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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 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.

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.

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.

Recently, there is an increased interest in the function of polyamine (PA) catabolism during fruit ripening, but little is known about its role during the ripening of peach (Prunus persica L.). In this study, the contents of both free and conjugated PAs markedly decreased as the peach fruit ripened. However, RNA-seq analysis showed that the transcript levels of PA synthesis–related genes increased, suggesting that the decreasing PA content during peach ripening was associated with PA catabolism. To better understanding the function of PA catabolism in peach ripening, genes encoding potential polyamine oxidases (PAOs), which are involved in PA catabolism, were identified across the peach genome. Four putative PAO genes (PpePAO1-PpePAO4) were identified in peach using the recently released genome database. The mRNA level of PpePAO1 was significantly increased during peach fruit ripening. Detection of free PAs in tobacco leaves transiently over-expressing PpePAO1 suggested that this gene is probably involved in terminal catabolism of PA. Treatment with the PAO inhibitor guazatine significantly reduced ethylene production and flesh softening of peach fruit, decreased the expression levels of fruit ripening-related genes, and significantly decreased the expression level of PpePAO1 and PAO activity, while PA contents were dramatically higher compared to control treatments. Our results suggests that PpePAO1-mediated PA catabolism is associated with decreased PA content during peach fruit ripening. These data provide valuable knowledge for better understanding the roles of PA catabolism in peach development and ripening.

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.

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.

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.

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.