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Peach encodes 14 INDETERMINATE DOMAIN (IDD) transcription factors. PpIDD4, -12 and -13 mediated PpDELLA1 binding to the PpGA20ox1 promoter. Each of these three PpIDD-DELLA1 complexes activated transcription of PpGA20ox1. PpTPR1 and -4 interrupted the interaction of PpIDDs with PpDELLA1. The plant growth regulator gibberellin (GA) plays an important role in the rapid growth of annual shoots in peach. Our previous study showed that the peach cultivar 'FenHuaShouXingTao' (FHSXT), a gibberellic acid receptor (gid1) mutant, accumulates active GAs in annual shoot tips. This mutant enhances GA feedback regulation in peach. The results of this study suggested that the PpIDD-DELLA1 complex is the underlying mechanism of GA feedback regulation in peach. Fourteen IDD genes were identified in peach, and three PpIDDs (PpIDD4, -12 and -13, all from group IV) interacted with PpDELLA1, an important component in GA signaling pathway. Truncation, segmentation and site mutation of the promoter of PpGA20ox1 (a GA biosynthesis gene) showed that all three PpIDD proteins recognized the core motif TTGTC. PpIDD4 and -13 mainly bind to site 3, while PpIDD12 binds to site 5 of the PpGA20ox1 promoter. All three PpIDD-DELLA1 complexes activated the PpGA20ox1 promoter-LUC fusion. These data suggested that PpIDDs bridge PpDELLA1 and the promoter of PpGA20ox1, which then activated the transcription of PpGA20ox1. In addition, PpTPR1 and -4 disrupted the interaction of PpIDDs with PpDELLA1. Our research will be helpful for understanding and possibly modifying the regulation of annual shoot growth and GA biosynthesis.

Controlling the tree size of fruit species such as peach can reduce the amount of labor and input needed for orchard management. The phytohormone gibberellin (GA) positively regulates tree size by inducing degradation of the GA signaling repressor DELLA. The N-terminal DELLA domain in this protein is critical for its GA-dependent interaction with the GA receptor GID1 and the resulting degradation of the DELLA protein, which allows for growth-promoting GA signaling. In this study, a DELLA family member, PpeDGYLA, contains a DELLA domain but has amino acid changes in three conserved motifs (DELLA into DGYLA, LEQLE into LERLE, and TVHYNP into AVLYNP). In the absence or presence of GA3, the PpeDGYLA protein did not interact with PpeGID1c and was stable in 35S-PpeDGYLA peach transgenic callus. The overexpression of PpeDGYLA in both polar and Arabidopsis showed an extremely dwarfed phenotype, and these transgenic plants were insensitive to GA3 treatment. PpeDGYLA could interact with PpeARF6-1 and -2, supposed growth-promoting factors. It is suggested that the changes in the DELLA domain of PpeDGYLA may, to some extent, account for the severe dwarf phenotype of poplar and Arabidopsis transgenic plants. In addition, our study showed that the DELLA family contained three clades (DELLA-like, DELLA, and DGLLA). PpeDGYLA clustered into the DGLLA clade and was expressed in all of the analyzed tissues. These results lay the foundation for the further study of the repression of tree size by PpeDGYLA.

Fruit ripening includes several metabolic changes that lead to sweeter and softer fruit. Pectin depolymerization is one of major factors that softens developing grape berries. Pectin lyases (PLs) play important roles in pectin degradation in the grape berry. However, little is known about the temporal and spatial expression of grapevine (Vitis spp.) pectin lyase genes (VvPLs) or their function during fruit ripening and softening. In this study, 18 individual VvPL genes were identified in the grape genome. All VvPL genes were sorted into group I and group II, except VvPL12 which demonstrated higher and similar expression trends in different tissues and organs. In grape berry, VvPL1, 5, 7, 11 and 16 were highly expressed, whereas VvPL18, 15, 2, 13, 10, 14, 17, 6 and 8 showed lower expression levels at different berry developmental stages. Expression of VvPL11 firstly increased and then decreased, and the highest expression was shown at 6 weeks after full bloom (WAFB) during berry development. Over-expression of the VvPL11 gene in tomato caused higher ethylene production and lower firmness compared to wild-type fruit. Moreover, decreased propectin and increased water-soluble pectin (WSP) levels were observed in VvPL11 transgenic tomato fruit. Consistent with this result, the expression levels of SlPG2, SlEXP, and SlPME1, all of which are genes involved in fruit softening, were up-regulated in VvPL11-OE tomato fruit, which supported the idea that VvPL11 plays an important role in fruit ripening and softening. This study provided a comprehensive analysis of the grapevine PL family and advanced our knowledge of the functions of VvPLs during fruit softening.

The heat shock protein 20 (HSP20) proteins comprise an ancient, diverse, and crucial family of proteins that exists in all organisms. As a family, the HSP20s play an obvious role in thermotolerance, but little is known about their molecular functions in addition to heat acclimation. In this study, 42 PpHSP20 genes were detected in the peach genome and were randomly distributed onto the eight chromosomes. The primary modes of gene duplication of the PpHSP20s were dispersed gene duplication (DSD) and tandem duplication (TD). PpHSP20s in the same class shared similar motifs. Based on phylogenetic analysis of HSP20s in peach, Arabidopsis thaliana, Glycine max, and Oryza sativa, the PpHSP20s were classified into 11 subclasses, except for two unclassified PpHSP20s. cis-elements related to stress and hormone responses were detected in the promoter regions of most PpHSP20s. Gene expression analysis of 42 PpHSP20 genes revealed that the expression pattern of PpHSP20-32 was highly consistent with shoot length changes in the cultivar ‘Zhongyoutao 14’, which is a temperature-sensitive semi-dwarf. PpHSP20-32 was selected for further functional analysis. The plant heights of three transgenic Arabidopsis lines overexpressing PpHSP20-32 were significantly higher than WT, although there was no significant difference in the number of nodes. In addition, the seeds of three over-expressing lines of PpHSP20-32 treated with high temperature showed enhanced thermotolerance. These results provide a foundation for the functional characterization of PpHSP20 genes and their potential use in the growth and development of peach.

The heat shock protein 20 (HSP20) proteins comprise an ancient, diverse, and crucial family of proteins that exists in all organisms. As a family, the HSP20s play an obvious role in thermotolerance, but little is known about their molecular functions in addition to heat acclimation. In this study, 42

Apple (Malus × domestica Borkh.) is one of the most cultivated fruit crops in China. Apple trees frequently encounter waterlogging stress, mainly due to excess rainfall, soil compaction, or poor soil drainage, results in yellowing leaves and declined fruit quality and yield in some regions. However, the mechanism underlying the response to waterlogging has not been well elucidated. Therefore, we performed a physiological and transcriptomic analysis to examine the differential responses of two apple rootstocks (waterlogging-tolerant M. hupehensis and waterlogging-sensitive M. toringoides) to waterlogging stress. The results showed that M. toringoides displayed more severe leaf chlorosis during the waterlogging treatment than M. hupehensis. Compared with M. hupehensis, the more severe leaf chlorosis induced by waterlogging stress in M. toringoides was highly correlated with increased electrolyte leakage and superoxide radicals, hydrogen peroxide accumulation, and increased stomata closure. Interestingly, M. toringoides also conveyed a higher ethylene production under waterlogging stress. Furthermore, RNA-seq revealed that a total of 13,913 common differentially expressed genes (DEGs) were differentially regulated between M. hupehensis and M. toringoides under waterlogging stress, especially those DEGs involved in the biosynthesis of flavonoids and hormone signaling. This suggests a possible link of flavonoids and hormone signaling to waterlogging tolerance. Taken together, our data provide the targeted genes for further investigation of the functions, as well as for future molecular breeding of waterlogging-tolerant apple rootstocks.

Phytoplasma can infect thousands of plants and caused huge economic losses around the world. The large-scale spread and serious lethality of phytoplasma prompt the urgent need for sensitive, accurate, visual and rapid detection of these pathogens. Current molecular assays used for detecting phytoplasma are expensive and time consuming. Here, we established a novel All-In-One Dual (AIOD) CRISPR detection platform based on CRISPR/LbCas12a technology and Recombinase Polymerase Amplification (RPA) for the diagnosis of multiple phytoplasma. The protocol is simple, requiring one vessel, rapid and sensitive, and the output is visual. Cas12a/crRNAs complexes are added into a reaction containing RPA Mix, RPA Primers and single-stranded DNA fluorophore-quencher (ssDNA-FQ). All components, including 1 μL of sample DNA, are added together and then incubated in one tube at 37 °C. Phytoplasma was detected after 15 min or less from leaf harvest. Positive results can be observed by the naked eye via fluorescent signals. We optimized the amounts of crRNA, LbCas12a and the ssDNA fluorophore in the detection system. Finally, an optimized system was established containing 1,000 nM ssDNA-FQ and a 2:1:1 ratio of LbCas12a/crRNA1/crRNA2 complex with a 0.8 μM concentration as 1. In the optimized reaction, the AIOD-CRISPR detection system exhibited high sensitivity, with limits of detection reaching 3.37E + 2 copies of phytoplasma DNA per reaction. Field tests indicated the AIOD-CRISPR detection system possessed high specificity and reached the 100% accuracy when compared with PCR detection. In conclusion, the AIOD-CRISPR detection system is a ideal selection with high specificity and sensitivity for phytoplasma detection. Our work provides a technique that can be potentially used to rapidly and simultaneously detect more pathogens.

Jujube production is threatened by jujube witches’ broom (JWB) disease, which is caused by JWB phytoplasma. The jujube TCP transcription factor (TF) ZjTCP16 may be involved in the interaction of jujube plants with JWB phytoplasma. In this study, qRT-PCR proved that the expression pattern of ZjTCP16 was altered by JWB phytoplasma. The gene functions of ZjTCP16 were analyzed by its overexpression in Arabidopsis and jujube, as well as knock-down in. The overexpression of ZjTCP16 in Arabidopsis and jujube resulted in dwarfism and small leaves, while the zjtcp16 CRISPR mutants were higher than the WT. Microscopic observation of paraffin sections of jujube stems showed that ZjTCP16 affected the size of cells. The interactions of ZjTCP16 with ZjAS2 and ZjLOB in both the cytoplasm and nucleus were demonstrated by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Yeast one-hybrid (Y1H) assays and qRT-PCR further confirmed that ZjTCP16 affected the expression of genes involved in leaf morphogenesis and cell proliferation (ZjAS1, ZjKNAT1, ZjKNAT2 and ZjKNAT6) at the mRNA level through the ZjAS2 and ZjLOB pathways. In conclusion, ZjTCP16 regulates plant growth and cell size by altering the expression pattern of morphogenesis-related genes in jujube.

Background The rapid growth of annual shoots is detrimental to peach production. While gibberellin (GA) promotes the rapid growth of peach shoots, there is limited information on the identity and expression profiles of GA-metabolism genes for this species. Results All six GA biosynthetic gene families were identified in the peach genome, and the expression profiles of these family members were determined in peach shoots. The upstream biosynthetic gene families have only one or two members (1 CPS, 2 KSs, and 1 KO), while the downstream gene families have multiple members (7 KAOs, 6 GA20oxs, and 5 GA3oxs). Between the two KS genes, PpKS1 showed a relatively high transcript level in shoots, while PpKS2 was undetectable. Among the seven KAO genes, PpKAO2 was highly expressed in shoots, while PpKAO1 and − 6 were weakly expressed. For the six GA20ox genes, both PpGA20ox1 and − 2 were expressed in shoots, but PpGA20ox1 levels were higher than PpGA20ox2. For the five GA3ox genes, only PpGA3ox1 was highly expressed in shoots. Among these biosynthesis genes, PpGA20ox1 and PpGA3ox1 showed a gradual decrease in transcript level along shoots from top to bottom, and a similar trend was observed in bioactive GA1 and GA4 distribution. Among the GA-deactivation genes, PpGA2ox6 was highly expressed in peach shoots. PpGA2ox1 and − 5 transcripts were relatively lower and showed a similar pattern to PpGA20ox1 and PpGA3ox1 in peach shoots. Overexpression of PpGA20ox1, − 2, or PpGA2ox6 in Arabidopsis or tobacco promoted or depressed the plant growth, respectively, while PpGA3ox1 did not affect plant height. Transient expression of PpGA20ox1 in peach leaves significantly increased bioactive GA1 content. Conclusions Our results suggest that PpGA20ox and PpGA2ox expression are closely associated with the distribution of active GA1 and GA4 in peach annual shoots. Our research lays a foundation for future studies into ways to effectively repress the rapid growth of peach shoot.

The plant growth regulator ethylene influences rachis browning in grape (Vitis vinifera L.). Although the ethylene signaling pathway is well defined, there is limited knowledge on its mode of action during rachis browning. Here, we show that an ethylene response factor (VvERF111) positively regulates chlorophyll degradation in rachis by binding to a DRE motif in the promoter of VvCLH1. The expression of VvERF111 and VvCLH1 in rachis was induced by ethylene and inhibited by 1-methylcyclopropene (1-MCP). VvERF111 belongs to the ERF IX subfamily of the APETALA2/ethylene responsive factor (AP2/ERF) superfamily, shows transcriptional activity in yeast, and is localized in the nucleus and membrane. The transient overexpression of VvERF111 or chlorophyllase (VvCLH1) in grape leaves accelerated chlorophyll degradation. In VvERF111-overexpressing leaves, transcript levels of VvCLH1 were also increased. Our findings offer a deeper understanding of the transcriptional regulation of chlorophyll degradation during the rachis browning of grape.

As a predominant phenolic compound in apple fruits, chlorogenic acid (CGA) benefits human health due to its various antioxidant properties. However, little has been known regarding the molecular mechanism underlying the CGA accumulation in apple fruits. In this study, we measured the CGA content and relative enzymes’ activities during fruit development in two different flesh-colored cultivars ‘Huashuo’ and ‘Red Love’. The CGA content in both cultivars decreased sharply from 30 days after full bloom (DAFB) to 60 DAFB. Notably, the CGA content in fruit flesh was relatively higher than that in the peel. Further, the activities of C3H and HCT enzymes downstream of the CGA biosynthesis showed the similar changing trend as CGA content. Based on the transcriptome data of ‘Huashuo’ fruit at 30 DAFB and 60 DAFB, 23 differentially expressed CGA synthesis-related genes were screened. Gene expression analyses further showed that MdHCT1/2/4/5/6 and MdC3H1/2/3 were positively correlated to the variation of CGA content in two cultivars. These findings establish a theoretical foundation for further mechanism study on CGA biosynthesis and provide guidance for nutrient improvement in apple breeding programs.

The jujube witches’ broom (JWB) phytoplasma is associated with witches’ broom, dwarfism, and smaller leaves in jujube, resulting in yield losses. In this study, eight putative JWB effector proteins were identified from potential mobile units of the JWB genome. Among them, Zaofeng6 induced witches’ broom symptoms in Arabidopsis and jujube. Zaofeng6-overexpressing Arabidopsis and unrooted jujube transformants displayed witches’ broom-like shoot proliferation. Transient expression of Zaofeng6 induced hypersensitive response like cell death and expression of hypersensitive response marker genes, like harpin-induced gene 1 (H1N1), and the pathogenesis-related genes PR1, PR2, and PR3 in transformed Nicotiana benthamiana leaves, suggesting that Zaofeng6 could be a virulence effector. Yeast two-hybrid library screening and bimolecular fluorescence complementation confirmed that Zaofeng6 interacts with ZjTCP7 through its first two α-helix domains in the cell nuclei. ZjTCP7 mRNA and protein abundance decreased in Zaofeng6 transgenic jujube seedlings. The expression of some genes in the strigolactone signaling pathway (ZjCCD7, ZjCCD8, and CYP711A1) were down-regulated in jujube shoots overexpressing Zaofeng6 and in zjtcp7 CRISPR/Cas9 mutants. Zaofeng6 induces shoot proliferation through decreased expression of ZjTCP7 at the transcriptional and translational levels.

Rapid growth of branches in a peach tree restricts the light penetration and air ventilation within the orchard, which lowers fruit quality and promotes the occurrence of diseases and insects. Our previous works showed that PpDELLA1 and PpDELLA2 repress the rapid growth of annual shoots. Proteins that interact with DELLA are vital for its function. In this study, seven PpPIFs (PpPIF1, -2, -3, -4, -6, -7 and -8) were identified in the peach genome and contain a conserved bHLH domain. Among the seven PpPIFs, PpPIF8 interacted with PpDELLA2 through an unknown motif in the C-terminal and/or the bHLH domain. Overexpression of PpPIF8 in Arabidopsis promotes plant height and branch numbers. Hypocotyl elongation was significantly enhanced by PpPIF8 under weak light intensity. PpPIF8 overexpressed in Arabidopsis and transiently expressed in peach seedlings upregulated the transcription of YUCCA and SAUR19 and downregulated SHY1 and -2. Additionally, PpPIF4 and -8 were significantly induced by weak light. Phylogentic analysis and intron patterns of the bHLH domain strongly suggested that PIFs from six species could be divided into two groups of different evolutionary origins. These results lay a foundation for the further study of the repression of shoot growth by PpDELLA2 through protein interaction with PpPIF8 in peach.

Cold stress limits plant growth, development and yields, and the C-repeat binding factors (CBFs) function in the cold resistance in plants. However, how pomegranate CBF transcription factors respond to cold signal remains unclear. Considering the significantly up-regulated expression of PgCBF3 and PgCBF7 in cold-tolerant Punica granatum ‘Yudazi’ in comparison with cold-sensitive ‘Tunisia’ under 4 °C, the present study focused on the two CBF genes. PgCBF3 was localized in the nucleus, while PgCBF7 was localized in the cell membrane, cytoplasm, and nucleus, both owning transcriptional activation activity in yeast. Yeast one-hybrid and dual-luciferase reporter assay further confirmed that PgICE1 could specifically bind to and significantly enhance the activation activity of the promoters of PgCBF3 and PgCBF7. Compared with the wild-type plants, the PgCBF3 and PgCBF7 transgenic Arabidopsis thaliana lines had the higher survival rate after cold treatment; exhibited increased the contents of soluble sugar and proline, while lower electrolyte leakage, malondialdehyde content, and reactive oxygen species production, accompanying with elevated enzyme activity of catalase, peroxidase, and superoxide dismutase; and upregulated the expression of AtCOR15A, AtCOR47, AtRD29A, and AtKIN1. Collectively, PgCBFs were positively regulated by the upstream PgICE1 and mediated the downstream COR genes expression, thereby enhancing freezing tolerance.

Pre-planting testing of seeds and plantlets for the existence of quarantine pathogens is an important phytosanitary measure. The CRISPR-mediated molecular diagnostic methodologies are being developed for pathogens detection, but many challenges remain. Here, we profiled an engineered Crispr/LbCas12a variant (LbCas12a-5M) that has more robust trans-cleavage activity and a wider PAM sequences (TNTN) preference than wild type. We developed a procedure for screening specific sequences of bacterial plant pathogens, and the designed species-specific crRNA displayed no cross-reactions with other bacterial species. Combined with a simple extraction of bacterial DNA, an LbCas12a-5M-based visual detection technique was established and optimized for detecting quarantine pathogens Erwinia amylovora and Acidovorax citrulli with detection limits up to 40 CFU/reaction and a sensitivity consistent with qPCR assay. This protocol was faster and simpler than qPCR, requiring 40 min or less from sample preparation. We further validated the potential application of the method by showing that it can be used for rapid and accurate diagnosis of A. citrulli on seeds of watermelon, with 100% agreement with the results of qPCR assay. The developed method simplifies the detection of pathogens and provides cost-effective countermeasures to quarantine interventions.

Apple (Malus domestica Borkh.), an economically important fruit crop, is widely cultivated worldwide. However, apple trees often suffer from environmental stressors which affect the quality and yield of apple fruit. Acetylserotonin methyltransferase (ASMT) is a key enzyme in melatonin biosynthesis and is known to play critical roles in melatonin production and stress responses of plants. In this study, a total of 37 ASMT genes in apple were identified and unequally distributed on 10 apple chromosomes, with fragment duplication detected in four pairs of genes. The ASMT genes were classified into three groups based on their sequence compositions and phylogenetic relationship. Sequence analysis showed that apple ASMT genes were generally conserved in each group, with minor variations in gene structure and motif distribution. Transcriptome analysis revealed that most apple ASMT genes were nearly unchanged or downregulated under heat, cold, drought, and NaCl stress, whereas MdASMT6, MdASMT11, MdASMT14 and MdASMT19 were highly induced by these stressors. Subsequent qRT-PCR of four apple ASMT genes showed that MdASMT11 and MdASMT14 were extremely up-regulated under heat, cold, drought, and NaCl stress. They were expressed in all tissues, especially in the roots. Subcellular localization showed that MdASMT11 mainly distributed on cell membranes and MdASMT14 mainly anchored to the nucleus and cell membrane, indicating that MdASMT11 and MdASMT14 are potential genes regulating abiotic stress resistance in apple. These results facilitate further investigation to the functional characterization of MdASMT genes in the synthesis of melatonin and the response to abiotic stress and in apple.