Circular RNAs (circRNAs) are covalently closed RNA molecules that regulate various biological processes in plants. However, the functions of most identified circRNAs remain unclear. Here, we report a nucleoplasmic-localized circRNA, Vv-circRCD1, derived from exons 2 and 3 of the grape VvRCD1 gene. Overexpression of Vv-circRCD1 significantly shortened primary root length and increased root hair number and length, notably, and improved the salt tolerance in Arabidopsis. Transient overexpression also significantly enhanced salt tolerance of grapevines. In silico analyses confirmed direct sequence complementarity between Vv-circRCD1 and the Vvi-miR399 family, and Vv-circRCD1 and Vvi-miR399 target genes (involved in salt stress responses) showed consistent expression patterns under salt stress, indicating a Vv-circRCD1–Vvi-miR399–target gene regulatory module may mediate salt tolerance. These results not only identified Vv-circRCD1 as a novel regulator of grapevine salt tolerance, but also highlighted its potential in improving crop stress resistance, providing a practical reference for crop breeding.

Polyploidization can generate morphological and physiological variation in plants, but the molecular basis underlying leaf trait changes after genome doubling in grapevine remains insufficiently understood. This study aimed to characterize phenotypic, physiological, transcriptomic, and metabolomic differences between diploid and induced autotetraploid plants of ‘Thompson Seedless’ and to identify biological processes potentially associated with the observed leaf trait variation. In this study, autotetraploid plants were induced from axillary buds of ‘Thompson Seedless’ using colchicine treatment, and ploidy levels were confirmed by flow cytometry and chromosome counting. Phenotypic, physiological, transcriptomic, and metabolomic analyses were performed to compare diploid and tetraploid plants. Compared with diploids, tetraploids exhibited enlarged leaves, reduced plant stature, larger but less dense stomata, increased chloroplast number in guard cells, and higher total chlorophyll and carotenoid contents. Fv/Fm remained unchanged, whereas increased Vj and decreased ψEo and φEo suggested differences in electron transport-related characteristics beyond QA. Transcriptomic analysis identified 1564 differentially expressed genes, and metabolomic profiling detected 618 differentially accumulated metabolites. Integrated analyses highlighted coordinated molecular differences associated mainly with cell-wall processes, secondary metabolism, redox-related functions, and carbon-related pathways. These findings identify candidate biological processes for future functional validation and provide a basis for evaluating the potential value of autotetraploid germplasm in grapevine breeding.

Terroir leaves its mark on the accumulation of flavours in grape berries, triggering biochemical reactions and ultimately shaping wine styles. However, the complicated relationship between terroir and grape volatiles remains not fully understood. We investigated volatile profiles of ripened berries in two major grape cultivars (i.e., ‘Cabernet Sauvignon’ and ‘Merlot’) during two consecutive vintages across the Ningxia region in combination with analyzation of the corresponding terroirs. The study revealed that maturity had a broad and significant impact on the accumulation of compounds in grape berries, determining the final aroma profile. In the Ningxia region, (E)-2-Octenal and nerol oxide in grapes can serve as stable marker volatile compounds that distinguish ‘Cabernet Sauvignon’ from ‘Merlot’. Specifically, (E)-2-Octenal is sensitive to ripeness but exhibits a weak response to environmental factors, while nerol oxide shows a significant differential response to terroir factors across cultivars and vintages. Beyond climate and soil, viticultural factors such as water status and trellising also significantly affect the nerol oxide content. Our results provide novel insights into the linkage between terroir factors and grape volatile profiles, and can help growers to better understand and control the production of fruits that maximize their aromatic typicity.

Terroir leaves its influence on the flavor build-up in grape berries by triggering biochemical reactions that ultimately shape the typicality of the produced wines. However, the complex relationship between terroir, carbon stable isotopic composition, and the biochemical composition of grapes remains poorly understood. To fill these gaps, grape berries were harvested at maturity from two grape cultivars, Cabernet Sauvignon and Merlot ( Vitis vinifera L.), across 32 sites within the Eastern Foothills of the Helan Mountains region over two vintages. The climate and soil data for these sites were collected, and cultivation practices were surveyed. In parallel, the grape quality indicators were measured at maturity to explore their variations among sub-regions and the correlations between terroir factors and grape quality. Moreover, the carbon stable isotopes (delta 13C) were measured to evaluate the water status of vines at different sites. Results indicated that organic acids and anthocyanins exhibited a strong vintage effect, with the cooler vintage exhibiting higher levels of these compounds relative to the warmer vintage. Specifically, the Hongsipu sub-region, with a cooler climate, consistently demonstrated higher anthocyanin concentrations than other sub-regions in both vintages of the study. Further analysis revealed that the cultivar Cabernet Sauvignon displayed a higher ratio of di-hydroxylated to tri-hydroxylated anthocyanins in the cooler year, whereas Merlot exhibited the opposite trend. The Mantel test identified several critical factors influencing anthocyanin concentration, including total soil phosphorus (Tp), total soil potassium (Tk), soil thickness (Thickness), precipitation (April-September precipitation, Pre4-9), April-September average temperature (4-9 Tave), April-September active accumulated temperature (4-9 Aa), April-June active accumulated temperature (4-6 Aa), July-September active accumulated temperature (7-9 Aa), temperature diurnal range (May, July, August, September, DR-5 and DR 7-9), trellis system, vine density, and delta 13 C. Finally, structural equation modeling (SEM) was used to analyze and summarize the relationships between terroir factors, anthocyanin concentration, and delta 13 C. The results indicated that June and September precipitation had a positive impact on anthocyanin concentrations, while soil total potassium content had a negative impact. On the other hand, the July to September temperature diurnal range, trellis system, vine perimeter, and canopy height were the main drivers of delta 13 C, which further influenced grape anthocyanin concentrations. This research contributes to a scientific foundation for grape cultivation in the Eastern Foothills of the Helan Mountains and provides valuable insights for practices in other regions.

Salt stress represents a major abiotic constraint impairing grapevine growth and productivity. While our previous work demonstrated that melatonin (MT) alleviates salt stress in grapes, the synergistic crosstalk between MT and diverse signaling pathways—particularly the hydrogen peroxide (H₂O₂)—remains poorly characterized. To address this, the present study combined MT with inhibitors of key signaling pathways to elucidate the mechanism underlying MT-mediated salt tolerance via H₂O₂ signaling, and further explored the role of endogenous H₂O₂ in this process through RBOHA overexpression. Results showed that MT significantly mitigated salt-induced impairments. Among inhibitor treatments, the auxin (IAA) transport inhibitor TIBA (Triiodobenzoic Acid) resulted in the lowest salt tolerance score, followed by the H₂O₂ inhibitor DPI (diphenyleneiodonium). Promoter analysis revealed that VvRBOH genes contain hormone and stress-responsive cis-elements. Overexpression of VvRBOHA enhanced salt tolerance in Arabidopsis thaliana and tomato, concomitant with increased endogenous hormone biosynthesis. In conclusion, MT alleviates salt stress in grapes partially through a VvRBOHA-dependent H₂O₂ signaling pathway that promotes hormone synthesis. Additionally, H₂O₂ may act upstream of MT to regulate salt tolerance-related ethylene (ET) and IAA pathways. This study provides valuable insights into dissecting plant adaptive responses to abiotic stress.

Grapes (Vitis spp.) are a globally significant fruit crop with a long history of cultivation and substantial cultivar diversity. Their high genetic differentiation and complex evolutionary history make them a valuable system for studying plant evolution. The chloroplast genome, known for its structural conservation and uniparental inheritance, offers a reliable molecular marker for phylogenetic reconstruction. In this study, we sequenced and assembled the complete chloroplast genomes of nine representative grape cultivars, analyzed their phylogenetic relationships, and compared structural variations. All chloroplast genomes displayed a typical quadripartite structure, with high conservation in genomic architecture, gene order and content, codon usage, and simple sequence repeats (SSRs). However, additional sequence comparisons revealed seven regions with high variation, including the genes rbcL and ndhF, and the intergenic regions rps16-trnQ, ndhC-trnV, accD-psaI, ndhF-rpl32, and trnL-ccsA. At the same time, seven natural variation sites were identified in the amino acid sequences of rbcL and ndhF. Additionally, the study’s maximum likelihood (ML) phylogenetic trees and photosynthetic index measurements suggest that developmental characteristics of grape photosynthesis may be related to the evolutionary origins of different populations. This phylogenetic classification not only elucidates the evolutionary origins of these germplasm resources but also provides a foundation for molecular-assisted breeding by identifying distinct genetic groups.

Grape (Vitis vinifera spp.) accessions exhibit rich diversity, and understanding their genetic variation and evolutionary relationships is crucial for cultivar selection and utilization. A highly representative SNP marker set was developed in this study based on re-sequencing data analysis, to clarify the phylogenetic relationships among 96 grape accessions and to evaluate the genetic resolution of core markers. Using PN40024 as the reference genome, high-quality SNP loci were screened from resequencing data of the 96 accessions. A phylogenetic tree was constructed, and genetic diversity was analyzed using PCA and population structure analysis. The results showed that the 96 accessions were mainly divided into four groups: European (‘Merlot’, ‘Chardonnay’), American (‘Beta’, ‘Concord’), Euro-American hybrids (‘Vidal’, ‘Miguang’), and wild populations along with their hybrid progeny (‘Zuoyouhong’, ‘Huajia 8’). PCA and ADMIXTURE validated population differentiation, revealing clear separation between wild and cultivated accessions. Through screening of core SNP markers, 384,304 candidate SNPs suitable for probe design were identified. Further refinement yielded 2000 and 10,000 SNP markers. Detailed analysis of core marker characteristics showed that their minor allele frequency (MAF) was predominantly between 0.1 and 0.3, with the majority distributed in CDS (38.65%), intronic (30.2%), and intergenic regions. The most common mutation types were [A/G] (35%) and [C/T] (34%) transitions. The 2000 core SNPs were associated with 1220 functional genes and were significantly enriched in pathways such as protein binding, RNA transport, and plant–pathogen interaction. These findings provide an efficient tool for grape genetic diversity analysis, cultivar identification, and molecular breeding, laying the groundwork for the precise utilization of grape germplasm resources.

With global warming, heat stress has been recognized as a significant factor limiting grapevine development and fruit quality. MicroRNAs (miRNAs) are a class of small non-coding RNAs known to play crucial regulatory roles in stress resistance. Hence, there is an immediate requirement to cultivate and identify grapevine varieties that are resistant to heat and explore miRNA-mediated heat stress defense mechanisms. In this study, we assessed the thermal resistance of 38 grape germplasm resources and identified a series of miRNAs involved in heat stress resistance. The CK (25°C) and HS (45°C) groups of “Shenyue” cuttings of grapes were used as experimental materials for next-generation sequencing and construct libraries of small RNAs. A total of 177 known and 20 novel miRNAs were detected in the libraries. Differential expression analysis identified 65 differentially expressed miRNAs (DEMs) using the DE-Seq procedure. Furthermore, RT-qPCR validation confirmed complementary expression profiles of eight DEMs and their target genes between the HS and CK groups. Heterologous transformation further identified the function of Vvi-miR3633a downregulated under heat stress in Arabidopsis. In the heterologous expression lines, the survival rate was reduced by high temperature treatment indicating the ability of Vvi-miR3633a to regulate heat resistance. Assessing the heat resistance of grape species and the expression patterns of miRNA in response to high temperatures may reveal the molecular processes of heat resistance regulation mediated by miRNA in grapes under heat stress.

High temperatures affect grape yield and quality. Grapes can develop thermotolerance under extreme temperature stress. However, little is known about the changes in transcription that occur because of high-temperature stress. The heat resistance indices and transcriptome data of five grape cultivars, ‘Xinyu’ (XY), ‘Miguang’ (MG), ‘Summer Black’ (XH), ‘Beihong’ (BH), and ‘Flame seedless’ (FL), were compared in this study to evaluate the similarities and differences between the regulatory genes and to understand the mechanisms of heat stress resistance differences. High temperatures caused varying degrees of damage in five grape cultivars, with substantial changes observed in gene expression patterns and enriched pathway responses between natural environmental conditions (35 °C ± 2 °C) and extreme high temperature stress (40 °C ± 2 °C). Genes belonging to the HSPs, HSFs, WRKYs, MYBs, and NACs transcription factor families, and those involved in auxin (IAA) signaling, abscisic acid (ABA) signaling, starch and sucrose pathways, and protein processing in the endoplasmic reticulum pathway, were found to be differentially regulated and may play important roles in the response of grape plants to high-temperature stress. In conclusion, the comparison of transcriptional changes among the five grape cultivars revealed a significant variability in the activation of key pathways that influence grape response to high temperatures. This enhances our understanding of the molecular mechanisms underlying grape response to high-temperature stress.

The grapevine, a globally significant fruit and an essential fruit tree species in China, is vulnerable to the adverse effects of high temperatures. Understanding the roles of microRNA and transcription factors in plant development and stress resistance is crucial for mitigating the impact of high temperature on grape growth and yield. This study investigates the response of miRNA to high-temperature stress in grape leaves. The expression level of Vvi-miR3633a was found to be inhibited under heat treatment in both Thompson seedless and Shen yue varieties, while its potential target genes (Vv-Atg36 and Vv-GA3ox2) were induced. Through transgenic overexpression experiments, it was demonstrated that Vvi-miR3633a plays a role in thermal response by affecting the expression of target genes. Furthermore, under heat stress conditions, overexpression of Vvi-miR3633a in grape callus decreased heat resistance compared to the control group (CK). The study also revealed that the target genes of Vvi-miR3633a regulate the expression of oxidase synthesis genes VvSOD and VvCAT, leading to reduced oxidase synthesis which may compromise the oxidation system. Additionally, the expression level of heat shock proteins in the transgenic lines was changed compared to the control (CK). Overall, this research provides valuable insights into understanding the molecular mechanisms involved in different crossing/breeding programs to produce heat-resistant grape varieties. Such varieties can be appropriate to propagate in warm climate areas with high temperature conditions.

In grape (Vitis vinifera L.) cultivation, high temperatures (HTs) usually reduce the accumulation of anthocyanins. In order to elucidate the regulatory mechanism of anthocyanin biosynthesis under high-temperature environments, we investigated the effects of HT stress at veraison (5% coloring of grape ears) on fruit coloration and anthocyanin biosynthesis in ‘Summer Black’ (XH) and ‘Flame seedless’ (FL) grapevines. Compared to the control group (35 °C), the total anthocyanin content of XH and FL grapes subjected to a high-temperature (HT) treatment group (40 °C) decreased significantly as the HT treatment continued, but showed an upward trend with fruit development. However, the concentration of procyanidins increased significantly following HT treatment but decreased with fruit development. Nonetheless, FL grapes showed some resistance to the HT condition, producing anthocyanin content at ripeness comparable to the control group, demonstrating a greater adaptability to HT conditions than XH grapes. Based on the CIRG index, at stage S4, the fruit of FL was classified as dark red, while XH was classified as blue-black in the control group. Anthocyanin-targeted metabonomics identified eight different types of anthocyanins accumulating in the peels of XH and FL grapes during ripening, including cyanidins, delphinidins, malvidins, pelargonidins, peonidins, petunidins, procyanidins, and flavonoids. Malvidins were the most abundant in the two grape varieties, with malvidin-3-O-glucoside being more sensitive to high temperatures. HT treatment also down-regulated the expression of structural genes and regulators involved in the anthocyanin synthesis pathways. We used the WGCNA method to identify two modules that were significantly correlated with total anthocyanin and procyanidin contents. Among them, MYBCS1, bHLH137, WRKY65, WRKY75, MYB113-like, bZIP44, and GST3 were predicted to be involved in grape anthocyanin biosynthesis. In conclusion, this study conducted in-depth research on the HT inhibition of the biosynthesis of anthocyanins in XH and FL grapes, for reference.

Circular RNAs (circRNAs) exist widely in animals and plants. Most researches focused on the identification and annotation of circRNA in plants, while the biological functions of circRNA are still limited. Based on our previous whole transcriptomic analysis of grapevines, in this study, 15 circRNAs were further selected to validate in grapevine through investigating their origin and back-splicing sites. The results showed that only four circRNAs (Vv-circRNA_0490, Vv-circRNA_1292, Vv-circRNA_2094 and Vv-circRNA_2451) followed the AG/GT canonical splicing signal. Moreover, we found that most of circRNAs were specifically expressed in different tissues and were preferential accumulation in cytoplasm and nucleus by nuclear-cytoplasmic fractionation assay. Furthermore, the expression cassettes of circRNAs were constructed and found that all circRNAs were precisely back-spliced in Nicotiana benthamiana, indicating that the conserved splicing mechanism of circRNA. In addition, Vv-circPAS1 a circRNA processed from host gene VvPAS1 which is a member of the FK506-binding protein (FKBP) gene family, were focused because of association with root development. The promoter analysis of Vv-circPAS1 showed that multiple cis-acting elements related to defense, light, phytohormone and plant development were found. Finally, Vv-circPAS1 was introduced into Arabidopsis and found that Vv-circPAS1 significantly inhibited the elongation of primary roots and increased the number and length of roots hair. Meanwhile, overexpression of Vv-circPAS1 improved salt tolerance in grapevine callus, while the linear fragments didn't. These results broaden our understanding of circRNAs in plants, and provide new insight into plant circRNAs.

The chloroplast genome plays an important role in exploring the origin and evolution of grapes. In this study, the chloroplast genomes of eight grape germplasms were analyzed. The results show that the sequence arrangement was highly conserved and has a typical annular quadripartite structure. At the same time, GC content, gene sequence, repeat type and codon bias pattern are highly conserved. Whereas, 19 mutation hotspot regions were identified by the comparative analysis, including 9 protein-coding genes (atpF, rpoC2, rps18, psbC, atpB, rbcL, rps20, ycf1 and ycf15) and 10 non-coding regions (trnK – rps16, rps16 – trnQ, trnE – trnT, psbZ - trnG, ndhC – trnV, accD – psaI, ycf2 – trnL, ndhF – rpl32, ccsA - ndhD, trnL - ycf2). Most of these highly variable genes were involved in the physiological process of photosynthesis. Further examination of photosynthetic fluorescence showed an obvious difference among those species under high-temperature conditions. In addition, maximum likelihood (ML) evolutionary trees depend on the chloroplast genome and CDS sequences suggest that heat tolerance may be related to the phylogeny of grapevine. In the future, the highly variable sequences and SSR loci were also identified by the chloroplast genome comparison, and those results may be used for the DNA molecular marker in taxonomic studies and contribute to the heat stress resistance breeding of grapevine.

MiR399 plays an important role in plant growth and development. The objective of the present study was to elucidate the evolutionary characteristics of the MIR399 gene family in grapevine and investigate its role in stress response. To comprehensively investigate the functions of miR399 in grapevine, nine members of the Vvi-MIR399 family were identified based on the genome, using a miRBase database search, located on four chromosomes (Chr 2, Chr 10, Chr 15, and Chr 16). The lengths of the Vvi-miR399 precursor sequences ranged from 82 to 122 nt and they formed stable stem–loop structures, indicating that they could produce microRNAs (miRNAs). Furthermore, our results suggested that the 2 to 20 nt region of miR399 mature sequences were relatively conserved among family members. Phylogenetic analysis revealed that the Vvi-MIR399 members of dicots (Arabidopsis, tomato, and sweet orange) and monocots (rice and grapevine) could be divided into three clades, and most of the Vvi-MIR399s were closely related to sweet orange in dicots. Promoter analysis of Vvi-MIR399s showed that the majority of the predicted cis-elements were related to stress response. A total of 66.7% (6/9) of the Vvi-MIR399 promoters harbored drought, GA, and SA response elements, and 44.4% (4/9) of the Vvi-MIRR399 promoters also presented elements involved in ABA and MeJA response. The expression trend of Vvi-MIR399s was consistent in different tissues, with the lowest expression level in mature and young fruits and the highest expression level in stems and young leaves. However, nine Vvi-MIR399s and four target genes showed different expression patterns when exposed to low light, high light, heat, cold, drought, and salt stress. Interestingly, a putative target of Vvi-MIR399 targeted multiple genes; for example, seven Vvi-MIR399s simultaneously targeted VIT_213s0067g03280.1. Furthermore, overexpression of Vvi_MIR399e and Vvi_MIR399f in Arabidopsis enhanced tolerance to drought compared with wild-type (WT). In contrast, the survival rate of Vvi_MIR399d-overexpressed plants were zero after drought stress. In conclusion, Vvi-MIR399e and Vvi-MIR399f, which are related to drought tolerance in grapevine, provide candidate genes for future drought resistance breeding.

The adverse effects of high temperatures on fruit quality and yield have been reported in many studies of grapevines. MiRNAs play crucial roles in plant growth and development and also fulfill functions in regulating the high-temperature response. In this research, miRNA-based SSR (simple sequence repeat) polymorphisms were analyzed according to the grape genome sequence and then combined with polymerase chain reaction (PCR) amplification and Sanger sequencing methods to analyze the miRNA-SSR diversity of different heat-resistant grape varieties. A statistical analysis of SSR sequences showed that 391 and 12 SSRs were specific to pri-miRNA and pre-miRNA, respectively. In pri-miRNA containing SSR, hexa-nucleotide repeats were the most abundant (52.69%), followed by tetra-nucleotide (13.04%) and minimum penta-nucleotide (4.09%), which were also observed in pre-miRNA sequences. On the other hand, differences in heat resistance among grape varieties were observed. Based on the results of leaf Fv/Fm images, values and phenotypic changes under high-temperature stress, 20 heat-resistant (e.g., Niagara Rosada and Grand Noir) and 20 heat-sensitive (e.g., Shine Muscat and Jumeigui) grape varieties were identified. Further, PCR-Sanger sequencing was used to screen SSR differences in four thermos-tolerant and four thermos-sensitive grape varieties, and finally, eight SSR differential primers were found to be able to distinguish these varieties. Of these, four pairs of primers were selected for validation in 40 grape germplasm resources (20 thermo-tolerance and 20 thermo-sensitivity). The VMIRSSR167c3 highlights that the ratio of SSR (CT) repeat values greater than 17 in heat-resistant varieties was 90%. In conclusion, the VMIRSSR167c3 marker can accurately distinguish between heat-resistant and heat-sensitive grape varieties. The results provide a novel molecular marker for the genetic improvement of grape germplasm resources and will be beneficial to the breeding of heat-resistant varieties in the future.

In this study, we analyzed the effects of treatments with titanium dioxide nanoparticles (NPs-TiO2) and ethylene on anthocyanin biosynthesis and reactive oxygen species (ROS) metabolism during light exposure in ripe 'red delicious' apples. Both treatments led to improved anthocyanins biosynthesis in detached mature apples, while the NPs-TiO2 had less impact on the fruit firmness, TSS, TA, and TSS/TA ratio. Furthermore, the effects of both treatments on the expression of anthocyanin-related enzymes and transcription factors in the apple peel were evaluated at the gene level. The differentially expressed genes induced by the two treatments were highly enriched in the photosynthesis and flavonoid biosynthesis pathways. The expression of structural genes involved in anthocyanin biosynthesis and ethylene biosynthesis was more significantly upregulated in the ethylene treatment group than in the NPs-TiO2 treatment group, and the opposite pattern was observed for the expression of genes encoding transcription factors involved in plant photomorphogenesis pathways. In addition, the ROS levels and antioxidant capacity were higher and the membrane lipid peroxidation level was lower in fruit in the NPs-TiO2 treatment group than in the ethylene treatment group. The results of this study reveal differences in the coloration mechanisms induced by NPs-TiO2 and ethylene in apples, providing new insights into improving the color and quality of fruits.

It is very important to promote plant growth and decrease the nitrogen leaching in soil, to improve nitrogen (N) utilization efficiency. In this experiment, we designed a new fertilization strategy, fruit tree hole storage brick (FTHSB) application under subsurface drip irrigation, to characterise the effects of FTHSB addition on N absorption and utilization in grapes. Three treatments were set in this study, including subsurface drip irrigation (CK) control, fruit tree hole storage brick A (T1) treatment, and fruit tree hole storage brick B (T2) treatment. Results showed that the pore number and size of FTHSB A were significantly higher than FTHSB B. Compared with CK, T1 and T2 treatments significantly increased the biomass of different organs of grape, N utilization and 15 N content in the roots, stems and leaves, along with more prominent promotion at T1 treatment. When the soil depth was 15–30 cm, the FTHSB application significantly increased the soil 15 N content. But when the soil depth was 30–45 cm, it reduced the soil 15 N content greatly. T1 and T2 treatments obviously increased the activities of nitrite reductase (NR) and glutamine synthetase (GS) in grape leaves, also the urease activity(UR) in 30 cm of soil. Our findings suggest that FTHSB promoted plant N utilization by reducing N loss in soil and increasing the enzyme activity related to nitrogen metabolism. In addition, this study showed that FTHSB A application was more effective than FTHSB B in improving nitrogen utilization in grapes.

Nitrogen loss after urea application and the low nitrogen utilization rate of plants are major issues in fertilizer application. We therefore adopted a combination of urea and 3,4-dimethylpyrazole phosphate (DMPP) applications in order to investigate the response of DMPP in ‘sweet sapphire’ (V. vinifera L.) grape seedlings growth. Three combinations of DMPP and urea were tested to screen suitable DMPP application concentrations for grape seedlings’ growth. Transcriptome differential expression analysis was adopted to elucidate the regulation mechanism of DMPP. The results showed that the application of DMPP with urea significantly increased grape seedlings’ root dry weight, as well as the above-ground dry weight. The application of DMPP with urea significantly improved the total root length, surface area, volume, and root vigor. The application of urea nitrogen content with 1% of DMPP (T2) showed optimum effects. The application of DMPP can also significantly increase the net photosynthetic rate, photosynthetic pigments, and fluorescence intensity of grape leaves. Furthermore, the transcriptome differential expression analysis under T2 treatment revealed that members of the Nar (7) and NRT (12) gene families were up-regulated, which promotes nitrogen uptake and metabolism. Moreover, the LHC (11), Psa (7), Pet (4), and Psb (5) genes were up-regulated, which promotes photosynthesis.

High temperature stress is one of the primary abiotic stresses that restrict fruit tree production. Grapevine (Vitis vinifera) with high economic value throughout the world is a cultivated fruit crop, and its growth and development is often influenced by high temperature stress. Studying the heat stress-response mechanism of grapevine has great significance for understanding the acclimation to heat stress. In this study, we identified a series of heat stress responsive miRNAs and analyzed their function during the heat tolerance response. CK (control group, 25 °C) and heat treatment stress (TS, 45 °C) small RNA (sRNA) libraries were constructed and sequenced by high-throughput sequencing in ‘Thompson seedless’ grapevine. 873 known-miRNAs and 86 novel-miRNAs were identified, of which 88 known and three novel miRNAs were expressed differentially under heat stress. 322 genes were predicted to be targeted by the miRNAs. Eight selected miRNAs and its targets were confirmed by real time quantitative PCR (RT - qPCR), indicating that these “miRNA - target” were responsive to heat stress. In addition, most of the predicted target genes were negatively regulated by corresponding miRNAs. Gene function and pathway analyses indicated that these genes probably play crucial roles in heat stress tolerance. Vvi-miR167b transiently overexpression in grapevine leaves decreased target gene vvARF6, vvARF6-like and vvARF8 expression. The function of vvi-miR167 was verified by ectopic transformation in Arabidopsis thaliana, and the heat tolerance in transgenic lines was enhanced significantly, suggesting that the vvi-miR167 plays a positive regulatory role in grape thermostability. Comparison of miRNA expression patterns between heat treatment stress and CK can help elucidate the heat stress response and resistance mechanisms in grapes. In conclusion, these results gave us useful information to better understand the heat stress-response during domestication as well as for breeding new cultivars with heat stress resistance in fruit trees.

Root restriction is a physical and ecological cultivation mode which restricts plant roots into a limited container to regulate vegetative and reproduction growth by reshaping root architecture. However, little is known about related molecular mechanisms. To uncover the root-related regulatory network of endogenous RNAs under root restriction cultivation (referred to RR), transcriptome-wide analyses of mRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) involved in root development were performed. During root development, RR treatment had a positive effect on root weight, typically, young roots were significantly higher than conventional cultivation (referred to NR) treatment, suggesting that root architecture reconstruction under RR was attributed to the vigorous induction into lateral roots. Furthermore, a total of 26,588 mRNAs, 1971 lncRNAs, and 2615 circRNAs were identified in root of annual “Muscat Hamburg” grapevine by the transcriptomic analyses. The expression profile of mRNAs, lncRNAs and circRNA were further confirmed by the quantitative real-time PCR (RT-qPCR). Gene ontology enrichment analysis showed that a majority of the differentially expressed mRNAs, lncRNAs and circRNAs were enriched into the categories of cellular process, metabolic process, cell part, binding, and catalytic activity. In addition, the regulatory network of endogenous RNAs was then constructed by the prediction of lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA network, implying that these RNAs play significant regulatory roles for root architecture shaping in response to root restriction. Our results, for the first time, the regulatory network of competitive endogenous RNAs (ceRNAs) functions of lncRNA and circRNA was integrated, and a basis for studying the potential functions of non-coding RNAs (ncRNAs) during root development of grapevine was provided.