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.

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.

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.

MdbZIP44-MdCPRF2-like-Mdα-GP2 regulate starch and sugar metabolism in apple under nitrogen supply.

Nitrogen (N) is regarded as an essential macronutrient and is tightly associated with carbon (C) metabolism in plants. The transcriptome data obtained from this study showed that the expression level of the apple basic leucine zipper (bZIP) transcription factor (TF) MdbZIP44 was up-regulated in 'Oregon Spur Delicious' (Malus domestica Borkh.) apple fruits under nitrogen supply. MdbZIP44 bound to the promoter of Mdα-GP2 gene and inhibited its expression, thereby promoting starch accumulation and decreasing glucose content in apple and tomato fruits. Besides, overexpression of MdbZIP44 promoted sucrose accumulation by regulating the activities of sucrose metabolism-related enzymes and the expression of sugar metabolism-related genes in apple callus and tomato fruits. Furthermore, biochemical assays indicated that MdbZIP44 directly interacted with MdCPRF2-like, another bZIP gene in apple. Meanwhile, this study found that MdCPRF2-like, along with the MdbZIP44 and MdCPRF2-like complex, could activate the expression of Mdα-GP2, respectively. In conclusion, this study provides a new reference for potential mechanisms underlying that MdbZIP44-MdCPRF2-like-Mdα-GP2 regulates starch and sugar metabolism under nitrogen supply.

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.

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.

Regulatory mechanism of GA3 application on grape (Vitis vinifera L.) berry size.

Gibberellin A3 (GA3) is often used as a principal growth regulator to increase plant size. Here, we applied Tween-20 (2%)-formulated GA3 (T1:40 mg/L; T2:70 mg/L) by dipping the clusters at the initial expansion phase of 'Red Globe' grape (Vitis vinifera L.) in 2018 and 2019. Tween-20 (2%) was used as a control. The results showed that GA3 significantly increased fruit cell length, cell size, diameter, and volume. The hormone levels of auxin (IAA) and zeatin (ZT) were significantly increased at 2 h (0 d) -1 d after application (DAA0-1) and remained significantly higher at DAA1 until maturity. Conversely, ABA exhibited an opposite trend. The mRNA and non-coding sequencing results yielded 436 differentially expressed mRNA (DE_mRNAs), 79 DE_lncRNAs and 17 DE_miRNAs. These genes are linked to hormone pathways like cysteine and methionine metabolism (ko00270), glutathione metabolism (ko00480) and plant hormone signal transduction (ko04075). GA3 application reduced expression of insensitive dwarf 2 (GID2, VIT_07s0129g01000), small auxin-upregulated RNA (SAUR, VIT_08s0007g03120) and 1-aminocyclopropane-1-carboxylate synthase (ACS, VIT_18s0001g08520), but increased SAUR (VIT_04s0023g00560) expression. These four genes were predicted to be negatively regulated by vvi-miR156, vvi-miR172, vvi-miR396, and vvi-miR159, corresponding to specific lncRNAs. Therefore, miRNAs could affect grape size by regulating key genes GID2, ACS and SAUR. The R2R3 MYB family member VvRAX2 (VIT_08s0007g05030) was upregulated in response to GA3 application. Overexpression of VvRAX2 in tomato transgenic lines increased fruit size in contrast to the wild type. This study provides a basis and genetic resources for elucidating the novel role of ncRNAs in fruit development.

[Identification and expression analysis of citrate synthase 3 gene family members in apple].

As one of the key enzymes in cell metabolism, the activity of citrate synthase 3 (CS3) regulates the substance and energy metabolism of organisms. The protein members of CS3 family were identified from the whole genome of apple, and bioinformatics analysis was performed and expression patterns were analyzed to provide a theoretical basis for studying the potential function of CS3 gene in apple. BLASTp was used to identify members of the apple CS3 family based on the GDR database, and the basic information of CS3 protein sequence, subcellular localization, domain composition, phylogenetic relationship and chromosome localization were analyzed by Pfam, SMART, MEGA5.0, clustalx.exe, ExPASy Proteomics Server, MEGAX, SOPMA, MEME, WoLF PSORT and other software. The tissue expression and inducible expression characteristics of 6 CS3 genes in apple were determined by acid content and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Apple CS3 gene family contains 6 members, and these CS3 proteins contain 473-608 amino acid residues, with isoelectric point distribution between 7.21 and 8.82. Subcellular localization results showed that CS3 protein was located in mitochondria and chloroplasts, respectively. Phylogenetic analysis divided them into 3 categories, and the number of genes in each subfamily was 2. Chromosome localization analysis showed that CS3 gene was distributed on different chromosomes of apple. The secondary structure of protein is mainly α-helix, followed by random curling, and the proportion of ß-angle is the smallest. The 6 members were all expressed in different apple tissues. The overall expression trend from high to low was the highest relative expression content of MdCS3.4, followed by MdCS3.6, and the relative expression level of other members was in the order of MdCS3.3 > MdCS3.2 > MdCS3.1 > MdCS3.5. qRT-PCR results showed that MdCS3.1 and MdCS3.3 genes had the highest relative expression in the pulp of 'Chengji No. 1' with low acid content, and MdCS3.2 and MdCS3.3 genes in the pulp of 'Asda' with higher acid content had the highest relative expression. Therefore, in this study, the relative expression of CS3 gene in apple cultivars with different acid content in different apple varieties was detected, and its role in apple fruit acid synthesis was analyzed. The experimental results showed that the relative expression of CS3 gene in different apple varieties was different, which provided a reference for the subsequent study of the quality formation mechanism of apple.

Genome-wide identification of grape ANS gene family and expression analysis at different fruit coloration stages.

BACKGROUND: Anthocyanin synthase (ANS) is the enzyme downstream of the anthocyanins synthesis pathway and the rate-limiting enzyme of the synthesis pathway. It catalyzes the conversion of colorless anthocyanins to anthocyanins and plays an important role in plant color presentation and stress resistance. However, ANS gene is rarely studied in grapes. RESULTS: In this study, 121 VvANS genes were identified and distributed on 18 chromosomes, VvANS family members were divided into 8 subgroups. Secondary structure prediction showed mainly irregular coils and α-helices, and subcellular localization indicated that VvANS gene family is mainly located in chloroplast, cytoplasm and nucleus. The promoter region of the VvANS gene family contains multiple cis-acting elements that are associated with light, abiotic stress, and hormones. Intraspecific collinearity analysis showed that there were 13 pairs of collinearity between VvANS genes. Interspecific collinearity analysis showed that there was more collinearity between grape, apple and Arabidopsis, but less collinearity between grape and rice. Microarray data analysis showed that VvANS17, VvANS23 and VvANS75 had higher expression levels in flesh and peel, while VvANS25, VvANS64 and VvANS106 had higher expression levels in flower. The results of qRT-PCR analysis showed that VvANS genes were expressed throughout the whole process of fruit coloring, such as VvANS47 and VvANS55 in the green fruit stage, VvANS3, VvANS64 and VvANS90 in the initial fruit color turning stage. The expression levels of VvANS21, VvANS79 and VvANS108 were higher at 50% coloring stage, indicating that these genes play an important role in the fruit coloring process. VvANS4, VvANS66 and VvANS113 had the highest expression levels in the full maturity stage. CONCLUSIONS: These results indicated that different members of VvANS gene family played a role in different coloring stages, and this study laid a foundation for further research on the function of ANS gene family.

Identification of Flavanone 3-Hydroxylase Gene Family in Strawberry and Expression Analysis of Fruit at Different Coloring Stages.

The color of strawberry fruit is an important appearance quality index that affects the marketability of fruit, and the content and type of anthocyanin are two of the main reasons for the formation of fruit color. At present, the research on anthocyanin synthesis mainly focuses on the phenylpropane metabolic pathway, and the F3H gene family is an important member of this metabolic pathway. Therefore, in order to clarify the role of flavanone 3-hydroxylase (F3H) in regulating anthocyanin accumulation in strawberry, we identified F3H gene family members in strawberry and analyzed their bioinformatics and expression at different fruit color stages. The results showed that the strawberry F3H family contains 126 members, which are distributed on seven chromosomes and can be divided into six subgroups. The promoter region of strawberry F3H gene family contains light response elements, abiotic stress response elements and hormone response elements. Intraspecic collinearity analysis showed that there were six pairs of collinearity of the F3H gene. Interspecific collinearity analysis showed that there were more collinearity relationships between strawberry and apple, grape and Arabidopsis, but less collinearity between strawberry and rice. Via tissue-specific expression analysis, we found that the expression levels of FvF3H48, FvF3H120 and FvF3H74 were higher in the stages of germination, growth, flowering and fruit setting. The expression levels of FvF3H42 and FvF3H16 were higher in seeds. The expression levels of FvF3H16 and FvF3H11 were higher in the ovary wall of stage 1, stage 2, stage 3 and stage 5. FvF3H15 and FvF3H48 were highly expressed in the pericardium, anther, receptacle and anther. Real-time fluorescence quantitative PCR showed the expression changes in F3H in the fruit coloring process. The results indicate that the expression levels of most members were higher during the S3 stage, such as FvF3H7, FvF3H16, FvF3H32, FvF3H82, FvF3H89, FvF3H92 and FvF3H112. FvF3H63 and FvF3H104 exhibited particularly high expression levels during the S1 stage, with some genes also showing elevated expression during the S4 stage, including FvF3H13, FvF3H27, FvF3H66 and FvF3H103. FvF3H58, FvF3H69, FvF3H79 and FvF3H80 showed higher expression levels during the S2 stage. These findings lay the groundwork for elucidating the biological functions of the strawberry F3H gene family and the selection of related genes.

[Identification and expression analysis of apple PDHB-1 gene family].

Pyruvate dehydrogenase E1 component subunit beta-1 (PDHB-1) is a gene encoding the ß-subunit of pyruvate dehydrogenase complex, which plays an important role in fruit acid accumulation. The aim of this study was to investigate the evolution characteristics of apple PDHB-1 family and its expression in apples with different acid contents. Bioinformatics analysis was performed using databases including NCBI, Pfam and software including ClustalX, MEGA, and TBtools. By combining titratable acid content determination and quantitative real-time PCR (qRT-PCR), the expression of this family genes in the peel and pulp of apple 'Asda' and 'Chengji No.1' with different acid content were obtained, respectively. The family members were mainly located in chloroplast, cytoplasm and mitochondria. α-helix and random coil were the main factors for the formation of secondary structure in this family. Tissue-specific expression profiles showed that the expression of most members were higher in fruit than in other tissues. qRT-PCR results showed that the expression profile of most members was consistent with the profile of titratable acid contents. In the peel, the expression levels of 14 members in 'Asda' apples with high acid content were significantly higher than that in 'Chengji No.1' apples with low acid content, where the expression difference of MdPDHB1-15 was the most significant. In the pulp, the expression levels of 17 members in 'Asda' apples were significantly higher than that in 'Chengji No.1' apples, where MdPDHB1-01 was the most highly expressed. It was predicted that PDHB-1 gene family in apple plays an important role in the regulation of fruit acidity.

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.

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.

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.

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.

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.

Comprehensive genomic identification and expression analysis 4CL gene family in apple.

To clarify the structural characteristics, phylogeny, biological function and regulation of 4-coumarate-CoAligase (4CL) in anthocyanin synthesis, the 4CL gene family members in apples were identified and bioinformatic analysis was performed. qRT-PCR was used to analyze the expression levels of this gene family members in different apple varieties, and the role of the 4CL gene in apple anthocyanin synthesis was preliminaries clarified, which provided a certain theoretical basis for the regulatory network of apple anthocyanin synthesis. The results showed that a total of 69 members of the 4CL gene family were identified in the apple (Malus domestica Brokh.), encoding amino acids ranging from 97 to 2310 with theoretical isoelectric points ranging from 5.28 to 9.84. The 69 4CL family members were distributed on 17 chromosomes in the apple, among which chromosome 17 had the largest distribution (9 members), followed by chromosome 9 (7 members), chromosomes 16 and 14 (6 members each), and chromosomes 15 and 13 (5 members each). The subcellular localization prediction showed that apple 4CL gene family members were mainly expressed in cytoplasm, chloroplast, nucleus and cell membrane, with a small amount of expression in mitochondria, vacuoles, peroxisomes, cytoskeleton, golgi and cell matrix, but not in endoplasmic reticulum. The secondary structures are mainly α-helices and irregular coils. Microarray expression profile analysis showed that the expression levels of each member in apple were related to fruit variety and tissue structure, and the expression levels were mainly higher in fruit, flower and leaf. Real-time PCR analysis showed that the expression level of each member was directly proportional to the degree of fruit coloring and anthocyanin accumulation. The expression levels of Md4CL10 and Md4CL23 in 'Astar' (G4) apple fruit skin with the highest anthocyanin content were 516, 20 and 2 times higher than those in 'Chengji NO.1' (G1), 'Golden Delicious' (G2) and 'Ruixue' (G3), respectively.

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 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.

Grape BES1 transcription factor gene VvBES1-3 confers salt tolerance in transgenic Arabidopsis.

BRI1-EMS-Suppressor 1 (BES1) regulates plant growth, development, and stress resistance, and plays a pivotal role in the brassinosteroid (BR) signal transduction pathway. In this study, a total of 12 BES1 genes were identified in the grape (Vitis vinifera) genome. Phylogenetic, structure, and motif sequence analyses of these genes provided insights into their evolutionary characteristics. Hormone-, stress-, and light-responsive and organ-specific cis-acting elements were identified in VvBES1 gene promoters. Microarray data analysis showed that VvBES1 family members exhibit diverse expression patterns in different organs. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression levels of VvBES1 genes differed in response to BR, methyl jasmonate (MeJA), cold (4 °C), NaCl, and polyethylene glycol (PEG) treatments. The expression of VvBES1-3 was 29-fold higher under salt stress than control at 12 h. Moreover, VvBES1-3-overexpessing Arabidopsis thaliana plants showed lower malondialdehyde content, higher proline content, enhanced antioxidant enzyme (catalase, superoxide dismutase, peroxidase) activities, and higher salt-responsive gene expression levels than wild-type plants under salt stress, indicating that VvBES1-3 overexpression enhances salt stress tolerance in transgenic Arabidopsis. These results will contribute to further understanding the functions of BES1 transcription factors in the abiotic stress response.