Protoplast isolation can be described as a stress-inducing procedure that causes a range of physiological and molecular changes. This stress response can affect the expression of totipotency in protoplast culture. Plant growth, development, and stress responses are all affected by microRNAs (miRNAs). The mechanism by which miRNAs are involved in protoplast totipotency is still not clear. High-throughput sequencing technology was employed to sequence two populations of small RNAs from Citrus reticulata Blanco calli and callus-derived protoplasts. There are 67 miRNAs known from 35 families, and 277 new miRNAs. Analysis of differentially expressed miRNAs (“DEMs”) identified 18 miRNAs and 64 new miRNAs among these miRNAs. qRTPCR was used to verify the expression patterns of eight DEMs. Target prediction revealed that most targets of miRNAs were transcription factor.
High-throughput sequencing of microRNAs from Citrus reticulata Blanco
Half of the targets had a lower expression level than those of miRNAs. The physiological analysis also showed that isolated protoplasts had high levels of antioxidant activity. Our results suggested that miRNAs might play an important role in protoplast-isolation responses. It has been shown that miRNAs can negatively regulate gene expression post-transcriptionally by either translational repression or direct transcript cleavage [1-3]. Many miRNAs have been discovered in animals, plants and viruses since the discovery of the first miRNA, Lin-4, in Caenorhabditis-elegans. According to miRBase 21 July 2014, 8,496 mature miRNAs have been identified in 73 species of plants, including 53 dicotyledons and 12 monocotyledons. There is increasing evidence that plant miRNAs are involved in almost all biological and metabolic processes including leaf development, stem branching , root development [7-9], control over flowering time , control over floral organ identity [10-12], fruit growth, developmental phase transitions, auxin signals, programmed cell deaths  and response to biotic and abiotic stress.
MiRNA identification can be done using experimental or computational methods, including deep sequencing, deep sequencing, computational method and homologue-based analyses. Deep sequencing is the most efficient because it can quickly generate millions of reads at a predetermined length. Deep sequencing is widely used in plants such as Arabidopsis thaliana, Citrus sinensis, Cucumis sativus, Medicago truncatula , Oryza sativa and Populus trichocarpa. Deep sequencing has been reported for Citrus grandis , Citrus reduta, Citrus sinensis, Citrus trifoliate.
Protoplast Culture can be described as spherical, naked cells that are obtained by enzymatic digestion. Protoplasts are homogeneous cells with no cell wall and have been extensively used in fundamental research as well as plant genetic improvement such as cell division, cell wall synthesis and membrane function. Protoplast Culture are able to regenerate to make whole plants. This is a prerequisite for protoplast-based techniques, particularly in plant genetic improvement research. Protoplasts of most agriculturally important species such as grape and rice have shown recalcitrance in regeneration. It is not known what the reasons are for protoplast recalcitrance. Therefore, more efforts are being made to understand the mechanisms that underlie protoplast totipotency.
Plant protoplast totipotency is achieved through protoplast culture, protoplast isolation and plant regeneration. Protoplast isolation results in morphological, physiological, or molecular changes. The time taken for protoplast separation can vary from several hours to one week depending on the species of the plant and the explant. Long-term treatment can further disrupt cellular redox homeostasis, causing oxidative stress. ROS was detected in enzymatically isolated oat mesophyll protoplasts. This was previously reported. The accumulation of ROS was also observed in protoplasts from other plants such as grapevine and tobacco. In isolated protoplasts from most plants, there was an increase in activity of the antioxidant machinery. This might indicate that protoplast-isolation may have a physiological effect on protoplast regeneration. Researchers have been paying much more attention to the molecular mechanisms that control the expression of protoplast totipotency in plants. Global chromatin condensation was followed by wide-ranging transcriptional and proteomic modifications. These studies revealed a variety of differentially expressed genes in isolated protoplasts. They included heat shock factor A2, MYB protein 7, and bZIP63. These TFs can be used to further investigate the molecular mechanisms that underlie plant protoplast totipotency.
We believe that protoplast isolation might be regulated by miRNAs, given the changes in gene expression. However, we are not aware of any protoplast-isolation sensitive microRNAs that have been identified in plants. This study attempted to identify protoplast-isolation sensitive miRNAs in Citrus. The physiological response to protoplast-isolation has been characterized by detection of H2O2 levels and MDA levels and analysis of antioxidant enzyme activities (SOD, POD). These results will provide a basis for further research on the miRNAs that are associated with protoplast separation and reveal the mechanism of protoplast totipotency.