Host-induced gene silencing in wild apple germplasm Malus hupehensis confers resistance to the fungal pathogen Botryosphaeria dothidea.

Host-induced gene silencing (HIGS) is an inherent mechanism of plant resistance to fungal pathogens, resulting from cross-kingdom RNA interference (RNAi) mediated by small RNAs (sRNAs) delivered from plants into invading fungi. Introducing artificial sRNA precursors into crops can trigger HIGS of selected fungal genes, and thus has potential applications in agricultural disease control. To investigate the HIGS of apple (Malus sp.) during the interaction with Botryosphaeria dothidea, the pathogenic fungus causing apple ring rot disease, we evaluated whether apple miRNAs can be transported into and target genes in B. dothidea. Indeed, miR159a from Malus hupehensis, a wild apple germplasm with B. dothidea resistance, silenced the fungal sugar transporter gene BdSTP. The accumulation of miR159a in extracellular vesicles (EVs) of both infected M. hupehensis and invading B. dothidea suggests that this miRNA of the host is transported into the fungus via the EV pathway. Knockout of BdSTP caused defects in fungal growth and proliferation, whereas knockin of a miR159a-insensitive version of BdSTP resulted in increased pathogenicity. Inhibition of miR159a in M. hupehensis substantially enhanced plant sensitivity to B. dothidea, indicating miR159a-mediated HIGS against BdSTP being integral to apple immunity. Introducing artificial sRNA precursors targeting BdSTP and BdALS, an acetolactate synthase gene, into M. hupehensis revealed that double-stranded RNAs were more potent than engineered MIRNAs in triggering HIGS alternative to those natural of apple and inhibiting infection. These results provide preliminary evidence for cross-kingdom RNAi in the apple-B. dothidea interaction and establish HIGS as a potential disease control strategy in apple.

MicroRNA candidate miRcand137 in apple is induced by Botryosphaeria dothidea for impairing host defense.

MicroRNA (miRNA)-mediated gene silencing is a master gene regulatory pathway in plant-pathogen interactions. The differential accumulation of miRNAs among plant varieties alters the expression of target genes, affecting plant defense responses and causing resistance differences among varieties. Botryosphaeria dothidea is an important phytopathogenic fungus of apple (Malus domestica). Malus hupehensis (Pamp.) Rehder, a wild apple species, is highly resistant, whereas the apple cultivar "Fuji" is highly susceptible. Here, we identified a 22-nt miRNA candidate named miRcand137 that compromises host resistance to B. dothidea infection and whose processing was affected by precursor sequence variation between M. hupehensis and "Fuji." miRcand137 guides the direct cleavage of and produced target-derived secondary siRNA against Ethylene response factor 14 (ERF14), a transcriptional activator of pathogenesis-related homologs that confers disease resistance to apple. We showed that miRcand137 acts as an inhibitor of apple immunity by compromising ERF14-mediated anti-fungal defense and revealed a negative association between miRcand137 expression and B. dothidea sensitivity in both resistant and susceptible apples. Furthermore, MIRCAND137 was transcriptionally activated by the invading fungi but not by the fungal elicitor, implying B. dothidea induced host miRcand137 as an infection strategy. We propose that the inefficient miRcand137 processing in M. hupehensis decreased pathogen-initiated miRcand137 accumulation, leading to higher resistance against B. dothidea.

Comparative Analysis of Alternative Splicing in Two Contrasting Apple Cultivars Defense against Alternaria alternata Apple Pathotype Infection.

Alternaria blotch disease, caused by the Alternaria alternata apple pathotype (A. alternata AP), is one of the most serious fungal diseases in apples. Alternative splicing (AS), one of the pivotal post-transcriptional regulatory mechanisms, plays essential roles in various disease resistance responses. Here, we performed RNA-Seq for two apple cultivars (resistant cultivar 'Jonathan' (J) and susceptible cultivar 'Starking Delicious' (SD)) infected by A. alternata AP to further investigate their AS divergence. In total, 1454, 1780, 1367 and 1698 specifically regulated differential alternative splicing (DAS) events were detected in J36, J72, SD36 and SD72 groups, respectively. Retained intron (RI) was the dominant AS pattern. Conformably, 642, 764, 585 and 742 uniquely regulated differentially spliced genes (DSGs) were found during A. alternata AP infection. Comparative analysis of AS genes in differential splicing and expression levels suggested that only a small proportion of DSGs overlapped with differentially expressed genes (DEGs). Gene ontology (GO) enrichment analysis demonstrated that the DSGs were significantly enriched at multiple levels of gene expression regulation. Briefly, the specific AS was triggered in apple defense against A. alternata AP. Therefore, this study facilitates our understanding on the roles of AS regulation in response to A. alternata AP infection in apples.

The regulation of DKGA2ox1 and miR171f_3 in scion dwarfing with Diospyros kaki Thunb. cv. 'Nan-tong-xiao-fang-shi' as interstocks.

MAIN CONCLUSION: High-throughput sequencing and yeast one and two-hybrid library screening reveal that DKGA2ox1 and miR171f_3 are involved in the regulation of scion dwarfing with 'Nan-tong-xiao-fang-shi' as interstocks. Diospyros kaki Thunb. cv. Nan-tong-xiao-fang-shi ('Nan-tong-xiao-fang-shi') interstocks play a critical role in the scion dwarfing. However, the understanding of the molecular signaling pathways that regulate the scion dwarfing with 'Nan-tong-xiao-fang-shi' as interstocks remain unclear. In this work, the regulatory network in the scion dwarfing with 'Nan-tong-xiao-fang-shi' as interstocks was identified. Using a yeast one-hybrid library screening, luciferase activity analysis, luciferase complementation imaging assays and GFP signal detection, a SPL transcription factor named Diospyros kaki SPL (DKSPL), potentially functioning as a transcriptional activator of the Diospyros kaki GA2ox1 (DKGA2ox1) gene, was identified as a key stimulating factor in the persimmon growth and development. The DKSPL was found in the nucleus, and might play a role in the transcriptional regulation system. A microRNA named miR171f_3 was identified, which might act as a negative regulator of Diospyros kaki SCR (DKSCR) in persimmon. The interactions between DKSCR and seven proteins were experimentally validated with a yeast two-hybrid library screening. Compared to the non-grafted wildtype persimmon, the tissue section of graft union healed well due to the increased expression of cinnamyl-alcohol dehydrogenase. These results indicate that DKGA2ox1 and miR171f_3 may co-promote the scion dwarfing by plant hormone signal transduction pathways.

SWATH-MS-facilitated proteomic profiling of fruit skin between Fuji apple and a red skin bud sport mutant.

BACKGROUND: Apple is one of the most popular fruit crops world-wide and its skin color is an important quality consideration essential for commercial value. However, the strategy on genetic breeding for red skin apple and the genetic basis of skin color differentiation is very limited and still largely unknown. RESULTS: Here, we reported a bud sport mutant of Fuji apple with red skin color and enhanced anthocyanins accumulation. Quantitative SWATH-MS (sequential window acquisition of all theoretical spectra-mass spectrometry) proteomics investigations revealed proteome changes in the apple red skin bud mutation and a total of 451 differentially expressed proteins were identified in apple skin. The mutant showed significantly increased expression levels of photosynthesis-related proteins, stress-related proteins as well as anthocyanins biosynthesis pathway. On the other hand, substantial downregulation of mitogen-activated protein kinase 4 (MAPK4) and mevalonate kinase (MVK) were detected, indicating a promising role for the red skin color development in the mutant. Furthermore, we also hypothesize that a post-transcriptional regulation of the skin color formation occurs in the mutant through the advanced SWATH-MS analysis. CONCLUSION: Our work provides important information on the application of proteomic methods for analysing proteomes changes in Fuji apple and highlights a clade of regulatory proteins potentially contributing for the molecular breeding of fruit skin color.

The role of miR319a and its target gene TCP4 in the regulation of pistil development in Prunus mume.

The microRNAs (miRNAs) comprise a broad class of non-coding small endogenous RNAs that are associated with many biological processes through the regulation of target genes, such as leaf morphogenesis and polarity, biotic and abiotic stress responses, and root and flower development. We identified a miRNA that affects flower development, miR319a, in Prunus mume. The Pm-miR319a target, Pm-TCP4, was validated by 5'RACE. The higher expression of Pm-TCP4 in imperfect flowers showed that Pm-TCP4 might promote pistil abortion. Further experiments showed that Pm-miR319a negatively regulates the expression of Pm-TCP4 mRNAs and affected pistil development. Sixteen downstream genes of Pm-TCP4 related to flower development were predicted. Previous studies have shown that they have an impact on the development of pistils. In this study it was established that Pm-miR319a indirectly regulates the development of pistils by regulating its target gene Pm-TCP4.

Malus hupehensis miR168 Targets to ARGONAUTE1 and Contributes to the Resistance against Botryosphaeria dothidea Infection by Altering Defense Responses.

MicroRNA (miRNA)-mediated post-transcriptional regulation plays a fundamental role in various plant physiological processes, including responses to pathogens. MicroRNA168 has been implicated as an essential factor of miRNA pathways by targeting ARGONAUTE1 (AGO1), the core component of the RNA-induced silencing complex (RISC). A fluctuation in AGO1 expression influences various plant-pathogen interactions, and the homeostasis of AGO1 and miR168 accumulation is maintained by a complicated feedback regulatory loop. In this study, the connection between miR168 and the resistance of Malus hupehensis to Botryosphaeria dothidea is revealed. The induction of both the mature miR168 and its precursor in plants subjected to B. dothidea infection indicate the transcriptional activation of MIR168a. MIR168a promoter analysis demonstrates that the promoter can be activated by B. dothidea and salicylic acid (SA). However, the direct target of miR168, M. hupehensis ARGONAUTE1 (MhAGO1), is shown to be induced under the infection. Expression and transcription activity analysis demonstrate the transcriptional activation and the post-transcriptional suppression of MhAGO1 in response to B. dothidea infection. By inhibiting reactive oxygen species (ROS) production and enhancing SA-mediated defense responses, miR168a delays the symptom development of leaves inoculated with B. dothidea and impedes the pathogen growth, while MhAGO1 is found to have the opposite effects. Collectively, these findings suggest that the expression of miR168 and MhAGO1 in M. hupehensis in response to B. dothidea infection is regulated by a complicated mechanism. Targeting to MhAGO1, a negative regulator, miR168 plays a positive role in the resistance by alterations in diverse defense responses.

Overexpression of the Malus hupehensis MhNPR1 gene increased tolerance to salt and osmotic stress in transgenic tobacco.

Earlier, we have reported that overexpression of Malus hupehensis Non-expressor of pathogenesis related gene 1 (MhNPR1) gene in tobacco could induce the expression of pathogenesis-related genes and enhance resistance to fungus Botrytis cinerea. In this study, we showed that MhNPR1 can be induced by NaCl, PEG6000, low temperature (4 °C), abscisic acid and apple aphids' treatments in M. hupehensis. Heterogonous expression of MhNPR1 gene in tobacco conferred enhanced resistance to NaCl at the stage of seed germination, and conferred resistance to mannitol at the stage of seed germination and to PEG6000 at the stage of seedlings. Furthermore, overexpression of MhNPR1 in transgenic tobacco led to higher expression levels of osmotic-stress related genes compared with wild-type plants. This was the first report of a novel function of NPR1 that overexpression of MhNPR1 gene has a positive effect on salt and osmotic stress in tobacco, which differs from the function that overexpressing of AtNPR1 gene has a negative effect on dehydration and salt stress in rice.

Apple ring rot-responsive putative microRNAs revealed by high-throughput sequencing in Malus × domestica Borkh.

MicroRNAs (miRNAs) are small non-coding RNAs, which silence target mRNA via cleavage or translational inhibition to function in regulating gene expression. MiRNAs act as important regulators of plant development and stress response. For understanding the role of miRNAs responsive to apple ring rot stress, we identified disease-responsive miRNAs using high-throughput sequencing in Malus × domestica Borkh.. Four small RNA libraries were constructed from two control strains in M. domestica, crabapple (CKHu) and Fuji Naga-fu No. 6 (CKFu), and two disease stress strains, crabapple (DSHu) and Fuji Naga-fu No. 6 (DSFu). A total of 59 miRNA families were identified and five miRNAs might be responsive to apple ring rot infection and validated via qRT-PCR. Furthermore, we predicted 76 target genes which were regulated by conserved miRNAs potentially. Our study demonstrated that miRNAs was responsive to apple ring rot infection and may have important implications on apple disease resistance.

Whole-genome resequencing identifies candidate genes and allelic variation in the MdNADP-ME promoter that regulate fruit malate and fructose contents in apple.

Soluble sugar and organic acids are key determinants of fruit organoleptic quality and directly affect the commodity value and economic returns of fruit crops. We performed whole-genome sequencing of the apple varieties Gala and Xiahongrou, along with their F1 hybrids, to construct a high-density bin map. Our quantitative genetic analysis pinpointed 53 quantitative trait loci (QTLs) related to 11 sugar and acid traits. We identified a candidate gene, MdNADP-ME, responsible for malate degradation, in a stable QTL on linkage group 15. Sequence analysis revealed an A/C SNP in the promoter region (MEp-799) that influences binding of the MdMYB2 transcription factor, thereby affecting MdNADP-ME expression. In our study of various apple genotypes, this SNP has been demonstrated to be linked to malate and fructose levels. We also developed a dCAPS marker associated with fruit fructose content. These results substantiate the role of MdNADP-ME in maintaining the equilibrium between sugar and acid contents in apple fruits.

The Mh-miR393a-TIR1 module regulates Alternaria alternata resistance of Malus hupehensis mainly by modulating the auxin signaling.

miRNAs govern gene expression and regulate plant defense. Alternaria alternata is a destructive fungal pathogen that damages apple. The wild apple germplasm Malus hupehensis is highly resistant to leaf spot disease caused by this fungus. Herein, we elucidated the regulatory and functional role of miR393a in apple resistance against A. alternata by targeting Transport Inhibitor Response 1. Mature miR393 accumulation in infected M. hupehensis increased owing to the transcriptional activation of MIR393a, determined to be a positive regulator of A. alternata resistance to either 'Orin' calli or 'Gala' leaves. 5' RLM-RACE and co-transformation assays showed that the target of miR393a was MhTIR1, a gene encoding a putative F-box auxin receptor that compromised apple immunity. RNA-seq analysis of transgenic calli revealed that MhTIR1 upregulated auxin signaling gene transcript levels and influenced phytohormone pathways and plant-pathogen interactions. miR393a compromised the sensitivity of several auxin-signaling genes to A. alternata infection, whereas MhTIR1 had the opposite effect. Using exogenous indole-3-acetic acid or the auxin synthesis inhibitor L-AOPP, we clarified that auxin enhances apple susceptibility to this pathogen. miR393a promotes SA biosynthesis and impedes pathogen-triggered ROS bursts by repressing TIR1-mediated auxin signaling. We uncovered the mechanism underlying the miR393a-TIR1 module, which interferes with apple defense against A. alternata by modulating the auxin signaling pathway.

miR164-NAC21/22 module regulates the resistance of Malus hupehensis against Alternaria alternata by controlling jasmonic acid signaling.

Apple leaf spot disease caused by Alternaria alternata apple pathotype (A. alternata AP) is one of the most severe fungal diseases affecting apple cultivation. Transcription factors are involved in various disease-resistance responses, and many of them are regulated by miRNAs. Here, we performed RNA-Seq to investigate gene expression changes during the defense response of Malus hupehensis against A. alternata AP. NAC21/22 was induced upon A. alternata AP infection and silenced by miR164 via direct mRNA cleavage. Contrasting expression patterns were noted between mature miR164 and NAC21/22 during infection. Contrary to NAC21/22 silencing, transiently overexpressing NAC21/22 in M. hupehensis alleviated disease symptoms on 'gala' leaves, impeded A. alternata AP growth, and promoted jasmonic acid (JA) signaling-related gene expression. Importantly, transient miR164f overexpression in 'gala' leaves enhanced A. alternata AP sensitivity, due perhaps to NAC21/22 downregulation, whereas miR164 suppression produced an opposite effect. In summary, the miR164-NAC21/22 module plays a pivotal role in apple resistance against A. alternata AP by regulating JA signaling.

Difference in calcium accumulation in the fruit of two apple varieties and its relationship with vascular bundle development in the pedicel.

Calcium (Ca) is an essential mineral element for plant growth and development that plays a key role in fruit growth and quality formation. To study the absorption and transport of Ca in 'Tonami' (susceptible to bitter pit (BP)) and 'Fuji' (resistant to BP), fruiting branches of 'Tonami' and 'Fuji' were injected with 0.05% 44Ca at the fruitlet stage (37 days after full bloom (DAFB)) and fruit expansion stage (72 DAFB). At the fruitlet and fruit expansion stages, the 44Ca content of fruiting branches increased from high to low in leaves, shoots, and fruit. In fruit, the 44Ca content was highest in the peel and lowest in the flesh. In both 'Tonami' and 'Fuji', Ca uptake was more efficient at the fruitlet stage than at the fruit expansion stage. 'Tonami' had a shorter growth and development time and earlier loss of fruit pedicel xylem structure and functionality than 'Fuji', resulting in less Ca accumulation in the fruit. The low Ca uptake efficiency of 'Tonami' fruit, the short Ca accumulation time, and the high Ca dilution resulted in low 44Ca content in the fruit, which may explain the susceptibility of 'Tonami' fruit to BP disease.

Overexpressing MhNPR1 in transgenic Fuji apples enhances resistance to apple powdery mildew.

Fuji is susceptible to fungal diseases like apple powdery mildew. Non-expressor of pathogenesis-related gene 1 (NPR1) plays a key role in regulating salicylic acid (SA)-mediated systemic acquired resistance (SAR). Previous studies show that overexpressing the Malus hupehensis-derived NPR1 (MhNPR1) gene in tobacco induces the transcript expression of pathogenesis-related genes (PRs) and resistance to the fungus Botrytis cinerea. In this study we introduced the MhNPR1 gene into the 'Fuji' apple via Agrobacterium-mediated transformation. Four transgenic apple lines were verified by PCR and RT-PCR. The semi-quantitative RT-PCR results showed that transcript overexpression of the MhNPR1 gene induced the expression of MdPRs and MdMLO genes known to interact with powdery mildew. Furthermore, the transgenic apple plants resisted infection by apple powdery mildew better than the wild-type plants. As a result, transcript overexpression of the MhNPR1 gene induced SAR and enhanced the Fuji apple's resistance to fungal disease.

Analysis of expressed sequence tags from grapevine flower and fruit and development of simple sequence repeat markers.

A total of 6,230 EST sequences were produced from 7,561 clones in a cDNA library generated from grapevine (Vitis vinifera cv. 'Summer Black') flower and fruit tissues in this study. After cluster and assembly analysis of the datasets, 3,582 unigenes (GenBank accession numbers GW836604-GW840185) were established, among which 381 were new grapevine EST sequences. Out of the 381 new ESTs, 289 could be mapped on the 19 grapevine chromosomes. 913 unique ESTs with known or putative functions were assigned to 11 putative cellular roles. 540 potentially workable grapevine EST-SSRs were developed from 3,582 unigenes and about 42.6% of these unigenes were identified as true-to-type SSR loci and could amplify polymorphic bands from 22 individual plants of V. vinifera L, indicating that grapevine EST datasets are a valuable source for the development of functional simple sequence repeat (SSR) markers.

Comparative transcriptomics analysis reveals MdGRAS53 contributes to disease resistance against Alternaria blotch of apple.

Alternaria blotch disease, caused by Alternaria alternata apple pathotype (AAAP), is one of the most prevalent diseases in apple production. To identify AAAP resistance-related genes and provide a theoretical basis for Alternaria blotch disease resistance breeding, we used two apple cultivars, 'Jonathan', a variety resistant to AAAP infection, and 'Starking Delicious', a variety susceptible to AAAP infection, as materials to perform transcriptome sequencing of apple leaves 72 h after AAAP infection. A Venn diagram showed that a total of 5229 DEGs of 'Jonathan' and 4326 DEGs of 'Starking Delicious' were identified. GO analysis showed that these DEGs were clustered into 25 GO terms, primarily "metabolic process" and "catalytic activity." Functional classification analyses of the DEGs indicated that "MAPK signaling pathway-plant pathway" is the most significant metabolic pathway among the top 15 KEGG pathways, followed by the "plant hormone signal transduction" pathway. There are more DEGs in 'Jonathan' that are significantly classified GO terms and KEGG pathways than in 'Starking Delicious'. Specifically, 13 DEGs were identified as involved in the GA-GID1-DELLA module, and the expression of MdGRAS53, a homologous gene of DELLA, was significantly upregulated in 'Jonathan' compared with 'Starking Delicious'. Phenotype analysis revealed that exogenous hormone GA3 suppressed apple resistance to AAAP infection and reduced the expression of MdGRAS53. The opposite result was observed for exogenous spraying of paclobutrazol (PAC), an inhibitor of gibberellin synthesis. Overexpression of MdGRAS53 in apple leaves by transient transformation decreased lesion area and the number of spores in leaves infected with AAAP, while silencing MdGRAS53 showed the opposite result. Meanwhile, SA/JA signaling pathway-related genes were upregulated significantly in MdGRAS53-overexpressed leaves and downregulated significantly in MdGRAS53-silenced leaves. The findings suggest that the GA-GID1-DELLA module is involved in apple resistance to AAAP, and MdGRAS53, a DELLA homologous gene, may play a positive role in this resistance by modulating cooperative JA- and SA-dependent pathways.

Differential expression of ferritin genes in response to abiotic stresses and hormones in pear (Pyrus pyrifolia).

In this study, the expression patterns of four ferritin genes (PpFer1, PpFer2, PpFer3, and PpFer4) in pear were investigated using quantitative real-time PCR. Analysis of tissue-specific expression revealed higher expression level of these genes in leaves than in other tested tissues. These ferritin genes were differentially expressed in response to various abiotic stresses and hormones treatments. The expression of ferritin wasn't affected by Fe(III)-citrate treatment. Abscisic acid significantly enhanced the expression of all four ferritin genes, especially PpFer2, followed by N-benzylyminopurine, gibberellic acid, and indole-3-acetic acid. The expression peaks of PpFer1 and PpFer3 in leaves appeared at 6, 6, and 12 h, respectively, after pear plant was exposed to oxidative stress (5 mM H(2)O(2)), salt stress (200 mM NaCl), and heat stress (40°C). A significant increase in PpFer4 expression was detected at 6 h after salt stress or heat stress. The expression of ferritin genes was not altered by cold stress. These results suggested that ferritin genes might be functionally important in acclimation of pear to salt and oxidative stresses. Hormone treatments had no significant effect on expression of ferritin genes compared to abiotic stresses. This showed accumulation of ferritin genes could be operated by different transduction pathways under abiotic stresses and hormones treatments.

Using artificial neural network in predicting the key fruit quality of loquat.

The formation and regulation of loquat fruit quality have always been an important research field to improve fruit quality, commodities, and market value. Fruit size, soluble solids content, and titratable acid content represent the most important quality factors in loquat. Mineral nutrients in abundance or deficiency are among the most important key factor that affect fruit quality. In the present study, we use artificial neural network (ANN) to explore the effects of mineral nutrients in soil and leaves on the key fruit quality of loquat. The results show that the ANN model with the structure of 12-12-1 can predict the single fruit weight with the highest accuracy (R 2 = .91), the ANN model with the structure of 10-11-1 can predict the soluble solid content with the highest accuracy (R 2 = .91), and the ANN model with the structure of 9-10-1 can predict the titratable acid content with the highest accuracy (R 2 = .95). Meanwhile, we also conduct sensitivity analysis to analyze the relative contribution of mineral nutrients in soils and leaves to determine of the key fruit quality. In terms of relative contribution, Ca, Fe, and Mg content in soils and Zn, K, and Ca content in leaves contribute relatively largely to a single fruit weight, Mn, Fe, and Mg content in soils and the N content in leaves contribute relatively largely to the soluble solid content, and the P, Ca, N, Mg, and Fe in leaves contribute relatively largely to the titratable acid content of loquat. The established artificial neural network prediction models can improve the quality of loquat fruit by optimizing the content of mineral elements in soils and leaves.

The regulatory role of gibberellin related genes DKGA2ox1 and MIR171f_3 in persimmon dwarfism.

'Nantongxiaofangshi' (Diospyros kaki Thunb., D. kaki Thunb.) is a local cultivar of persimmon with dwarf-like traits in Jiangsu, China. Closely spaced planting afforded by dwarfism is usually one of the most important ways to promote fruit cultivation and production. However, the understanding of dwarfism in D. kaki Thunb. is very limited at the molecular level, which hinders the further increase of the fruit production. In this work, a persimmon transgenic system was successfully established, and the field experiments of grafting phenotype were carried out. The results showed that D. kaki Thunb. could be used as an interstock to induce dwarfing in grafted scions, and the dwarf character was better when interstock lengths were between 20 and 25 cm. Furthermore, the key genes related to dwarfism in D. kaki Thunb. were screened and verified, and subsequently, the regulatory role of related genes in persimmon dwarfism was figured out. It was found that the gene encoding gibberellin 2-oxidase-1 (DkGA2ox1) involved in GA biosynthesis was associated with the dwarfing in D. kaki Thunb. Overexpression of DkGA2ox1 in Diospyros lotus resulted in a typical dwarf phenotype. Meanwhile, the microRNA data showed that the miR171f_3 demonstrated the active involvement in GA pathway response in persimmon dwarfism. DkGA2ox1 and MIR171f_3, as two highly expressed genes in D. kaki Thunb. interstock, could be used as stimulus signals to affect the content of GA in scion, however, the specific transmission mechanism still needs to be further explored. Ultimately, the bioactive GA level was decreased, resulting in the scion dwarfism.

The R2R3-type MYB transcription factor MdMYB90-like is responsible for the enhanced skin color of an apple bud sport mutant.

The anthocyanin content in apple skin determines its red coloration, as seen in a Fuji apple mutant. Comparative RNA-seq analysis was performed to determine differentially expressed genes at different fruit development stages between the wild-type and the skin color mutant. A novel R2R3-MYB transcription factor, MdMYB90-like, was uncovered as the key regulatory gene for enhanced coloration in the mutant. The expression of MdMYB90-like was 21.3 times higher in the mutant. MdMYB90-like regulates anthocyanin biosynthesis directly through the activation of anthocyanin biosynthesis genes and indirectly through the activation of other transcription factors that activate anthocyanin biosynthesis. MdMYB90-like bound to the promoters of both structural genes (MdCHS and MdUFGT) and other transcription factor genes (MdMYB1 and MdbHLH3) in the yeast one-hybrid system, electrophoretic mobility shift assay, and dual-luciferase assay. Transgenic analysis showed that MdMYB90-like was localized in the nucleus, and its overexpression induced the expression of other anthocyanin-related genes, including MdCHS, MdCHI, MdANS, MdUFGT, MdbHLH3, and MdMYB1. The mutant had reduced levels of DNA methylation in two regions (-1183 to -988 and -2018 to -1778) of the MdMYB90-like gene promoter, which might explain the enhanced expression of the gene and the increased anthocyanin content in the mutant apple skin.