Molecular evolution of Phytocyanin gene and analysis of expression at different coloring periods in apple (Malus domestica).

BACKGROUND: PC (phytocyanin) is a class of copper-containing electron transfer proteins closely related to plant photosynthesis, abiotic stress responses growth and development in plants, and regulation of the expression of some flavonoids and phenylpropanoids, etc., however, compared with other plants, the PC gene family has not been systematically characterized in apple. RESULTS: A total of 59 MdPC gene members unevenly distributed across 12 chromosomes were identified at the genome-wide level. The proteins of the MdPC family were classified into four subfamilies based on differences in copper binding sites and glycosylation sites: Apple Early nodulin-like proteins (MdENODLs), Apple Uclacyanin-like proteins (MdUCLs), Apple Stellacyanin-like proteins (MdSCLs), and Apple Plantacyanin-like proteins (MdPLCLs). Some MdPC members with similar gene structures and conserved motifs belong to the same group or subfamily. The internal collinearity analysis revealed 14 collinearity gene pairs among members of the apple MdPC gene. Interspecific collinearity analysis showed that apple had 31 and 35 homologous gene pairs with strawberry and grape, respectively. Selection pressure analysis indicated that the MdPC gene was under purifying selection. Prediction of protein interactions showed that MdPC family members interacted strongly with the Nad3 protein. GO annotation results indicated that the MdPC gene also regulated the biosynthesis of phenylpropanoids. Chip data analysis showed that (MdSCL3, MdSCL7 and MdENODL27) were highly expressed in mature fruits and peels. Many cis-regulatory elements related to light response, phytohormones, abiotic stresses and flavonoid biosynthetic genes regulation were identified 2000 bp upstream of the promoter of the MdPC gene, and qRT-PCR results showed that gene members in Group IV (MdSCL1/3, MdENODL27) were up-regulated at all five stages of apple coloring, but the highest expression was observed at the DAF13 (day after fruit bag removal) stage. The gene members in Group II (MdUCL9, MdPLCL3) showed down-regulated or lower expression in the first four stages of apple coloring but up-regulated and highest expression in the DAF 21 stage. CONCLUSION: Herein, one objective of these findings is to provide valuable information for understanding the structure, molecular evolution, and expression pattern of the MdPC gene, another major objective in this study was designed to lay the groundwork for further research on the molecular mechanism of PC gene regulation of apple fruit coloration.

The asymmetric expression of plasma membrane H+-ATPase family genes in response to pulvinus-driven leaf phototropism movement in Vitis vinifera.

Phototropism movement is crucial for plants to adapt to various environmental changes. Plant P-type H+-ATPase (HA) plays diverse roles in signal transduction during cell expansion, regulation of cellular osmotic potential and stomatal opening, and circadian movement. Despite numerous studies on the genome-wide analysis of Vitis vinifera, no research has been done on the P-type H+-ATPase family genes, especially concerning pulvinus-driven leaf movement. In this study, 55 VvHAs were identified and classified into nine distinct subgroups (1 to 9). Gene members within the same subgroups exhibit similar features in motif, intron/exon, and protein tertiary structures. Furthermore, four pairs of genes were derived by segmental duplication in grapes. Cis-acting element analysis identified numerous light/circadian-related elements in the promoters of VvHAs. qRT-PCR analysis showed that several genes of subgroup 7 were highly expressed in leaves and pulvinus during leaf movement, especially VvHA14, VvHA15, VvHA16, VvHA19, VvHA51, VvHA52, and VvHA54. Additionally, we also found that the VvHAs genes were asymmetrically expressed on both sides of the extensor and flexor cell of the motor organ, the pulvinus. The expression of VvHAs family genes in extensor cells was significantly higher than that in flexor cells. Overall, this study serves as a foundation for further investigations into the functions of VvHAs and contributes to the complex mechanisms underlying grapevine pulvinus growth and development.

Overexpression of VaBAM3 from Vitis amurensis enhances seedling cold tolerance by promoting soluble sugar accumulation and reactive oxygen scavenging.

KEY MESSAGE: The VaBAM3 cloned from Vitis amurensis can enhance the cold tolerance of overexpressed plants, but VaBAM3 knock out by CRISPR/Cas9 system weakened grape callus cold tolerance. In grape production, extreme cold conditions can seriously threaten plant survival and fruit quality. Regulation of starch content by ß-amylase (BAM, EC: 3.2.1.2) contributes to cold tolerance in plants. In this study, we cloned the VaBAM3 gene from an extremely cold-tolerant grape, Vitis amurensis, and overexpressed it in tomato and Arabidopsis plants, as well as in grape callus for functional characterization. After exposure to cold stress, leaf wilting in the VaBAM3-overexpressing tomato plants was slightly less pronounced than that in wild-type tomato plants, and these plants were characterized by a significant accumulation of autophagosomes. Additionally, the VaBAM3-overexpressing Arabidopsis plants had a higher freezing tolerance than the wild-type counterparts. Under cold stress conditions, the activities of total amylase, BAM, peroxidase, superoxide dismutase, and catalase in VaBAM3-overexpressing plants were significantly higher than those in the corresponding wild-type plants. Furthermore, sucrose, glucose, and fructose contents in these lines were similarly significantly higher, whereas starch contents were reduced in comparison to the levels in the wild-type plants. Furthermore, we detected high CBF and COR gene expression levels in cold-stressed VaBAM3-overexpressing plants. Compared with those in VaBAM3-overexpressing grape callus, the aforementioned indicators tended to change in the opposite direction in grape callus with silenced VaBAM3. Collectively, our findings indicate that heterologous overexpression of VaBAM3 enhanced cold tolerance of plants by promoting the accumulation of soluble sugars and scavenging of excessive reactive oxygen species. These findings provide a theoretical basis for the cultivation of cold-resistant grape and support creation of germplasm resources for this purpose.

Grape SnRK2.7 Positively Regulates Drought Tolerance in Transgenic Arabidopsis.

In this study, we obtained and cloned VvSnRK2.7 by screening transcriptomic data to investigate the function of the grape sucrose non-fermenting kinase 2 (SnRK2) gene under stress conditions. A yeast two-hybrid (Y2H) assay was used to further screen for interaction proteins of VvSnRK2.7. Ultimately, VvSnRK2.7 was heterologously expressed in Arabidopsis thaliana, and the relative conductivity, MDA content, antioxidant enzyme activity, and sugar content of the transgenic plants were determined under drought treatment. In addition, the expression levels of VvSnRK2.7 in Arabidopsis were analyzed. The results showed that the VvSnRK2.7-EGFP fusion protein was mainly located in the cell membrane and nucleus of tobacco leaves. In addition, the VvSnRK2.7 protein had an interactive relationship with the VvbZIP protein during the Y2H assay. The expression levels of VvSnRK2.7 and the antioxidant enzyme activities and sugar contents of the transgenic lines were higher than those of the wild type under drought treatment. Moreover, the relative conductivity and MDA content were lower than those of the wild type. The results indicate that VvSnRK2.7 may activate the enzyme activity of the antioxidant enzyme system, maintain normal cellular physiological metabolism, stabilize the berry sugar metabolism pathway under drought stress, and promote sugar accumulation to improve plant resistance.

Mechanism of the Pulvinus-Driven Leaf Movement: An Overview.

Leaf movement is a manifestation of plant response to the changing internal and external environment, aiming to optimize plant growth and development. Leaf movement is usually driven by a specialized motor organ, the pulvinus, and this movement is associated with different changes in volume and expansion on the two sides of the pulvinus. Blue light, auxin, GA, H+-ATPase, K+, Cl-, Ca2+, actin, and aquaporin collectively influence the changes in water flux in the tissue of the extensor and flexor of the pulvinus to establish a turgor pressure difference, thereby controlling leaf movement. However, how these factors regulate the multicellular motility of the pulvinus tissues in a species remains obscure. In addition, model plants such as Medicago truncatula, Mimosa pudica, and Samanea saman have been used to study pulvinus-driven leaf movement, showing a similarity in their pulvinus movement mechanisms. In this review, we summarize past research findings from the three model plants, and using Medicago truncatula as an example, suggest that genes regulating pulvinus movement are also involved in regulating plant growth and development. We also propose a model in which the variation of ion flux and water flux are critical steps to pulvinus movement and highlight questions for future research.

Shading Treatment Reduces Grape Sugar Content by Suppressing Photosynthesis-Antenna Protein Pathway Gene Expression in Grape Berries.

To explore the impact of shade treatment on grape berries, 'Marselan' grape berries were bagged under different light transmission rates (100% (CK), 75% (A), 50% (B), 25% (C), 0% (D)). It was observed that this treatment delayed the ripening of the grape berries. The individual weight of the grape berries, as well as the content of fructose, glucose, soluble sugars, and organic acids in the berries, was measured at 90, 100, and 125 days after flowering (DAF90, DAF100, DAF125). The results revealed that shading treatment reduced the sugar content in grape berries; the levels of fructose and glucose were higher in the CK treatment compared to the other treatments, and they increased with the duration of the shading treatment. Conversely, the sucrose content exhibited the opposite trend. Additionally, as the weight of the grape berries increased, the content of soluble solids and soluble sugars in the berries also increased, while the titratable acidity decreased. Furthermore, 16 differentially expressed genes (DEGs) were identified in the photosynthesis-antenna protein pathway from the transcriptome sequencing data. Correlation analysis revealed that the expression levels of genes VIT_08s0007g02190 (Lhcb4) and VIT_15s0024g00040 (Lhca3) were positively correlated with sugar content in the berries at DAF100, but negatively correlated at DAF125. qRT-PCR results confirmed the correlation analysis. This indicates that shading grape clusters inhibits the expression of genes in the photosynthesis-antenna protein pathway in the grape berries, leading to a decrease in sugar content. This finding contributes to a deeper understanding of the impact mechanisms of grape cluster shading on berry quality, providing important scientific grounds for improving grape berry quality.

Physiological, biochemical and molecular responses associated with drought tolerance in grafted grapevine.

BACKGROUND: Grafting is one of the promising techniques for improving abiotic stress tolerance in horticultural crops, but the underlying regulatory mechanisms of drought on grafted grapevine are largely unexplored. RESULTS: Herein, we investigated the phenotypic, physiologic, biochemical, and drought related genes change of self-rooted 1103P (1103 Paulsen), SM (Shine Muscat) and grafted SM/1103P (SM shoot/1103P root) under drought stress condition. The results indicated that grafted grapevine effectively alleviated drought damage in grape leaves by higher RWC, water potential and free water content. Drought stress led to the alterations of chlorophyll, carotenoid, photosynthetic parameters and chlorophyll fluorescence in grapevine leaves after drought treatment indicated grafted plants improved the photosystem response to drought stress. Moreover, grafted plants under drought stress exhibited higher levels of abscisic acid (ABA), indoleacetic acid (IAA) and soluble protein, but less contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) both in leaves and roots. Drought stress also increased the activities of antioxidant enzymes (SOD, POD and CAT) and activated the transcript expression of VvCu/ZnSOD, VvPOD4 and VvCAT1) in both leaves and roots. Further expression analysis by real-time PCR indicated that the expression levels of ABA-dependent and ABA-independent related genes could be activated in grafted grape after drought treatment. CONCLUSIONS: Taken together, our findings demonstrated that grafting onto 1103P enhanced tolerance against drought stress in grape by improving water content, photosynthesis and antioxidant defense capacity, which provided a valuable information for understanding the mechanisms of drought tolerance regulated by grafting plants.

Whole-genome resequencing and transcriptome analyses of four generation mutants to reveal spur-type and skin-color related genes in apple (Malus domestica Borkh. Cv. Red delicious).

BACKGROUND: Bud sport is a kind of somatic mutation that usually occurred in apple. 'Red Delicious' is considered to be a special plant material of bud sport, whereas the genetic basis of plant mutants is still unknown. In this study, we used whole-genome resequencing and transcriptome sequencing to identify genes related to spur-type and skin-color in the 'Red Delicious' (G0) and its four generation mutants including 'Starking Red' (G1), 'Starkrimson' (G2), 'Campbell Redchief' (G3) and 'Vallee Spur' (G4). RESULTS: The number of single nucleotide polymorphisms (SNPs), insertions and deletions (InDels) and structural variations (SVs) were decreased in four generation mutants compared to G0, and the number of unique SNPs and InDels were over 9-fold and 4-fold higher in G1 versus (vs.) G2 and G2 vs. G3, respectively. Chromosomes 2, 5, 11 and 15 carried the most SNPs, InDels and SVs, while chromosomes 1 and 6 carried the least. Meanwhile, we identified 4,356 variation genes by whole-genome resequencing and transcriptome, and obtained 13 and 16 differentially expressed genes (DEGs) related to spur-type and skin-color by gene expression levels. Among them, DELLA and 4CL7 were the potential genes that regulate the difference of spur-type and skin-color characters, respectively. CONCLUSIONS: Our study identified potential genes associated with spur-type and skin-color differences in 'Red Delicious' and its four generation mutants, which provides a theoretical foundation for the mechanism of the apple bud sport.

Insights on the stem elongation of spur-type bud sport mutant of 'Red Delicious' apple.

MAIN CONCLUSION: The decreased capacity of auxin-, CTK-, and BR-mediated cell division and cell enlargement pathways, combined with the enhanced capacity of GA and ETH-, JA-, ABA-, SA-mediated stress-resistant pathways were presumed to be the crucial reasons for the formation of spur-type 'Red Delicious' mutants. Vallee Spur', which exhibit short internodes and compact tree shape, is the fourth generation of the spur-type bud sport mutant of 'Red Delicious'. However, the underlying molecular mechanism of these properties remains unclear. Here, comparative phenotypic, full-length transcriptome and phytohormone analyses were performed between 'Red Delicious' (NSP) and 'Vallee Spur' (SP). The new shoot internode length of NSP was ˃ 1.53-fold higher than that of the SP mutant. Cytological analysis showed that the stem cells of the SP mutant were smaller and more tightly arranged relative to the NSP. By Iso-Seq, a total of 1426 differentially expressed genes (DEGs) were detected, including 808 upregulated and 618 downregulated genes in new shoot apex with 2 leaves of the SP mutant. Gene expressions involved in auxin, cytokinin (CTK), and brassinosteroid (BR) signal transduction were mostly downregulated in the SP mutant, whereas those involved in gibberellin (GA), ethylene (ETH), jasmonate (JA), ABA, and salicylic acid (SA) signal transduction were mostly upregulated. The overall thermogram analysis of hormone levels in the shoot apex carrying two leaves detected by LC-MS/MS absolute quantification showed that the levels of IAA-Asp, IAA, iP7G, OPDA, and 6-deoxyCS were significantly upregulated in the SP mutant, while the remaining 28 hormones were significantly downregulated. It is speculated that the decreased capacity of auxin, CTK, and BR-mediated cell division and cell enlargement pathways is crucial for the formation of the SP mutant. GA and stress-resistant pathways of ETH, JA, ABA, and SA also play vital roles in stem elongation. These results highlight the involvement of phytohormones in the formation of stem elongation occurring in 'Red Delicious' spur-type bud sport mutants and provide information for exploring its biological mechanism.

Transcriptome and metabolites analysis of water-stressed grape berries at different growth stages.

Drought is one of the main abiotic factors affecting grape quality. However, the impacts of drought stress on sugar and related gene expression during grape berry ripening remain unclear. In this experiment, the grapes were subjected to different levels of continuous water stress from 45 to 120 days after flowering (DAA) to study the changes in berry sugar content and the expression of genes related to sugar metabolism under different water stresses. Data supported that glucose, fructose, sucrose, and soluble sugars increased from 45 DAA. Combined with previous research results, T1, T2, and Ct grape berries with 60 ~ 75 DAA and large differences in sucrose, fructose, glucose and soluble sugars compared with the Ct were selected for RNA sequencing (RNA-seq). Through transcriptome analysis, 4471 differentially expressed genes (DEGs) were screened, and 65 genes in photosynthesis, ABA signaling pathway and photosynthetic carbon metabolism pathway were analyzed further by qRT-PCR. At 60 DAA, the relative expression levels of CAB1R, PsbP, SNRK2, and PYL9 were significantly upregulated in response to water stress, while AHK1, At4g02290 were down-regulated. At 75 DAA, the relative expression levels of ELIP1, GoLS2, At4g02290, Chi5, SAPK, MAPKKK17, NHL6, KINB2, and AHK1 were upregulated. And CAB1R, PsbA, GoLS1, SnRK2, PYL9, and KINGL were significantly downregulated under moderate water stress. In addition, PsbA expression was down-regulated in response to water stress. These results will help us to fully understand the potential connections between glucose metabolism and gene expression in grapes under drought stress.

Nitrogen application improves salt tolerance of grape seedlings via regulating hormone metabolism.

Salt stress is a dominant environmental factor that restricts the growth and yield of crops. Nitrogen is an essential mineral element for plants, regulates various physiological and biochemical processes, and has been reported to enhance salt tolerance in plants. However, the crosstalk between salt and nitrogen in grapes is not well understood. In this study, we found that nitrogen supplementation (0.01 and 0.1 mol L-1 NH4 NO3 ) significantly increased the accumulation of proline, chlorophyll, Na+ , NH4 + , and NO3 - , while it reduced the malondialdehyde content and inhibited photosynthetic performance under salt stress conditions (200 mmol L-1 NaCl). Further transcriptome and metabolome analyses showed that a total of 4890 differentially expressed genes (DEGs) and 753 differently accumulated metabolites (DAMs) were identified. Joint omics results revealed that plant hormone signal transduction pathway connected the DEGs and DAMs. In-depth analysis revealed that nitrogen supplementation increased the levels of endogenous abscisic acid, salicylic acid, and jasmonic acid by inducing the expression of 11, 4, and 13 genes related to their respective biosynthesis pathway. In contrast, endogenous indoleacetic acid content was significantly reduced due to the remarkable regulation of seven genes of its biosynthetic pathway. The modulation in hormone contents subsequently activated the differential expression of 13, 10, 12, and 29 genes of the respective downstream hormone signaling transduction pathways. Overall, all results indicate that moderate nitrogen supplementation could improve salt tolerance by regulating grape physiology and endogenous hormone homeostasis, as well as the expression of key genes in signaling pathways, which provides new insights into the interactions between mineral elements and salt stress.

Integrated transcriptome and metabolome analysis reveals antioxidant machinery in grapevine exposed to salt and alkali stress.

Plant acclimation to salt and alkali stress is closely linked to the ability of the antioxidant system to mediate the scavenging of reactive oxygen species (ROS). In this study, we investigated the effects of salt stress and alkali stress on ROS, antioxidant enzymes, transcriptome, and metabolome. The results showed that the levels of superoxide anions, hydrogen peroxide, malondialdehyde, and electrolyte leakage increased under salt and alkali stress, with higher concentrations observed under alkali stress than salt stress. The activities of superoxide dismutase (EC 1.15.1.1), peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6), ascorbate peroxidase (EC 1.11.1.11), glutathione reductase (EC 1.6.4.2), dehydroascorbate reductase (EC 1.8.5.1), and monodehydroascorbate reductase (EC 1.6.5.4) varied under salt and alkali stress. The transcriptome analysis revealed the induction of signal transduction and metabolic processes and differential expression of genes encoding antioxidant enzymes in response to salt and alkali stress. The metabolome analysis demonstrated increased ascorbic acid and glutathione under salt stress, while most phenolic acids, flavonoids, and alkaloids increased under salt and alkali stress. Integrative analysis of the metabolome and transcriptome data revealed that the flavonoid biosynthesis pathway played a key role in the grapevine's response to salt stress. The total flavonoid content increased under salt and alkali stress, but the accumulation of flavonoids was higher under salt stress than alkali stress. In conclusion, our findings indicate significant differences in the antioxidant defense of grapevines under these two stresses, providing insight into distinct acclimation mechanisms in grapevine under salt and alkali stress.

Genome-wide characterization of Alfin-like (AL) genes in apple and functional identification of MdAL4 in response to drought stress.

KEY MESSAGE: Eleven Alfin-like (AL) genes were obtained from apple and MdAL4 was selected for improving drought stress tolerance of transgenic apple callus and Arabidopsis. Drought is an important environmental factor affecting plant growth all over the world. Alfin-like (AL) have well-documented functions in abiotic stress response, but their drought stress tolerance in apple (Malus domestica) are poorly understood. According to the transcriptome data, 11 MdAL genes containing conserved Alfin and PHD-finger domain were identified in apple and divided into three subgroups with a total of 35 members from different species. Subsequently, gene structures, conserved amino acid sequences, promoter cis-acting elements, and gene evolution events were analyzed. Based on differential expression of MdALs in response to abiotic stresses, MdAL4, which was highly expressed under drought, was further cloned and investigated. MdAL4 encoding nuclear-localized protein conferred enhanced drought tolerance in overexpressing transgenic calli of apple 'Orin'. Moreover, the ectopic expression of MdAL4 improved the drought tolerance of transgenic Arabidopsis, as judged from remarkably decreased malonaldehyde (MDA) content and electrolyte leakage in MdAL4 overexpressing plants relative to WT. Furthermore, MdAL4 possibly could bind to promoter regions of ROS-scavenging and stress-related genes to improve drought tolerance. Additionally, we found in silico evidence that three proteins containing the WD40 domain that interact with MdAL4. Based on these results, MdAL4 was identified as a positive regulator for improving drought stress of apple.

VaSUS2 confers cold tolerance in transgenic tomato and Arabidopsis by regulation of sucrose metabolism and ROS homeostasis.

KEY MESSAGE: VaSUS2 enhances cold tolerance of transgenic tomato and Arabidopsis by regulating sucrose metabolism and improving antioxidant enzymes activity. Sucrose synthetase (SUS) is a key enzyme of sugar metabolism, and plays an important role in response to abiotic stress in plant. However, the function of VaSUS2 remains unknown in cold tolerance. Here, the cloning and functional characterization of the plasma membrane-localized VaSUS2 gene isolated from Vitis amurensis was studied. The transcript level of VaSUS2 was up-regulated under cold stress in Vitis amurensis. Heterologous expression of VaSUS2 in tomato increased SUS activity, which promoted the accumulation of glucose and fructose under cold treatment. The transgenic tomato and Arabidopsis exhibited higher levels of antioxidant enzymes activity, lower relative electrolyte leakage (REL), malondialdehyde (MDA) and hydrogen peroxide (H2O2) content compared to wild type under cold stress. Importantly, the ability of scavenging reactive oxygen species (ROS) in transgenic plants was significantly improved. Moreover, yeast two-hybrid (Y2H) indicated that VaSnRK1 might be a potential interaction protein of VaSUS2. qRT-PCR showed that sucrose metabolism-related genes SlSUS, SlSPS and SlINV were significantly up-regulated in transgenic tomatoes. Meanwhile, the expression levels of antioxidant enzyme genes and cold-related genes CBF1, COR47 and ICE1 were up-regulated in transgenic plants. Taken together, these results suggested that VaSUS2 was involved in cold tolerance by increasing the levels of soluble sugars, improving the activity of antioxidant enzymes, and up-regulating the expression of cold-related genes in transgenic tomatoes and Arabidopsis.

Genome-Wide Identification and Expression Analysis of ANS Family in Strawberry Fruits at Different Coloring Stages.

To elucidate the structural characteristics, phylogeny and biological function of anthocyanin synthase (ANS) and its role in anthocyanin synthesis, members of the strawberry ANS gene family were obtained by whole genome retrieval, and their bioinformatic analysis and expression analysis at different developmental stages of fruit were performed. The results showed that the strawberry ANS family consisted of 141 members distributed on 7 chromosomes and could be divided into 4 subfamilies. Secondary structure prediction showed that the members of this family were mainly composed of random curls and α-helices, and were mainly located in chloroplasts, cytoplasm, nuclei and cytoskeletons. The promoter region of the FvANS gene family contains light-responsive elements, abiotic stress responsive elements and hormone responsive elements, etc. Intraspecific collinearity analysis revealed 10 pairs of FvANS genes, and interspecific collinearity analysis revealed more relationships between strawberries and apples, grapes and Arabidopsis, but fewer between strawberries and rice. Chip data analysis showed that FvANS15, FvANS41, FvANS47, FvANS48, FvANS49, FvANS67, FvANS114 and FvANS132 were higher in seed coat tissues and endosperm. FvANS16, FvANS85, FvANS90 and FvANS102 were higher in internal and fleshy tissues. Quantitative real-time PCR (qRT-PCR) showed that the ANS gene was expressed throughout the fruit coloring process. The expression levels of most genes were highest in the 50% coloring stage (S3), such as FvANS16, FvANS19, FvANS31, FvANS43, FvANS73, FvANS78 and FvANS91. The expression levels of FvANS52 were the highest in the green fruit stage (S1), and FvANS39 and FvANS109 were the highest in the 20% coloring stage (S2). These results indicate that different members of the FvANS gene family play a role in different pigmentation stages, with most genes playing a role in the expression level of the rapid accumulation of fruit coloring. This study lays a foundation for further study on the function of ANS gene family.

MdMYB66 Is Associated with Anthocyanin Biosynthesis via the Activation of the MdF3H Promoter in the Fruit Skin of an Apple Bud Mutant.

Skin color is an important trait that is mainly determined by the content and composition of anthocyanins in apples. In this study, a new bud mutant (RM) from 'Oregon Spur II' (OS) of Red Delicious apple was obtained to reveal the mechanism underlying red color formation. Results showed that the total anthocyanin content in RM was significantly higher than that in OS with the development of fruit. Through widely-targeted metabolomics, we found that cyanidin-3-O-galactoside was significantly accumulated in the fruit skin of RM. Transcriptome analysis revealed that the structural gene MdF3H and MdMYB66 transcription factor were significantly up-regulated in the mutant. Overexpression of MdMYB66 in apple fruit and apple callus significantly promoted anthocyanin accumulation and significantly increased the expression level of MdMYB66 and structural genes related to anthocyanin synthesis. Y1H and LUC analysis verified that MdMYB66 could specifically bind to the promoter of MdF3H. The results of the double luciferase activity test showed that MdMYB66 activated MdF3H 3.8 times, which led to increased anthocyanin contents. This might explain the phenotype of red color in RM at the early stage. Taken together, these results suggested that MdMYB66 was involved in regulating the anthocyanin metabolic pathways through precise regulation of gene expression. The functional characterization of MdMYB66 provides insight into the biosynthesis and regulation of anthocyanins.

Identification and Expression Analysis of the SKP1-Like Gene Family under Phytohormone and Abiotic Stresses in Apple (Malus domestica).

Ubiquitination participates in plant hormone signaling and stress response to adversity. SKP1-Like, a core component of the SCF (Skp1-Cullin-F-box) complex, is the final step in catalyzing the ubiquitin-mediated protein degradation pathway. However, the SKP1-Like gene family has not been well characterized in response to apple abiotic stresses and hormonal treatments. This study revealed that 17 MdSKP1-Like gene family members with the conserved domain of SKP1 were identified in apples and were unevenly distributed on eight chromosomes. The MdSKP1-Like genes located on chromosomes 1, 10, and 15 were highly homologous. The MdSKP1-like genes were divided into three subfamilies according to the evolutionary affinities of monocotyledons and dicotyledons. MdSKP1-like members of the same group or subfamily show some similarity in gene structure and conserved motifs. The predicted results of protein interactions showed that members of the MdSKP1-like family have strong interactions with members of the F-Box family of proteins. A selection pressure analysis showed that MdSKP1-Like genes were in purifying selection. A chip data analysis showed that MdSKP1-like14 and MdSKP1-like15 were higher in flowers, whereas MdSKP1-like3 was higher in fruits. The upstream cis-elements of MdSKP1-Like genes contained a variety of elements related to light regulation, drought, low temperature, and many hormone response elements, etc. Meanwhile, qRT-PCR also confirmed that the MdSKP1-Like gene is indeed involved in the response of the apple to hormonal and abiotic stress treatments. This research provides evidence for regulating MdSKP1-Like gene expression in response to hormonal and abiotic stresses to improve apple stress resistance.

Genome-Wide Identification and Abiotic Stress Response Analysis of PP2C Gene Family in Woodland and Pineapple Strawberries.

Protein phosphatase 2C (PP2C) is a negative regulator of serine/threonine residue protein phosphatase and plays an important role in abscisic acid (ABA) and abiotic-stress-mediated signaling pathways in plants. The genome complexity of woodland strawberry and pineapple strawberry is different due to the difference in chromosome ploidy. This study conducted a genome-wide investigation of the FvPP2C (Fragaria vesca) and FaPP2C (Fragaria ananassa) gene family. Fifty-six FvPP2C genes and 228 FaPP2C genes were identified from the woodland strawberry and pineapple strawberry genomes, respectively. FvPP2Cs were distributed on seven chromosomes, and FaPP2Cs were distributed on 28 chromosomes. The size of the FaPP2C gene family was significantly different from that of the FvPP2C gene family, but both FaPP2Cs and FvPP2Cs were localized in the nucleus, cytoplasm, and chloroplast. Phylogenetic analysis revealed that 56 FvPP2Cs and 228 FaPP2Cs could be divided into 11 subfamilies. Collinearity analysis showed that both FvPP2Cs and FaPP2Cs had fragment duplication, and the whole genome duplication was the main cause of PP2C gene abundance in pineapple strawberry. FvPP2Cs mainly underwent purification selection, and there were both purification selection and positive selection effects in the evolution of FaPP2Cs. Cis-acting element analysis found that the PP2C family genes of woodland and pineapple strawberries mainly contained light responsive elements, hormone responsive elements, defense and stress responsive elements, and growth and development-related elements. The results of quantitative real-time PCR (qRT-PCR) showed that the FvPP2C genes showed different expression patterns under ABA, salt, and drought treatment. The expression level of FvPP2C18 was upregulated after stress treatment, which may play a positive regulatory role in ABA signaling and abiotic stress response mechanisms. This study lays a foundation for further investigation on the function of the PP2C gene family.

Genome-Wide Identification and Analysis of the Genes Encoding Q-Type C2H2 Zinc Finger Proteins in Grapevine.

Q-type C2H2 zinc finger proteins (ZFPs), the largest family of transcription factors, have been extensively studied in plant genomes. However, the genes encoding this transcription factor family have not been explored in grapevine genomes. Therefore, in this study, we conducted a genome-wide identification of ZFP genes in three species of grapevine, namely Vitis vinifera, Vitis riparia, and Vitis amurensis, based on the sequence databases and phylogenetic and their conserved domains. We identified 52, 54, and 55 members of Q-type C2H2 ZFPs in V. vinifera, V. riparia, and V. amurensis, respectively. The physical and chemical properties of VvZFPs, VrZFPs, and VaZFPs were examined. The results showed that these proteins exhibited differences in the physical and chemical properties and that they all were hydrophobic proteins; the instability index showed that the four proteins were stable. The subcellular location of the ZFPs in the grapevine was predicted mainly in the nucleus. The phylogenetic tree analysis of the amino acid sequences of VvZFP, VaZFP, VrZFP, and AtZFP proteins showed that they were closely related and were divided into six subgroups. Chromosome mapping analysis showed that VvZFPs, VrZFPs, and VaZFPs were unevenly distributed on different chromosomes. The clustered gene analysis showed that the motif distribution was similar and the sequence of genes was highly conserved. Exon and intron structure analysis showed that 118 genes of ZFPs were intron deletion types, and the remaining genes had variable numbers of introns, ranging from 2 to 15. Cis-element analysis showed that the promoter of VvZFPs contained multiple cis-elements related to plant hormone response, stress resistance, and growth, among which the stress resistance elements were the predominant elements. Finally, the expression of VvZFP genes was determined using real-time quantitative PCR, which confirmed that the identified genes were involved in response to methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA), and low-temperature (4 °C) stress. VvZFP10-GFP and VvZFP46-GFP fusion proteins were localized in the nucleus of tobacco cells, and VvZFP10 is the most responsive gene among all VvZFPs with the highest relative expression level to MeJA, ABA, SA and low-temperature (4 °C) stress. The present study provides a theoretical basis for exploring the mechanism of response to exogenous hormones and low-temperature tolerance in grapes and its molecular breeding in the future.

Grapevine (Vitis vinifera) responses to salt stress and alkali stress: transcriptional and metabolic profiling.

BACKGROUND: Soil salinization and alkalization are widespread environmental problems that limit grapevine (Vitis vinifera L.) growth and yield. However, little is known about the response of grapevine to alkali stress. This study investigated the differences in physiological characteristics, chloroplast structure, transcriptome, and metabolome in grapevine plants under salt stress and alkali stress. RESULTS: We found that grapevine plants under salt stress and alkali stress showed leaf chlorosis, a decline in photosynthetic capacity, a decrease in chlorophyll content and Rubisco activity, an imbalance of Na+ and K+, and damaged chloroplast ultrastructure. Fv/Fm decreased under salt stress and alkali stress. NPQ increased under salt stress whereas decreased under alkali stress. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed the differentially expressed genes (DEGs) induced by salt stress and alkali stress were involved in different biological processes and have varied molecular functions. The expression of stress genes involved in the ABA and MAPK signaling pathways was markedly altered by salt stress and alkali stress. The genes encoding ion transporter (AKT1, HKT1, NHX1, NHX2, TPC1A, TPC1B) were up-regulated under salt stress and alkali stress. Down-regulation in the expression of numerous genes in the 'Porphyrin and chlorophyll metabolism', 'Photosynthesis-antenna proteins', and 'Photosynthesis' pathways were observed under alkali stress. Many genes in the 'Carbon fixation in photosynthetic organisms' pathway in salt stress and alkali stress were down-regulated. Metabolome showed that 431 and 378 differentially accumulated metabolites (DAMs) were identified in salt stress and alkali stress, respectively. L-Glutamic acid and 5-Aminolevulinate involved in chlorophyll synthesis decreased under salt stress and alkali stress. The abundance of 19 DAMs under salt stress related to photosynthesis decreased. The abundance of 16 organic acids in salt stress and 22 in alkali stress increased respectively. CONCLUSIONS: Our findings suggested that alkali stress had more adverse effects on grapevine leaves, chloroplast structure, ion balance, and photosynthesis than salt stress. Transcriptional and metabolic profiling showed that there were significant differences in the effects of salt stress and alkali stress on the expression of key genes and the abundance of pivotal metabolites in grapevine plants.