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.

VvJAZ13 Positively Regulates Cold Tolerance in Arabidopsis and Grape.

Cold stress adversely impacts grape growth, development, and yield. Therefore, improving the cold tolerance of grape is an urgent task of grape breeding. The Jasmonic acid (JA) pathway responsive gene JAZ plays a key role in plant response to cold stress. However, the role of JAZ in response to low temperatures in grape is unclear. In this study, VvJAZ13 was cloned from the 'Pinot Noir' (Vitis vinefera cv. 'Pinot Noir') grape, and the potential interacting protein of VvJAZ13 was screened by yeast two-hybrid (Y2H). The function of VvJAZ13 under low temperature stress was verified by genetic transformation. Subcellular localization showed that the gene was mainly expressed in cytoplasm and the nucleus. Y2H indicated that VvF-box, VvTIFY5A, VvTIFY9, Vvbch1, and VvAGD13 may be potential interacting proteins of VvJAZ13. The results of transient transformation of grape leaves showed that VvJAZ13 improved photosynthetic capacity and reduced cell damage by increasing maximum photosynthetic efficiency of photosystem II (Fv/Fm), reducing relative electrolyte leakage (REL) and malondialdehyde (MDA), and increasing proline content in overexpressed lines (OEs), which played an active role in cold resistance. Through the overexpression of VvJAZ13 in Arabidopsis thaliana and grape calli, the results showed that compared with wild type (WT), transgenic lines had higher antioxidant enzyme activity and proline content, lower REL, MDA, and hydrogen peroxide (H2O2) content, and an improved ability of scavenging reactive oxygen species. In addition, the expression levels of CBF1-2 and ICE1 genes related to cold response were up-regulated in transgenic lines. To sum up, VvJAZ13 is actively involved in the cold tolerance of Arabidopsis and grape, and has the potential to be a candidate gene for improving plant cold tolerance.

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.

Evolution of the 14-3-3 gene family in monocotyledons and dicotyledons and validation of MdGRF13 function in transgenic Arabidopsis thaliana.

KEY MESSAGE: The 14-3-3 family is more highly conserved among monocotyledons, and overexpression of MdGRF13 improved drought and salt tolerance in transgenic Arabidopsis thaliana. The 14-3-3 are highly conserved regulatory proteins found in eukaryotes and play an essential role in plant growth, development and stress response. However, the 14-3-3 gene family evolution in monocotyledons and dicotyledons and the biological functions of the MdGRF13 under abiotic stress remain unknown. In our study, 195 members of the 14-3-3 family were identified from 12 species and divided into ε group and the Non-ε group. Synteny analysis within the 14-3-3 family indicated that segmental duplication events contributed to the expansion of the family. Selective pressure analysis indicated that purifying selection was a vital force in the 14-3-3 genes evolution, and monocotyledons had a lower million years ago (Mya) mean values than dicotyledons. Meanwhile, the codon adaptation index (CAI) and frequency of optical codons (FOP) are higher and the effective number of codons (Nc) is lower in monocotyledons 14-3-3 genes compared to dicotyledons. Moreover, the yeast two-hybrid (Y2H) demonstrated that MdGRF13 interacts with MdRD22, MdLHP1a and MdMORF1. Significantly, the malondialdehyde (MDA) content and relative conductivity were decreased, while the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were increased in transgenic Arabidopsis compared to the wild type (WT) under drought and salt stress. These results suggest that overexpression of MdGRF13 significantly improved the tolerance to drought and salt stress in transgenic Arabidopsis. Thus, our results provide a theoretical basis for exploring the evolution and function of the 14-3-3 gene family in monocotyledons and dicotyledons.

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 expression analysis of the BURP domain-containing genes in Malus domestica.

The conserved BURP-containing proteins are specific to plants and play a crucial role in plant growth, development, and response to abiotic stresses. However, less is known about the systematic characterization of BURP-containing proteins in apple. This study aimed to identify and analyze all BURP-containing genes in the apple genome, as well as to examine their expression patterns through various bioinformatics methods. Eighteen members of BURP-containing genes were identified in apple, six members lacked signal peptides, and the secondary structure was mainly a Random coil of BURP-containing genes. Gene structure and Motif analysis showed that proteins have similar structures and are conserved at the C-terminal. Cis-acting element analysis revealed that the proteins contain phytohormone and stress response elements, and chromosomal localization revealed that the family is unevenly distributed across eight chromosomes, with duplication of fragments leading to the expansion of family proteins. Tissue expression showed that MdPG3 and MdPG4 were expressed in different tissues and different varieties, MdRD2 and MdRD7 were highly expressed in 'M74' fruits and MdRD7 in 'M49' leaves, while MdUSP1 was highly expressed in 'GD' roots. The quantitative real-time PCR analysis showed that the expressions of six and seven genes were significantly up-regulated under NaCl and PEG treatments, respectively, whereas MdRD7 was significantly up-regulated under NaCl and PEG treatment over time. This study offers a comprehensive identification and expression analysis of BURP-containing proteins in apple. The findings provide a theoretical foundation for further exploration of the functions of this protein family. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01393-7.

Genome-wide identification and expression analysis of PIN gene family under phytohormone and abiotic stresses in Vitis Vinifera L.

The auxin efflux transport proteins PIN-formed (PIN) has wide adaptability to hormone and abiotic stress, but the response mechanism of PINs in grape remains unclear. In this study, 12 members of VvPINs were identified and distributed on 8 chromosomes. The PIN genes of five species were divided into two subgroups, and the similarity of exons/introns and motifs of VvPIN genes were found in the same subgroup. Meanwhile, according to the examination of conserved motifs, the motif 3 included the conserved structure NPNTY. The promoter region of VvPIN gene family contained various cis-acting elements, which were related to light, abiotic stress, and hormones which are essential for growth and development. Additionally, VvPIN1, VvPIN9, and VvPIN11 proteins simultaneously interacted with the ARF, ABC, PINOID, GBF1, and VIT_08s0007g09010. The results of qRT-PCR revealed that the majority of the VvPINs were highly induced by NAA, GA3, ABA, MeJA, SA, NaCl, low-temperature (4 ℃), and PEG treatments, and the results were consistent with the prediction of the cis-acting elements. Moreover, the expression profile and quantitative real-time PCR (qRT-PCR) demonstrated that VvPIN genes were expressed in roots, stems, and leaves. The subcellular localization of VvPIN1 in Nicotiana benthamiana revealed that VvPIN1 was localized at the plasma membrane. Collectively, this study revealed that PIN genes could respond to various abiotic stresses, and provided a framework for regulating the expression of PIN genes to enhance the resistance of the grape. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01239-8.

Identification and expression analysis of the grape pentatricopeptide repeat (PPR) gene family in abiotic stress.

The pentatricopeptide repeat (PPR) is one of the largest gene family in plants, and play important role in regulating plant growth, development and abiotic stress response. However, PPR genes have been poorly studied in grapes. In this study, based on the grape genome database, bioinformatics methods and quantitative real-time PCR (qRT-PCR) were used to identify the VvPPR family and the response to abiotic stress. A total of 181 PPR genes were identified in grape and divided into two subfamilies. Subcellular localization predicted that this gene family mainly functions in chloroplasts, nucleus, and mitochondria. Protein-protein interaction prediction indicated that there may be interaction between VvPPR44,53 and VvPPR44. The promoter region of VvPPR gene family contained various cis-acting elements, which were related to light and hormone. Expression pattern analysis showed that the VvPPR gene family was highly expressed in grape leaves, buds and carpel tissues. qRT-PCR results showed that the expression of VvPPR genes in roots was higher than stems and leaves under NAA, SA, ABA, MeJA and GA3 treatments. VvPPR8 was significantly up-regulated after GA3 and MeJA treatment for 24 h, VvPPR53 was significantly up-regulated after SA, NAA, ABA and MeJA treatment. In addition, In grape leaves, VvPPR53 was up-regulated under PEG, Nacl and 4 â„ƒ treatments. These data indicate that VvPPR gene family members are responsive to hormones and abiotic stresses, and that there are some differences in the degree of response and expression in different grape tissues. This study provides a certain theoretical basis for grape resistance breeding.

Overexpression VaPYL9 improves cold tolerance in tomato by regulating key genes in hormone signaling and antioxidant enzyme.

BACKGROUND: Abscisic acid (ABA) has been reported in controlling plant growth and development, and particularly dominates a role in resistance to abiotic stress. The Pyrabactin Resistance1/PYR1-Like /Regulatory Components of ABA receptors (PYR1/PYL/RCAR) gene family, of which the PYL9 is a positive regulator related to stress response in ABA signaling transduction. Although the family has been identified in grape, detailed VaPYL9 function in cold stress remains unknown. RESULTS: In order to explore the cold tolerance mechanism in grape, VaPYL9 was cloned from Vitis amurensis. The subcellular localization showed that VaPYL9 was mainly expressed in the plasma membrane. Yeast two-hybrid (Y2H) showed VaPCMT might be a potential interaction protein of VaPYL9. Through the overexpression of VaPYL9 in tomatoes, results indicated transgenic plants had higher antioxidant enzyme activities and proline content, lower malondialdehyde (MDA) and H2O2 content, and improving the ability to scavenge reactive oxygen species than wild-type (WT). Additionally, ABA content and the ratio of ABA/IAA kept a higher level than WT. Quantitative real-time PCR (qRT-PCR) showed that VaPYL9, SlNCED3, SlABI5, and antioxidant enzyme genes (POD, SOD, CAT) were up-regulated in transgenic tomatoes. Transcriptome sequencing (RNA-seq) found that VaPYL9 overexpression caused the upregulation of key genes PYR/PYL, PYL4, MAPK17/18, and WRKY in transgenic tomatoes under cold stress. CONCLUSION: Overexpression VaPYL9 enhances cold resistance of transgenic tomatoes mediated by improving antioxidant enzymes activity, reducing membrane damages, and regulating key genes in plant hormones signaling and antioxidant enzymes.