used as an effective method to achieve the simultaneous separation and preliminary purification of phenolics from grape pomace.