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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.

Plant growth caused by ambient temperature is thought to be regulated by a complex transcriptional network. A temperature-sensitive peach (Prunus persica) was used to explore the mechanisms behind shoot internode elongation at elevated temperatures. There was a significantly positive correlation between the length of the terminal internode (TIL) and the maximum temperature three days prior to the measuring day. Four critical growth stages (initial period and initial elongation period at lower temperature, rapid growth period and stable growth period at higher temperature) were selected for comparative RNA-seq analysis. About 6.64G clean bases were obtained for each library, and 88.27% of the data were mapped to the reference genome. Differentially expressed gene (DEG) analysis among the three pairwise comparisons resulted in the detection of several genes related to the shoot elongation in temperature-sensitive peach. HSFAs were up-regulated in response to the elevated temperature, while the up-regulated expression of HSPs might influence hormone signaling pathways. Most of DEGs involved in auxin, abscisic acid and jasmonic acid were up-regulated, while some involved in cytokinin and brassinosteroid were down-regulated. Genes related to ethylene, salicylic acid and circadian rhythm were also differentially expressed. Genes related to aquaporins, expansins, pectinesterases and endoglucanase were up-regulated, which would promote cell elongation. These results lay a foundation for further dissection of the regulatory mechanisms underlying shoot elongation at elevated temperatures.

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.

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.

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.

Ethylene is important during the berry development and in the last stages of rachis development or rachis senescence. Since grapes develop in a cluster that comprises both the fruit berry and the nonfruit rachis, we measured the release of ethylene from both tissues. Detached berries from Vitis vinifera ‘Ruby Seedless’ and ‘Thompson Seedless’ showed that ethylene release peaks at the beginning of berry development and at veraison. Ethylene production in the rachis was higher than that in the berry and had an obvious peak before harvest in ‘Thompson Seedless’. In both cultivars, ethephon treatment induced ethylene production in the rachis but not in the berry. Expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO) genes showed diverse temporal and spatial patterns in ‘Thompson Seedless’ and ‘Ruby Seedless’. For most gene family members, the low ACS expression levels were observed in berry and rachis. Expression levels of most of the ACS and ACO genes did not correlate with ethylene released in the same organ. The transcriptional level of VvACS1 did correlate with ethylene evolution in rachis of ‘Thompson Seedless’ during berry development and storage, which suggested that VvACS1 may have important roles in rachis senescence. In berries of ‘Thompson Seedless’ and ‘Ruby Seedless’, the transcriptional levels of VvACO1, VvACS2, and VvACS6 coincided with ethylene production, indicating possible roles in berry development. Expression of VvACS2–VvACO9 and VvACO1–VvACO3 was not consistent with ethylene production during storage or in response to ethephon treatment, which suggests that the expression of ACS and ACO was affected by other stress factors after harvest.

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.

Resveratrol contributes to a plant’s tolerance of various abiotic and biotic stresses and is highly beneficial to human health. A search for elite alleles affecting resveratrol production was undertaken to find useful grapevine germplasm resources. Resveratrol levels in both berry skins and leaves were determined in 95 grapevine accessions (including 50 wild Chinese grapevine accessions and 45 cultivars) during two consecutive years. Resveratrol contents were higher in berry skins than in leaves and in wild Chinese grapevines than in grapevine cultivars. Using genotyping data, 79 simple sequence repeat (SSR) markers linked to 44 stilbene synthase (STS) genes were detected in the 95 accessions, identifying 40 SSR markers with higher polymorphisms. Eight SSR marker loci, encompassing 19 alleles, were significantly associated with resveratrol content on (P

The composition and content of anthocyanins were surveyed by HPLC–MS for assessing genotypic variation in berry skin of 110 grape cultivars, including 3 species and 5 interspecific hybrids. Twenty-nine anthocyanins were identified. For total anthocyanin content, Vitis vinifera and hybrids of Vitis labrusca and V. vinifera were low, and in general, wild species and rootstock were higher than interspecific hybrids, and wine grapes were higher than table grapes in the same species. As regards the composition of anthocyanins, malvidin-derivatives were the most abundant anthocyanins in the majority of germplasms. All anthocyanins were monoglucoside derivatives in V. vinifera , but all the other Vitis germplasms had both mono- and di-glucoside derivatives. Moreover, peonidin-derivatives and malvidin 3- O -glucoside were, respectively, main anthocyanins in table and wine grapes of V. vinifera . Via principal component analysis, the distribution of the cultivars in a scatter plot depended upon their total anthocyanins content, mono- and di-glucoside derivatives .

Effects of salicylic acid (SA), ultraviolet radiation (UV-B and UV-C) on the trans-resveratrol (Res) inducement of the skin of harvested grape berries were studied with three grape cultivars Takasuma, Tano Red and Carigane. Split plot design tests were adopted to compare the effects of UV-B and UV-C radiation on Res inducement of different cultivars. Results showed that spraying 100 mg·L−1 SA markedly enhanced Res contents in the skins of harvested berries for the three selected cultivars. However, the effect of SA varied with the cultivars, and Res inducement by SA was more effective to Tano Red than Takasuma and Carigane. UV-B or UV-C irradiation significantly increased Res contents in grape skins and UV-C was more effective than UV-B. The effects of UV types and dosages on Res inducement depended upon cultivars. In the range of 0–3.6 kJ·m−2, the Res contents in the skins of the three grape cultivars were enhanced along with the increase of dosages of UV-B and UV-C.

This study was conducted to develop a protocol for rapid callus induction and subsequent shoot regeneration in Zizyphus jujuba Mill. ‘Huizao’. The best callus induction (95%) was obtained from young expanding leaves between the third and fifth nodes in vitro jujube shoots in MS media supplemented with 0.5 mg/L benzylamino purine (6-BA), 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2, 4-D), and 0.5 mg/L silver nitrate (AgNO3). Incubation in darkness had non-significant effects on callus induction frequency of leaf explants, while callus formation of leaf was observed five to seven days earlier in one or two weeks of dark incubation. For shoot regeneration, the results showed that combination of zeatin (ZT) and, indole-3-acetic acid (IAA) was found to be more effective than that of 6-BA and indole-3-butyric acid (IBA). A maximum of 37.34% callus differentiation occurred in MS medium supplemented with2.0 mg/L ZT and 0.1 mg/L IAA. Key words: Callus induction, shoot regeneration, Zizyphus jujuba.