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Chinese jujube (Ziziphus jujuba) is an important fruit tree in China. Jujube witches' broom (JWB) disease, caused by JWB phytoplasma (‘Candidatus phytoplasma ziziphi’), is the most devastating infectious disease of the jujube tree. Lipoxygenase (LOX) plays important roles in the jasmonic acid (JA) biosynthesis pathway and plant defense to biotic stresses. In this study, we identified 13 LOX genes in the jujube genome distributed on 5 chromosomes. The ZjLOX proteins were classified into two categories based on phylogenetic analysis. We tracked gene duplication events and found two sets of tandem repeats on chromosome 2. We predicted the promoters of the ZjLOXs using PlantCARE and found a large number of cis elements that may respond to phytohormones, light, and biological stresses. Transcriptomic, proteomic, and qRT-PCR expression analyses showed that ZjLOX02, ZjLOX05, ZjLOX06 and ZjLOX08 may play important roles in the interaction between jujube and phytoplasma. This study provides a foundation for better understanding both the molecular evolution of the jujube LOX gene family and the response of the ZjLOX genes to JWB phytoplasma infection.

Apple (Malus × domestica Borkh.) is a perennial woody plant that often suffers from various biological stresses. Many harmful pathogens can infect apple trees and lead to reduced production. We comprehensively identified the WRKY genes in the apple genome and analyzed their expression in response to several biological stressors, including Alternaria alternata, Pythium ultimum, Botryosphaeria dothidea, Erwinia amylovora, Penicillium expansum, Gymnosporangium yamadae, and Apple replant disease. There were 113 MdWRKYs identified in the apple genome. Twenty-two MdWRKYs were differentially expressed in response to at least five pathogens. Promoter sequence analysis showed that these genes carried many defense- and stress-responsive elements, such as MeJA-response elements, salicylic acid-response elements, and W-box elements, in their promoters. Transient expression assays showed that MdWRKY40a and MdWRKY54h played negative roles in defense against B. dothidea infection. WRKY40 and WRKY60 and the MdWKRY33s might play important roles in responding to pathogens and are conserved in some plants. These differentially expressed MdWRKYs might play key roles in the apple response to multiple pathogens.

Summary Co‐infection of apple trees with several viruses/viroids is common and decreases fruit yield and quality. Accurate and rapid detection of these viral pathogens helps to reduce losses and prevent virus spread. Current molecular detection assays used for apple viruses require specialized and expensive equipment. Here, we optimized a CRISPR/Cas12a‐based nucleic acid detection platform for the diagnosis of the most prevalent RNA viruses/viroid in apple, namely Apple necrotic mosaic virus (ApNMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd). We detected each RNA virus/viroid directly from crude leaf extracts after simultaneous multiplex reverse transcription‐recombinase polymerase amplification (RT‐RPA) with high specificity. Positive results can be distinguished by the naked eye via oligonucleotide‐conjugated gold nanoparticles. The CRISPR/Cas12a‐RT‐RPA platform exhibited comparable sensitivity to RT‐qPCR, with limits of detection reaching 250 viral copies per reaction for ASPV and ASGV and 2500 copies for the others. However, this protocol was faster and simpler, requiring an hour or less from leaf harvest. Field tests showed 100% agreement with RT‐PCR detection for 52 samples. This novel Cas12a‐based method is ideal for rapid and reliable detection of apple viruses in the orchard without the need to send samples to a specialized laboratory.

Melatonin acts both as an antioxidant and as a growth regulatory substance in plants. Pseudomonas fluorescens endophytic bacterium has been shown to produce melatonin and increase plant resistance to abiotic stressors through increasing endogenous melatonin. However, in bacteria, genes are still not known to be melatonin-related. Here, we reported that the bacterial phenylalanine 4-hydroxylase (PAH) may be involved in the 5-hydroxytryptophan (5-HTP) biosynthesis and further influenced the subsequent production of melatonin in P. fluorescens. The purified PAH protein of P. fluorescens not only hydroxylated phenylalanine but also exhibited l-tryptophan (l-Trp) hydroxylase activity by converting l-Trp to 5-HTP in vitro. However, bacterial PAH displayed lower activity and affinity for l-Trp than l-phenylalanine. Notably, the PAH deletion of P. fluorescens blocked melatonin production by causing a significant decline in 5-HTP levels and thus decreased the resistance to abiotic stress. Overall, this study revealed a possible role for bacterial PAH in controlling 5-HTP and melatonin biosynthesis in bacteria, and expanded the current knowledge of melatonin production in microorganisms.

Background Heat shock factors (HSFs) play important roles during normal plant growth and development and when plants respond to diverse stressors. Although most studies have focused on the involvement of HSFs in the response to abiotic stresses, especially in model plants, there is little research on their participation in plant growth and development or on the HSF (PpHSF) gene family in peach (Prunus persica). Methods DBD (PF00447), the HSF characteristic domain, was used to search the peach genome and identify PpHSFs. Phylogenetic, multiple alignment and motif analyses were conducted using MEGA 6.0, ClustalW and MEME, respectively. The function of PpHSF5 was confirmed by overexpression of PpHSF5 into Arabidopsis. Results Eighteen PpHSF genes were identified within the peach genome. The PpHSF genes were nonuniformly distributed on the peach chromosomes. Seventeen of the PpHSFs (94.4%) contained one or two introns, except PpHSF18, which contained three introns. The in silico-translated PpHSFs were classified into three classes (PpHSFA, PpHSFB and PpHSFC) based on multiple alignment, motif analysis and phylogenetic comparison with HSFs from Arabidopsis thaliana and Oryza sativa. Dispersed gene duplication (DSD at 67%) mainly contributed to HSF gene family expansion in peach. Promoter analysis showed that the most common cis-elements were the MYB (abiotic stress response), ABRE (ABA-responsive) and MYC (dehydration-responsive) elements. Transcript profiling of 18 PpHSFs showed that the expression trend of PpHSF5 was consistent with shoot length changes in the cultivar ‘Zhongyoutao 14’. Further analysis of the PpHSF5 was conducted in 5-year-old peach trees, Nicotiana benthamiana and Arabidopsis thaliana, respectively. Tissue-specific expression analysis showed that PpHSF5 was expressed predominantly in young vegetative organs (leaf and apex). Subcellular localization revealed that PpHSF5 was located in the nucleus in N. benthamiana cells. Two transgenic Arabidopsis lines were obtained that overexpressed PpHSF5. The root length and the number of lateral roots in the transgenic seedlings were significantly less than in WT seedlings and after cultivation for three weeks. The transgenic rosettes were smaller than those of the WT at 2–3 weeks. The two transgenic lines exhibited a dwarf phenotype three weeks after transplanting, although there was no significant difference in the number of internodes. Moreover, the PpHSF5-OE lines exhibited enhanced thermotolerance. These results indicated that PpHSF5 might be act as a suppresser of growth and development of root and aerial organs.

Apple (Malus domestica Borkh.), an economically important tree fruit worldwide, frequently suffers from temperature stress during growth and development, which strongly affects the yield and quality. Heat shock protein 20 (HSP20) genes play crucial roles in protecting plants against abiotic stresses. However, they have not been systematically investigated in apple. In this study, we identified 41 HSP20 genes in the apple 'Golden Delicious' genome. These genes were unequally distributed on 15 different chromosomes and were classified into 10 subfamilies based on phylogenetic analysis and predicted subcellular localization. Chromosome mapping and synteny analysis indicated that three pairs of apple HSP20 genes were tandemly duplicated. Sequence analysis revealed that all apple HSP20 proteins reflected high structure conservation and most apple HSP20 genes (92.6%) possessed no introns, or only one intron. Numerous apple HSP20 gene promoter sequences contained stress and hormone response cis-elements. Transcriptome analysis revealed that 35 of 41 apple HSP20 genes were nearly unchanged or downregulated under normal temperature and cold stress, whereas these genes exhibited high-expression levels under heat stress. Subsequent qRT-PCR results showed that 12 of 29 selected apple HSP20 genes were extremely up-regulated (more than 1,000-fold) after 4 h of heat stress. However, the heat-upregulated genes were barely expressed or downregulated in response to cold stress, which indicated their potential function in mediating the response of apple to heat stress. Taken together, these findings lay the foundation to functionally characterize HSP20 genes to unravel their exact role in heat defense response in apple.

Adventitious root (AR) formation is indispensable for vegetative asexual propagation. Indole-3-butyric acid (IBA) functioned indirectly as precursor of IAA in regulating AR formation. Ethylene affects auxin synthesis, transport, and/or signaling processes. However, the interactions between auxin and ethylene that control AR formation in apple have not been elucidated. In this study, we investigated the effects of IBA and its interaction with ethylene on AR development in apple. The results revealed that IBA stimulated the formation of root primordia, increased the number of ARs, and upregulated expression of genes (MdWOX11, MdLBD16, and MdLBD29) involved in AR formation. Comparison of different periods of IBA application indicated that IBA was necessary for root primordium formation, while long time IBA treatment obviously inhibited root elongation. RNA-seq analysis revealed that many plant hormone metabolism and signal transduction related genes were differentially expressed. IBA stimulated the production of ethylene during AR formation. Auxin inhibiting ARs elongation depended on ethylene. Together, our results suggest that the inhibitory role of auxin on AR elongation in apples is partially mediated by stimulated ethylene production.

Witches’ broom disease, caused by phytoplasma, is currently the most destructive disease of jujube. Tetracycline derivatives have been used to treat witches’ broom, and can allow recovery from the phytoplasma infection. Genes related to jasmonic acid (JA) biosynthesis and JA-induced protein-like play roles in phytoplasma infection. Jujube shoots (Ziziphus jujuba Mill. ‘Huizao’) infected by phytoplasma were excised and grown in vitro before treatment with tetracycline. RNA-Seq and iTRAQ analyses of samples treated for different lengths of time were performed during recovery from jujube witches’ broom (JWB) symptoms and diminishing presence of phytoplasma. Phytoplasma was not detected by PCR in the shoots after 6 months of tetracycline treatment (MTT). RNA-Seq and iTRAQ analyses identified 26,790 genes and 6184 proteins from jujube shoot samples. There were 272 differentially expressed genes (DEGs) and 20 differentially expressed proteins (DEPs) between the 3 and 6 MTT samples, respectively. The largest number and the greatest changes of DEGs and DEPs were for those related to alpha-linolenic acid metabolism, jasmonate biosynthesis, and jasmonate induced protein-like (JIPs). JA content slightly decreased at the sixth month compared with the third month. The research avenues explored here showed that genes in the JA biosynthesis pathway, proteins that respond to JA (JIPs), and JA content itself were concurrently regulated during JWB recovery. Alpha-linolenic acid metabolism pathway had higher RichFactor in comparisons between the 3 and 6 MTT samples during JWB recovery, which suggesting JA played vital roles during JWB recovery. The results in this study will help us to understand the roles JA plays in host-phytoplasma interactions during recovery and infection of JWB.

Apple is one of the most important fruit trees in temperate zones, and is cultivated widely throughout the world. Drought stress affects the normal growth of apple tree, and further affects fruit yield and quality. The present study examined the effects of drought on photosynthesis and water use efficiency (WUE) of two apple cultivars (Honeycrisp and Yanfu 3) that differ in drought tolerance. The results showed that the photosynthetic rate decreased in response to drought stress for both cultivars, with significant differences in intensity. Values for net photosynthetic rate (Pn) in stressed Yanfu 3 remained significantly lower than in the controls, while, for Honeycrisp, only a slight drop in photosynthesis. Similarly, stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr) were markedly reduced in Yanfu 3 under drought stress. However, Honeycrisp showed only minor changes. Under drought stress, the contents of Chl a, Chl b and Chl t in Yanfu 3 were all decreased significantly compared with the control. However, little difference in Honeycrisp was noted between stressed plants and controls. Values for WUE in stressed Yanfu 3 remained higher than in the controls from day 3 until the end of the experiment, while no significant difference was observed in Honeycrisp. Furthermore, Honeycrisp also exhibited superior physiological traits, as indicated by its anatomical and morphological characteristics. Therefore, we conclude that the superior drought tolerance of Honeycrisp was due to its anatomical and morphological characteristics, which possibly contributed to the maintenance of higher photosynthetic capacity than Yanfu 3.

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.

Ethylene plays an important role in the grape rachis, where its production can be 10 times higher than in the berry. VvACS1 is the only rachis-specific ACC synthase (ACS) gene, and its expression is coincident with ethylene production in the rachis of Vitis vinifera 'Thompson seedless'. VvACS1 was cloned and ectopically expressed in tomato (Solanum lycopersicum 'Moneymaker'). Lateral buds were increased in two- or four-week-old 35s∷VvACS1 transgenic tomato plants after transplanting. Compared with wild-type (WT) plants, the transgenic tomato plants showed higher expression of the VvACS1 gene in the flowers, leaves, rachis, and fruits. There was no obvious difference of ACS activity in the fruit of tomato, and only increased ACS activity in the rachis of tomato. Ethylene production was decreased in flowers, leaves, and fruits (seven weeks after full bloom), while the relative expression of endogenous tomato ACS1 and ACS6 genes was not down-regulated by the ectopic expression of VvACS1. These results imply that post-transcriptional or post-translational regulation of ACS may occur, resulting in lower ethylene production in the transgenic tomato plants. Moreover, expression of VvACS1 in tomato resulted in decreased auxin and increased zeatin contents in the lateral buds, as well as reduced or delayed formation of adventitious roots in lateral bud cuttings. RNA-Seq and qRT-PCR analyses of rooted lateral bud cuttings indicated that the relative expression levels of the genes for zeatin O-glucosyltransferase-like, auxin repressed/dormancy-associated protein, and ERF transcription factors were higher in transgenic tomatoes than in WT, suggesting that ethylene may regulate auxin transport and distribution in shoots and that adventitious root formation employs coordination between auxin and ethylene.

Background Jujube witches' broom (JWB), caused by a phytoplasma, devastates jujube tree (Ziziphus jujuba) growth and production in Asia. Although host responses to phytoplasmas are studied at the phenotypic, physiological, biochemical and molecular levels, it remains unclear how a host plant responds at the molecular level during the primary stage of infection. Methods To understand the response of the jujube tree to JWB infection, leaves were sampled at different times during the phytoplasma infection. Transcriptomic analyses at six stages were performed to reveal how phytoplasma infection affects Chinese jujube gene expression through the determination of the key differentially expressed genes (DEGs), and their related pathways. Quantitative real-time PCR was applied to validate 10 differentially expressed genes at different JWB phytoplasma infection stages. Results A total of 25,067 unigenes were mapped to jujube genome reference sequences. In the first infection stage (0–2 weeks after grafting (WAG), a total of 582 jujube genes were differentially regulated but no visible symptoms appeared. Quite a few DEGs related to abscisic acid (ABA) and cytokinin (CTK) were down-regulated, while some related to jasmonic acid (JA) and salicylic acid (SA) were up-regulated, Genes related to plant-pathogen interaction were also differentially expressed. In the second infection stage (37–39WAG), witches' broom symptoms were visible, and a total of 4373 DEGs were identified. Genes involved in biosynthesis and signal transduction of ABA, brassinosteroid (BR), CTK, ethylene (ET), and auxin (IAA), GA, JA and SA, plant-pathogen interaction, flavonoid biosynthesis genes were significantly regulated, suggesting that jujube trees activated defense factors related to SA, JA, ABA and secondary metabolites to defend against phytoplasma infection. By the third infection stage (48–52WAG), serious symptoms occurred and 3386 DEGs were identified. Most DEGs involved in biosynthesis and signal transduction of JA, SA and GA were up-regulated, while those relating to ABA were down-regulated. Genes involved in plant-pathogen interaction were up- or down-regulated, while phenylpropanoid and flavonoid biosynthesis genes were significantly up-regulated. Meanwhile, DEGs involved in photosynthesis, chlorophyll and peroxisome biosynthesis, and carbohydrate metabolism were down-regulated. These results suggested that phytoplasma infection had completely destroyed jujube trees' defense system and had disturbed chlorophyll synthesis and photosynthetic activity in the infected leaves at the late stage, resulting in yellow leaves and other JWB symptoms. Discussion The results in this report suggested that phytohormone biosynthesis and signal transduction, photosynthesis, and secondary metabolism all played important roles in the battle between colonization and defense in the interaction between Ca. Phytoplasma ziziphi and jujube.

Fruit ripening is a complicated process that is influenced by diverse factors at different regulation levels. The microRNAs (miRNAs), a class of endogenous small noncoding RNAs, are recently demonstrated to regulate several critical biological processes in various aspects of the fruit's life cycle. In this paper, to gain more insights into the regulatory mechanism of miRNAs in apple fruit ripening, a genome-wide identification of the sRNAome was implemented in the postharvest ‘Qinguan’ apple fruit. The ripening process of the postharvest apple fruit was significantly inhibited via the application of 1-MCP (1 µL/L). Totally, 569 miRNAs were successfully identified, including 132 known and 437 novel miRNAs, and 29 of them were significantly differentially expressed. The mdm-miR398a, mdm-miR395i-3p, mdm-miR395b, mdm-miR395d-3p, mdm-miR395h, mdm-miR395g-3p, and the novel-miR156 were identified to be the differentially expressed miRNAs. Moreover, after parsing the targets of these miRNAs, several transcription factors, such as ERF and WRKY, were found to be involved in the apple fruit ripening. The above results provide new information for understanding the sophisticated coordinated regulatory network of apple fruit ripening.

Phytoplasmas are plant pathogenic bacteria that inhabit the phloem sieve elements in infected plants and lack a cell wall. Infection of the woody perennial Ziziphus jujuba with phytoplasma causes jujube witches’ broom (JWB), a disease that causes heavy crop losses. The TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) transcription factors (TFs) are plant-specific regulators of plant morphogenesis, development, and defense after phytoplasma infection. While the TCP gene family has been studied in many plants, there is no report on TCPs in jujube. In this study, 21 ZjTCP TFs were identified and bioinformatically analyzed based on the genome of the Dongzao cultivar. Another 2 ZjTCPs were found in the genome of the Junzao cultivar. Physicochemical properties of the ZjTCP proteins were quite varied, indicating possible versatility of functions. ZjTCP proteins ranged from 172 to 590 amino acids (aa), had isoelectric points (pIs) between 5.53 (ZjTCP1) and 9.81 (ZjTCP11), and molecular weights ranging from 19,279.98 (ZjTCP11) to 61,784.17 kDa (ZjTCP19). Interaction network mapping predicted several hubs, such as ZjTCP6, ZjTCP7, ZjTCP8, ZjTCP15, ZjTCP16, and ZjTCP19, among which ZjTCP6 and ZjTCP16 were predicted to function in plant defense and morphogenesis. Spatiotemporal expression analysis of the ZjTCPs showed that ten of the ZjTCP genes were detected after infection with ‘Candidatus Phytoplasma ziziphi’. ZjTCP6, ZjTCP7, ZjTCP16, and ZjTCP19 were up-regulated after phytoplasma infection. ZjTCP16 showed the most significant increase in transcript levels, after the emergence of disease symptoms. ZjTCP12, ZjTCP15, and ZjTCP18 were down-regulated after phytoplasma infection. We concluded that ZjTCP6 and ZjTCP16 were most likely regulatory factors with roles during the plant response to jujube witches’ broom.

Jujube witches’ broom (JWB) is caused by infection with a phytoplasma. A multi-omics approach was taken during graft infection of jujube by JWB-infected scion through the analysis of the plant transcriptome, proteome and phytohormone levels. A high number of differentially expressed genes (DEGs) were identified 37 weeks after grafting (WAG), followed by observation of typical symptoms of JWB at 48 WAG. At 37 WAG, the majority of the upregulated DEGs and differentially expressed proteins (DEPs) were related to flavonoid biosynthesis, phenylalanine metabolism and phenylpropanoid biosynthesis. Two of the four upregulated proteins were similar to jasmonate-induced protein-like. Among the downregulated genes, the two most populated GO terms were plant–pathogen interaction and plant hormone signal transduction (mainly for tryptophan metabolism). Moreover, phytoplasma infection resulted in reduced auxin content and increased jasmonate content, indicating that auxin and jasmonic acid have important roles in regulating jujube responses during the first and second stages of phytoplasma infection. At 48 WAG, the two largest groups of upregulated genes were involved in phenylpropanoid biosynthesis and flavonoid biosynthesis. Both genes and proteins involved in carbon metabolism and carbon fixation in photosynthetic organisms were downregulated, indicating that photosynthesis was affected by the third stage of phytoplasma infection. A multi-omics approach sheds light on the mechanisms of infection and defense at play between jujube trees and an enigmatic pathogen. Phytoplasmas are unculturable bacterial parasites discovered only a few decades ago, which cause deformed growths known as ‘witch's brooms’ in woody plants. A team at Henan Agricultural University, led by Jiancan Feng, explored the mechanisms of phytoplasma infection in the jujube tree (Ziziphus jujuba), by grafting healthy trees with infected branches. They employed a multi-pronged approach, analyzing the trees' gene activity, protein production, and hormone levels following infection. Their results suggest that the plant hormones auxin and jasmonic acid play important early roles in response to phytoplasma infection and that, as the witch's broom developed, processes such as photosynthesis were negatively impacted. These findings underpin future research towards preventing this devastating disease.