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Dormancy is a physiological state that plants enter for winter hardiness. Environmental-induced dormancy onset and release in temperate perennials coordinate growth cessation and resumption, but how the entire process, especially chilling-dependent dormancy release and flowering, is regulated remains largely unclear. We utilized the transcriptome profiles of floral buds from fall to spring in apricot (Prunus armeniaca) genotypes with contrasting bloom dates and peach (Prunus persica) genotypes with contrasting chilling requirements (CR) to explore the genetic regulation of bud dormancy. We identified distinct gene expression programming patterns in endodormancy and ecodormancy that reproducibly occur between different genotypes and species. During the transition from endo- to eco-dormancy, 1,367 and 2,102 genes changed in expression in apricot and peach, respectively. Over 600 differentially expressed genes were shared in peach and apricot, including three DORMANCY ASSOCIATED MADS-box (DAM) genes (DAM4, DAM5, and DAM6). Of the shared genes, 99 are located within peach CR quantitative trait loci, suggesting these genes as candidates for dormancy regulation. Co-expression and functional analyses revealed that distinctive metabolic processes distinguish dormancy stages, with genes expressed during endodormancy involved in chromatin remodeling and reproduction, while the genes induced at ecodormancy were mainly related to pollen development and cell wall biosynthesis. Gene expression analyses between two Prunus species highlighted the conserved transcriptional control of physiological activities in endodormancy and ecodormancy and revealed genes that may be involved in the transition between the two stages.

The genetic transformation of plants has become a necessary tool for fundamental plant biology research, as well as the generation of engineered plants exhibiting improved agronomic and industrial traits. However, this technology is significantly hindered by the fact that transgene expression is often highly variable amongst independent transgenic lines. Two of the major contributing factors to this type of inconsistency are inappropriate enhancer-promoter interactions and chromosomal position effects, which frequently result in mis-expression or silencing of the transgene, respectively. Since the precise, often tissue-specific, expression of the transgene(s) of interest is often a necessity for the successful generation of transgenic plants, these undesirable side effects have the potential to pose a major challenge for the genetic engineering of these organisms. In this review, we discuss strategies for improving foreign gene expression in plants via the inclusion of enhancer-blocking insulators, which function to impede enhancer-promoter communication, and barrier insulators, which block the spread of heterochromatin, in transgenic constructs. While a complete understanding of these elements remains elusive, recent studies regarding their use in genetically engineered plants indicate that they hold great promise for the improvement of transgene expression, and thus the future of plant biotechnology

Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium supplemented with sucrose. Therefore, SacB-SacR genes can be used as negative selection markers to suppress the overgrowth of A. tumefaciens in the plant tissue culture process. We generated a mutant A. tumefaciens strain GV2260 (recA-SacB/R) that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R) can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R) to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcription factor. Published version

The wild strawberry (Fragaria vesca) has recently emerged as an excellent model for cultivated strawberry (Fragaria × ananassa) as well as other Rosaceae fruit crops due to its short seed-to-fruit cycle, diploidy, and sequenced genome. Deep sequencing and parallel analysis of RNA ends were used to identify F. vesca microRNAs (miRNAs) and their target genes, respectively. Thirty-eight novel and 31 known miRNAs were identified. Many known miRNAs targeted not only conserved mRNA targets but also developed new target genes in F. vesca. Significantly, two new clusters of miRNAs were found to collectively target 94 F-BOX (FBX) genes. One of the miRNAs in the new cluster is 22 nucleotides and triggers phased small interfering RNA production from six FBX genes, which amplifies the silencing to additional FBX genes. Comparative genomics revealed that the main novel miRNA cluster evolved from duplications of FBX genes. Finally, conserved trans-acting siRNA pathways were characterized and confirmed with distinct features. Our work identified novel miRNA-FBX networks in F. vesca and shed light on the evolution of miRNAs/phased small interfering RNA networks that regulate large gene families in higher plants.

Background MicroRNAs (miRNAs) have recently emerged as important gene regulators in plants. MiRNAs and their targets have been extensively studied in Arabidopsis and rice. However, relatively little is known about the characterization of miRNAs and their target genes in peach (Prunus persica), which is a complex crop with unique developmental programs. Results We performed small RNA deep sequencing and identified 47 peach-specific and 47 known miRNAs or families with distinct expression patterns. Together, the identified miRNAs targeted 80 genes, many of which have not been reported previously. Like the model plant systems, peach has two of the three conserved trans-acting siRNA biogenesis pathways with similar mechanistic features and target specificity. Unique to peach, three of the miRNAs collectively target 49 MYBs, 19 of which are known to regulate phenylpropanoid metabolism, a key pathway associated with stone hardening and fruit color development, highlighting a critical role of miRNAs in the regulation of peach fruit development and ripening. We also found that the majority of the miRNAs were differentially regulated in different tissues, in part due to differential processing of miRNA precursors. Up to 16% of the peach-specific miRNAs were differentially processed from their precursors in a tissue specific fashion, which has been rarely observed in plant cells. The miRNA precursor processing activity appeared not to be coupled with its transcriptional activity but rather acted independently in peach. Conclusions Collectively, the data characterizes the unique expression pattern and processing regulation of peach miRNAs and demonstrates the presence of a complex, multi-level miRNA regulatory network capable of targeting a wide variety of biological functions, including phenylpropanoid pathways which play a multifaceted spatial-temporal role in peach fruit development. Published version

Background MicroRNAs (miRNAs) and their regulatory functions have been extensively characterized in model species but whether apple has evolved similar or unique regulatory features remains unknown. Results We performed deep small RNA-seq and identified 23 conserved, 10 less-conserved and 42 apple-specific miRNAs or families with distinct expression patterns. The identified miRNAs target 118 genes representing a wide range of enzymatic and regulatory activities. Apple also conserves two TAS gene families with similar but unique trans-acting small interfering RNA (tasiRNA) biogenesis profiles and target specificities. Importantly, we found that miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. These miRNA target sites are differentially conserved among MYBs, which is largely influenced by the location and conservation of the encoded amino acid residues in MYB factors. Finally, we found that 10 of the 19 miR828-targeted MYBs undergo small interfering RNA (siRNA) biogenesis at the 3' cleaved, highly divergent transcript regions, generating over 100 sequence-distinct siRNAs that potentially target over 70 diverse genes as confirmed by degradome analysis. Conclusions Our work identified and characterized apple miRNAs, their expression patterns, targets and regulatory functions. We also discovered that three miRNAs and the ensuing siRNAs exploit both conserved and divergent sequence features of MYB genes to initiate distinct regulatory networks targeting a multitude of genes inside and outside the MYB family. Published version

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The unveiling of small RNA (sRNA)-mediated gene regulatory pathways has profoundly shaped our understanding of the complexity of gene regulation. In eukaryotes, sRNAs have been found to control cellular metabolism, growth and differentiation, to maintain genome integrity, and to combat viruses and mobile genetic elements. To gain insight into the roles of small RNAs in apple and peach, we conducted sRNA-seq, computational analysis and molecular experiments to genome-widely characterize their microRNAs (miRNAs) and trans-acting siRNA (tasiRNA) pathways. We identified totally 75 miRNAs or families, including 23 conserved, 10 less-conserved and 42 apple-specific ones, and 118 miRNA target genes in apple. Two classical trans-acting siRNA (tasiRNA) pathways, miR390-TAS3 and miR828-TAS4, were characterized with similar but unique tasiRNA biogenesis profiles and target specificities. Importantly, miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. In contrast to the location of the miR159 target site in a sequence-divergent region, the target sites of miR828 and miR858 are located in the region encoding the conserved R3 repeat domain of MYB proteins. 10 out of the 19 miR828-targeted MYBs undergo the biogenesis of various phased siRNA (phasiRNA), which potentially regulate diverse genes outside the MYB family. In peach, totally 94 miRNAs or families and 80 target genes were identified. Similar pathways of tasiRNA (miR828-TAS4 and miR390-TAS3) or phasiRNA (miR828-MYB-siRNA) processing were also characterized in peach. Taking advantage of reverse computation and public available deep-sequencing data, we demonstrated that the miRNA-TAS-PPR-siRNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to instigate phasiRNA production from PPR �genes, which are triggered by different 22-nt miRNAs, including miR7122, miR1509, and fve-PPRtri1/2 and through distinct mechanistic strategies, like miRNA direct-targeting or indirect-targeting through TAS-like genes, one-hit or two-hit, or even two layers of tasiRNA-TAS interactions. We found that the MIRNA genes of these miRNA triggers show great identity with the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Combined results from phylogenetic analyses and conservation extent profiling revealed that the super-miR7122 was potentially evolved from another miRNA superfamily (super-miR4376), which probably originated from the miR390. Additionally, the miR482/2118-NB-LRR-siRNA pathway was found to be conserved, but evolved with distinct features, in apple and peach. Taken together, widespread and complex miRNA and tasiRNA regulatory networks have been adapted in apple and peach. They add another crucial layer of regulation on gene activity and stability, and must exert essential functions in all aspects of plant life. Ph. D.

Fruit thinning is critical to the success of apple industry because most apple trees are prone to biennial bearing characterized by heavy bloom and over-cropping in the "on" year and low or no cropping in the "off" year. Fruit thinning can improve fruit size, increase return bloom, and reduce alternate bearing habit of apple trees. Chemical thinning has been widely used as a horticultural practice by growers for years in apple and other fruit production. However, its thinning results are often variable and very difficult to predict since parameters as well as their interactions (e.g. concentration, environmental condition, cultivar response) that affect thinning effectiveness have been poorly defined and the regulatory mechanisms at molecular level remain to be illustrated. Therefore, the purpose of this study is to characterize the physiological and molecular responses of the apple trees to the thinning treatments, and then use key genes as molecular markers for screening potential thinning agents. The long-term goal is to understand how the applied chemicals and environmental factors interact and regulate key regulatory genes as well as the thinning effectiveness during thinning process, and establish a predictable model for the improvement of fruit thinning consistency and effectiveness in apple and other fruit trees. Effects of naphthaleneacetic acid (NAA), shading, amimoethoxyvinylglycine (AVG) and 1-methylcyclopropene (1-MCP) on young apple fruit abscission, leaf and fruit ethylene production, and the expression of genes related to ethylene biosynthesis, perception and cell wall degradation were examined in "Golden Delicious" apples (Malus x domestica borkh.). NAA at 15 mg·L-1 and shading increased fruit abscission and ethylene production of leaves and fruit when applied at a 12-mm stage of fruit development, whereas AVG, an inhibitor of ethylene biosynthesis, at 250 mg·L-1 reduced NAA-induced fruit abscission and ethylene production of leaves and fruit. 1-MCP at 160 mg·L-1 had no effect on fruit abscission but induced ethylene production by both leaves and fruit. Changes in the gene expression pattern responding to each treatment were analyzed by real-time quantitative PCR. NAA treatment was found to enhance the expression of genes related to ethylene biosynthesis (MdACS5A, MdACS5B and MdACO1) and perception (MdETR1, MdETR1b, MdETR2, MdERS1 and MdERS2). AVG reduced NAA-induced expression of these genes except for MdERS2 in the fruit abscission zone (FAZ). NAA increased the expression of a polygalacturonase gene (MdPG2) in the FAZ but not in the fruit cortex (FC), whereas AVG reduced NAA-enhanced expression of MdPG2 in the FAZ. These results suggest that ethylene biosynthesis, ethylene perception, and MdPG2 gene are involved in young fruit abscission caused by NAA. On the other hand, 1-MCP did not affect the expression of MdACS5A and MdACS5B in the FAZ, although it enhanced the expression of these two genes in the FC from 6 to 24 hours post-treatment. The expression of MdACO1 in both tissues was increased by 1-MCP after 3 or more days post-treatment. 1-MCP had only a small influence on the expression of most ethylene receptor genes, with the exception of MdETR1, which was up-regulated in the FC to a level similar to that observed for NAA treatment. In response to 1-MCP, in the FAZ, the expression of MdCel1 and MdPG2 was up-regulated at the beginning and the end, respectively, of the experiment, but otherwise remained at or below control levels. 1-MCP did not inhibit NAA-induced abscission of young apple fruit, suggesting that abscission does not solely depend on ethylene signal transduction, or that the periods of effectiveness for 1-MCP and ethylene were asynchronous in this study. Gene expression analysis also revealed that both NAA and shading enhanced the expression of gene related to ABA biosynthesis in the FAZ, which suggested the involvement of ABA in young fruit abscission. Global gene expression profile during young fruit abscission was analyzed using an apple oligonucleotide microarray. More than 700 genes were identified with reproducible changes in transcript abundance in the FAZ after NAA treatment. Genes associated with abscission, ethylene, ABA, cell wall degradation, mitochondrial activity, glycolysis, lipid catabolism, secondary metabolism, abiotic stress, and apoptosis were upregulated, while genes involved in regulation of cell cycle, cell wall biosynthesis, photosynthesis, carbon fixation, chromatin assembly, auxin transport/efflux, cytoskeleton function, and flower development were generally downregulated. Comparison of changes in the gene expression patterns during NAA-induced fruit abscission with shading-induced fruit abscission revealed similarities, but also considerable differences. The data suggested that young fruit were shutting down growth and energy production and undergoing a certain kind of programmed cell death (PCD) induced by these different stimuli. Gene expression analysis in abscising young fruit revealed that photosynthesis, carbon utilization, and ABA/ethylene pathways appeared to operate in both NAA- and shading-induced fruit abscission. However, more sugar signaling genes were regulated in shading-induced fruit abscission, compared to NAA-induced fruit abscission. Ph. D.