Overexpression of a Fragaria vesca NAM, ATAF, and CUC (NAC) Transcription Factor Gene (FvNAC29) Increases Salt and Cold Tolerance in Arabidopsis thaliana.

The NAC (NAM, ATAF1/2, CUC2) family of transcription factors (TFs) is a vital transcription factor family of plants. It controls multiple parts of plant development, tissue formation, and abiotic stress response. We cloned the FvNAC29 gene from Fragaria vesca (a diploid strawberry) for this research. There is a conserved NAM structural domain in the FvNAC29 protein. The highest homology between FvNAC29 and PaNAC1 was found by phylogenetic tree analysis. Subcellular localization revealed that FvNAC29 is localized onto the nucleus. Compared to other tissues, the expression level of FvNAC29 was higher in young leaves and roots. In addition, Arabidopsis plants overexpressing FvNAC29 had higher cold and high-salinity tolerance than the wild type (WT) and unloaded line with empty vector (UL). The proline and chlorophyll contents of transgenic Arabidopsis plants, along with the activities of the antioxidant enzymes like catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) under 200 mM NaCl treatment or -8 °C treatment, were higher than those activities of the control. Meanwhile, malondialdehyde (MDA) and the reactive oxygen species (ROS) content were higher in the WT and UL lines. FvNAC29 improves transgenic plant resistance to cold and salt stress by regulating the expression levels of AtRD29a, AtCCA1, AtP5CS1, and AtSnRK2.4. It also improves the potential to tolerate cold stress by positively regulating the expression levels of AtCBF1, AtCBF4, AtCOR15a, and AtCOR47. These findings suggest that FvNAC29 may be related to the processes and the molecular mechanisms of F. vesca response to high-salinity stress and LT stress, providing a comprehensive understanding of the NAC TFs.

Overexpression of a 'Beta' MYB Factor Gene, VhMYB15, Increases Salinity and Drought Tolerance in Arabidopsis thaliana.

'Beta' is a hybrid of Vitis riparia L. and V. labrusca and has a strong ability to adapt to adverse growth environments and is mainly cultivated and used as a resistant rootstock. At present, the most extensively studied MYB TFs are R2R3-type, which have been found to be involved in plant growth, development, and stress response processes. In the present research, VhMYB15, a key transcription factor for abiotic stress tolerance, was screened by bioinformatics in 'Beta' rootstock, and its function under salinity and drought stresses was investigated. VhMYB15 was highly expressed in roots and mature leave under salinity and drought stresses. Observing the phenotype and calculating the survival rate of plants, it was found that VhMYB15-overexpressing plants exhibited relatively less yellowing and wilting of leaves and a higher survival rate under salinity and drought stresses. Consistent with the above results, through the determination of stress-related physiological indicators and the expression analysis of stress-related genes (AtSOS2, AtSOS3, AtSOS1, AtNHX1, AtSnRK2.6, AtNCED3, AtP5CS1, and AtCAT1), it was found that transgenic Arabidopsis showed better stress tolerance and stronger adaptability under salinity and drought stresses. Based on the above data, it was preliminarily indicated that VhMYB15 may be a key factor in salinity and drought regulation networks, enhancing the adaptability of 'Beta' to adverse environments.

The Transcription Factor MbWRKY46 in Malus baccata (L.) Borkh Mediate Cold and Drought Stress Responses.

The living environment of plants is not static; as such, they will inevitably be threatened by various external factors for their growth and development. In order to ensure the healthy growth of plants, in addition to artificial interference, the most important and effective method is to rely on the role of transcription factors in the regulatory network of plant responses to abiotic stress. This study conducted bioinformatics analysis on the MbWRKY46 gene, which was obtained through gene cloning technology from Malus baccata (L.) Borkh, and found that the MbWRKY46 gene had a total length of 1068 bp and encodes 355 amino acids. The theoretical molecular weight (MW) of the MbWRKY46 protein was 39.76 kDa, the theoretical isoelectric point (pI) was 5.55, and the average hydrophilicity coefficient was -0.824. The subcellular localization results showed that it was located in the nucleus. After conducting stress resistance studies on it, it was found that the expression of MbWRKY46 was tissue specific, with the highest expression level in roots and old leaves. Low temperature and drought had a stronger induction effect on the expression of this gene. Under low temperature and drought treatment, the expression levels of several downstream genes related to low temperature and drought stress (AtKIN1, AtRD29A, AtCOR47A, AtDREB2A, AtERD10, AtRD29B) increased more significantly in transgenic Arabidopsis. This indicated that MbWRKY46 gene can be induced to upregulate expression in Arabidopsis under cold and water deficient environments. The results of this study have a certain reference value for the application of M. baccata MbWRKY46 in low-temperature and drought response, and provide a theoretical basis for further research on its function in the future.

Overexpression of a Grape MYB Transcription Factor Gene VhMYB2 Increases Salinity and Drought Tolerance in Arabidopsis thaliana.

In viticulture, the highly resistant rootstock 'Beta' is widely used in Chinese grape production to avoid the effects of soil salinization and drought on grape growth. However, the mechanism of high resistance to abiotic stress in the 'Beta' rootstock is not clear. In this study, we demonstrated that VhMYB2 as a transcription factor made a significant contribution to salinity and drought stress, which was isolated from the 'Beta' rootstock. The coding sequence of the VhMYB2 gene was 858 bp, encoding 285 amino acids. The subcellular localization of VhMYB2 was located in the nucleus of tobacco epidermal cells. Moreover, RT-qPCR found that VhMYB2 was predominantly expressed in the mature leaf and root of the grape. Under salinity and drought stress, overexpressing VhMYB2 showed a higher resistant phenotype and survival rates in A. thaliana while the transgenic lines had a survival advantage by measuring the contents of proline, chlorophyll, and MDA, and activities of POD, SOD, and CAT, and expression levels of related stress response genes. The results reveal that VhMYB2 may be an important transcription factor regulating 'Beta' resistance in response to abiotic stress.

MxFRO4 confers iron and salt tolerance through up-regulating antioxidant capacity associated with the ROS scavenging.

Iron is involved in various metabolic pathways of plants. Stress from iron deficiency and toxicity in the soil adversely affects plant growth. Therefore, studying the mechanism of iron absorption and transport by plants is of important for resistance to iron stress and to increase crop yield. In this study, Malus xiaojinensis (a Fe-efficient Malus plant) was used as research material. A ferric reduction oxidase (FRO) family gene member was cloned and named MxFRO4. The MxFRO4 encoded a protein of 697 amino acid residues with a predicted molecular weight of 78.54 kDa and a theoretical isoelectric point of 4.90. A subcellular localization assay showed that the MxFRO4 protein was localized on the cell membrane. The expression of MxFRO4 was enriched in immature leaves and roots of M. xiaojinensis, and was strongly affected by low-iron, high-iron, and salt treatments. After introduction of MxFRO4 into Arabidopsis thaliana, the iron and salt stress tolerance of transgenic A. thaliana was greatly improved. Under exposure to low-iron and high-iron stresses, the primary root length, seedling fresh weight, contents of proline, chlorophyll, and iron, and iron(III) chelation activity of the transgenic lines were significantly increased compared with the wild type. The contents of chlorophyll and proline, and the activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase were significantly higher in transgenic A. thaliana overexpressing MxFRO4 under salt stress compared with the wild type, whereas the malondialdehyde content was decreased. These results suggest that MxFRO4 contributes to alleviating the effects of low-iron, high-iron, and salinity stresses in transgenic A. thaliana.

MbMYBC1, a M. baccata MYB transcription factor, contribute to cold and drought stress tolerance in transgenic Arabidopsis.

Cold and drought stress considerably suppress the development of plants. In this study, a new MYB (v-myb avian myeloblastosis viral)TF gene, MbMYBC1, was isolated from the M. baccata and located in nucleus. MbMYBC1 has a positive response to low temperature and drought stress. After being introduced into Arabidopsis thaliana, the physiological indicators of transgenic Arabidopsis had corresponding changes under these two stresses, the activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) increased, electrolyte leakage rate (EL) and the content of proline increased, but the content of chlorophyll decreased. In addition, its overexpression can also activate the downstream expression of AtDREB1A, AtCOR15a, AtERD10B and AtCOR47 related to cold stress and AtSnRK2.4, AtRD29A, AtSOD1and AtP5CS1 related to drought stress. Based on these results, we speculate that MbMYBC1 can respond to cold and hydropenia signals, and can be used in transgenic technology to improve plant tolerance to low temperature and drought stress.

Overexpression of a Fragaria vesca 1R-MYB Transcription Factor Gene (FvMYB114) Increases Salt and Cold Tolerance in Arabidopsis thaliana.

The MYB (v-MYB avian myeloblastosis viral oncogene homolog) transcription factor (TF) family has numerous members with complex and diverse functions, which play an indispensable role in regulating the response of plants to stress. In this study, a new 1R-MYB TF gene was obtained from Fragaria vesca (a diploid strawberry) by cloning technology and given a new name, FvMYB114. According to the subcellular localization results, FvMYB114 protein was a nuclear localization protein. Overexpression of FvMYB114 greatly enhanced the adaptability and tolerance of Arabidopsis thaliana to salt and low temperature. Under salt and cold stress, the transgenic plants had greater proline and chlorophyll contents and higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than the wild-type (WT) and unloaded-line (UL) A. thaliana. However, malondialdehyde (MDA) was higher in the WT and UL lines. These results suggested that FvMYB114 may be involved in regulating the response of A. thaliana to salt stress and cold stress. FvMYB114 can also promote the expression of genes, such as the genes AtSOS1/3, AtNHX1 and AtLEA3 related to salt stress and the genes AtCCA1, AtCOR4 and AtCBF1/3 related to cold stress, further improving the tolerance of transgenic plants to salt and cold stress.

Isolation and preliminary functional analysis of FvICE1, involved in cold and drought tolerance in Fragaria vesca through overexpression and CRISPR/Cas9 technologies.

Cold and drought stresses are serious problems of strawberry cultivation in temperate and subtropical regions. In the molecular regulation system of cold and drought stresses, ICE transcription factors (TFs) are crucial. In this research, the FvICE1 was isolated from Fragaria vesca 'Hawaii 4', a bioinformatics analysis was conducted, overexpression vector and CRISPR/cas9 vector were constructed. The results showed that FvICE1 was a member of the bHLH TF family, with a length of 1608 bp, encoding 535 amino acids, and its molecular formula was C2504H3987N745O811S22. By observing the fusion protein 35S-FvICE1-GFP, it was found that FvICE1 was a nuclear protein. The qRT-PCR results demonstrated that FvICE1 was significantly upregulated in different tissues of Fragaria vesca after cold, drought, salt and heat treatments. The wild type (WT) strawberry was selected as the control group, FvICE1-overexpression strawberries showed high tolerance to cold and drought treatments at the phenotypic and physiological levels. On the contrary, fvice1 mutant strawberries obtained by CRISPR/cas9 editing technology had lower tolerance to cold and drought treatments. Moreover, the expression of FvCBF1, FvCBF2, FvCBF3, FvCOR413, FvRD22 and FvKIN1 was positively regulated in the FvICE1-overexpression strawberries and inhibited in fvice1 mutant strawberries. Overall, the current results suggested that FvICE1 functioned as a positively regulator of cold and drought resistances.

MbICE1 Confers Drought and Cold Tolerance through Up-Regulating Antioxidant Capacity and Stress-Resistant Genes in Arabidopsis thaliana.

Malus baccata (L.) Borkh is an apple rootstock with good drought and cold resistance. The ICE gene is a key factor in the molecular mechanisms of plant drought and cold resistance. In the present research, the function of drought- and cold-induced MbICE1 of Malus baccata was investigated in Arabidopsis. According to GFP fluorescence images, MbICE1 was determined to be a nuclear protein. The MbICE1 was transferred to Arabidopsis, showing enhanced tolerance to drought and cold stresses. Under drought and cold treatments, the transgenic Arabidopsis had higher chlorophyll content and free proline content than WT plants, but the Malondialdehyde (MDA) content and electrolyte leakage (EL) were lower than those of WT plants. In addition, drought and cold led to a large accumulation of ROS (H2O2 and O2-) content in Arabidopsis, while overexpression of MbICE1 enhanced the antioxidant enzyme activity in Arabidopsis and improved the plant's resistance to stresses. Moreover, the accumulation of MbICE1 promoted the expression of AtCBF1, AtCBF2, AtCBF3, AtCOR15a, AtCOR47 and AtKIN1 genes in Arabidopsis. These data indicate that MbICE1 is a key regulator of drought and cold and can be used as a backup gene for breeding Malus rootstocks.

Isolation and Functional Analysis of VvWRKY28, a Vitis vinifera WRKY Transcription Factor Gene, with Functions in Tolerance to Cold and Salt Stress in Transgenic Arabidopsis thaliana.

The grape (Vitis vinifera L.) not only has a long history of cultivation, but also has rich nutritional value and high economic value. However, grapes often face many threats in the growth process. For example, low temperature and salt stress restrict the growth status, yield, and geographical distribution of grapes. WRKY, as one of the largest transcription factor (TF) families in plants, participates in the response of plants to stress. VvWRKY28, a new zinc finger type transcriptional regulator gene, was isolated from Beichun (V. vinifera × V.amurensis) in this study. From the subcellular localization results, it can be concluded that VvWRKY28 was localized in the nucleus. The expression of VvWRKY28 was enriched in leaves (young and mature leaves), and cold and high salt conditions can induce high expression of VvWRKY28. After being transferred into Arabidopsis, VvWRKY28 greatly improved the tolerance of Arabidopsis to low temperature and high salt and also changed many physiological and biochemical indicators of transgenic Arabidopsis to cope with cold and high salt stimulation. The content of malondialdehyde (MDA) was decreased, but for chlorophyll and proline, their content increased, and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were improved. In addition, under cold stress, binding with cis-acting elements promotes the expression of downstream genes related to cold stress (RAB18, COR15A, ERD10, PIF4, COR47, and ICS1). Moreover, it also plays an active role in regulating the expression of genes related to salt stress (NCED3, SnRK2.4, CAT2, SOD1, SOS2, and P5CS1) under salt stress. Therefore, these results provide evidence that VvWRKY28 may play a role in the process of plant cold and salt stress tolerance.

Overexpression of a Malus baccata CBF transcription factor gene, MbCBF1, Increases cold and salinity tolerance in Arabidopsis thaliana.

CBFs play a crucial role when plants are in adverse environmental conditions for growth. However, there are few reports on the role of CBF gene in stress responses of Malus plant. In this experiment, a new CBF TF was separated from M. baccata which was named MbCBF1. MbCBF1 protein was found to be localized in the nucleus after subcellular localization. Furthermore, the expression of MbCBF1 was highly accumulated in new leaves and roots due to the high influence of cold and high salt in M. baccata seedlings. After introducing MbCBF1 into A. thaliana, transgenic A. thaliana can better adapt to the living conditions of cold and high salt. The increased expression of MbCBF1 in A. thaliana also increased the contents of proline, remarkablely improved the activities of SOD, POD and CAT, but the content of MDA was decreased. Although the chlorophyll content also decreased, it decreased less in transgenic plants. In short, above date showed that MbCBF1 has a positive effect on improving A. thaliana cold and high salt tolerance. MbCBF1 can regulate the expression of its downstream gene in transgenic lines, up-regulate the expression of key genes COR15a, RD29a/bandCOR6.6/47 related to low temperature under cold conditions and NCED3, CAT1, P5CS1, RD22, DREB2A,PIF1/4, SOS1 and SnRK2.4 related to salt stress under high salt conditions, so as to further improve the adaptability and tolerance of the transgenic plants to low temperature and high salt environment.

Isolation and Functional Analysis of MbCBF2, a Malus baccata (L.) Borkh CBF Transcription Factor Gene, with Functions in Tolerance to Cold and Salt Stress in Transgenic Arabidopsis thaliana.

CBF transcription factors (TFs) are key regulators of plant stress tolerance and play an integral role in plant tolerance to adverse growth environments. However, in the current research situation, there are few reports on the response of the CBF gene to Begonia stress. Therefore, this experiment investigated a novel CBF TF gene, named MbCBF2, which was isolated from M. baccata seedlings. According to the subcellular localization results, the MbCBF2 protein was located in the nucleus. In addition, the expression level of MbCBF2 was higher in new leaves and roots under low-temperature and high-salt induction. After the introduction of MbCBF2 into Arabidopsis thaliana, the adaptability of transgenic A. thaliana to cold and high-salt environments was significantly enhanced. In addition, the high expression of MbCBF2 can also change many physiological indicators in transgenic A. thaliana, such as increased chlorophyll and proline content, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity, and reduced malondialdehyde (MDA) content. Therefore, it can be seen from the above results that MbCBF2 can positively regulate the response of A. thaliana to low-temperature and osmotic stress. In addition, MbCBF2 can also regulate the expression of its downstream genes in transgenic lines. It can not only positively regulate the expression of the downstream key genes AtCOR15a, AtERD10, AtRD29a/b and AtCOR6.6/47, related to cold stress at low temperatures, but can also positively regulate the expression of the downstream key genes AtNCED3, AtCAT1, AtP5CS, AtPIF1/4 and AtSnRK2.4, related to salt stress. That is, the overexpression of the MbCBF2 gene further improved the adaptability and tolerance of transgenic plants to low-temperature and high-salt environments.

Overexpression of a Fragaria vesca MYB Transcription Factor Gene (FvMYB82) Increases Salt and Cold Tolerance in Arabidopsis thaliana.

The MYB transcription factor (TF) family is one of the largest transcription families in plants, which is widely involved in the responses to different abiotic stresses, such as salt, cold, and drought. In the present study, a new MYB TF gene was cloned from Fragaria vesca (a diploid strawberry) and named FvMYB82. The open reading frame (ORF) of FvMYB82 was found to be 960 bp, encoding 319 amino acids. Sequence alignment results and predictions of the protein structure indicated that the FvMYB82 contained the conserved R2R3-MYB domain. Subcellular localization analysis showed that FvMYB82 was localized onto the nucleus. Furthermore, the qPCR showed that the expression level of FvMYB82 was higher in new leaves and roots than in mature leaves and stems. When dealing with different stresses, the expression level of FvMYB82 in F. vesca seedlings changed markedly, especially for salt and cold stress. When FvMYB82 was introduced into Arabidopsis thaliana, the tolerances to salt and cold stress of FvMYB82-OE A. thaliana were greatly improved. When dealt with salt and cold treatments, compared with wild-type and unloaded line (UL) A. thaliana, the transgenic lines had higher contents of proline and chlorophyll, as well as higher activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). However, the transgenic A. thaliana had lower level of malondialdehyde (MDA) and electrolytic leakage (EL) than wild-type and UL A. thaliana under salt and cold stress. Meanwhile, FvMYB82 can also regulate the expression of downstream genes associated with salt stress (AtSnRK2.4, AtSnRK2.6, AtKUP6, and AtNCED3) and cold stress (AtCBF1, AtCBF2, AtCOR15a, and AtCOR78). Therefore, these results indicated that FvMYB82 probably plays an important role in the response to salt and cold stresses in A. thaliana by regulating downstream related genes.

Overexpression of MxbHLH18 Increased Iron and High Salinity Stress Tolerance in Arabidopsis thaliana.

In the life cycle of apple, it will suffer a variety of abiotic stresses, such as iron stress and salt stress. bHLH transcription factors (TFs) play an indispensable role in the response of plants to stress. In this study, a new bHLH gene named MxbHLH18 was separated from Malus xiaojinensis. According to the results of subcellular localization, MxbHLH18 was localized in the nucleus. Salt stress and iron stress affected the expression of MxbHLH18 in Malus xiaojinensis seedlings to a large extent. Due to the introduction of MxbHLH18, the resistance of Arabidopsis thaliana to salt, high iron and low iron was significantly enhanced. Under the environmental conditions of high iron and low iron, the overexpression of MxbHLH18 increased many physiological indexes of transgenic Arabidopsis compared to wild type (WT), such as root length, fresh weight and iron content. The high level expression of MxbHLH18 in transformed Arabidopsis thaliana can not only increased the content of chlorophyll and proline, as well as increasing the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT); it also reduced the content of malondialdehyde (MDA), which was more obvious under high salt conditions. In addition, the relative conductivity, H2O2 content and O2- content in transgenic Arabidopsis decreased under salt stress. Meanwhile, MxbHLH18 can also regulate the expression of downstream genes associated with salt stress (AtCBF1/2/3, AtKIN1 and AtCOR15a/b) and iron stress (AtIRT1, AtFRO2, AtNAS2, ATACT2, AtZIF1 and AtOPT3). Therefore, MxbHLH18 can actively promote the adaptability of plants to the growth environment of salt and low and/or iron.

Molecular Cloning and Characterization of MbMYB108, a Malus baccata MYB Transcription Factor Gene, with Functions in Tolerance to Cold and Drought Stress in Transgenic Arabidopsis thaliana.

The MYB transcription factor (TF) family is one of the largest transcription families in plants, which is widely involved in the responses of plants to biotic and abiotic stresses, as well as plant growth, development, and metabolic regulation. In the present study, a new MYB TF gene, MbMYB108, from Malus baccata (L.) Borkh, was identified and characterized. The open reading frame (ORF) of MbMYB108 was found to be 903 bp, encoding 300 amino acids. Sequence alignment results and predictions of the protein structure indicated that the MbMYB108 protein contained the conserved MYB domain. Subcellular localization showed that MbMYB108 was localized to the nucleus. The expression of MbMYB108 was enriched in young and mature leaves, and was highly affected by cold and drought treatments in M. baccata seedlings. When MbMYB108 was introduced into Arabidopsis thaliana, it greatly increased the cold and drought tolerances in the transgenic plant. Increased expression of MbMYB108 in transgenic A. thaliana also resulted in higher activities of peroxidase (POD) and catalase (CAT), higher contents of proline and chlorophyll, while malondialdehyde (MDA) content and relative conductivity were lower, especially in response to cold and drought stresses. Therefore, these results suggest that MbMYB108 probably plays an important role in the response to cold and drought stresses in A. thaliana by enhancing the scavenging capability for reactive oxygen species (ROS).

Effects of biodegradable films on melon quality and substrate environment in solar greenhouse.

With the increase in plastic pollution of farmland substrate, biodegradable mulch film research has become a hotspot. However, the degradation rate of biodegradable plastic film over the entire crop growth period is still unclear, as well as its impact on crop growth and product quality. Here, several properties of two kinds of composite biodegradable mulch films, PBAT/PLA-[S1] and PBAT/lignin-[S2], are studied with polyethylene-[PE] and uncovered substrate (CK) as controls. We tested the differences in morphological characterization, physical properties, and weight loss rate of the plastic films, as well as the effects of the different plastic films on melon yield and quality, substrate temperature and humidity, physical and chemical properties of the substrate, and substrate fungal species composition. Compared to PE, biodegradable plastic films S1 and S2 increased substrate temperature and the net photosynthetic rate of leaves. The results of substrate 18 s rDNA assay of CK, PE, S1, and S2 after 80 days of treatment and pre-treatment showed that a total of 12 fungal phylum, with 317 fungal genera were found, in which Ascomycota as the main phyla and Penicillium as the main genera. Compared with PE, the S2 treatment significantly increased the single fruit weight, central sugar content and soluble sugar of melon by 225.35 g, 1.26%, and 0.68%, respectively (p < 0.05). When buried for 240 d, the weight loss rate of S2 was significantly increased by 86.08% compared with PE (p < 0.05). From these results, we extrapolated that covering the substrate with the most biodegradable film, PBAT/lignin composite (10 µm), improved the yield and fruit quality of the melon in winter greenhouse production.

Overexpression of a Malus baccata MYB Transcription Factor Gene MbMYB4 Increases Cold and Drought Tolerance in Arabidopsis thaliana.

In the natural environment, plants often face unfavorable factors such as drought, cold, and freezing, which affect their growth and yield. The MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor family is widely involved in plant responses to biotic and abiotic stresses. In this study, Malus baccata (L.) Borkh was used as the research material, and a gene MbMYB4 of the MYB family was cloned from it. The open reading frame (ORF) of MbMYB4 was found to be 762 bp, encoding 253 amino acids; sequence alignment results and predictions of the protein structure indicated that the MbMYB4 protein contained the conserved MYB domain. Subcellular localization showed that MbMYB4 was localized in the nucleus. In addition, the use of quantitative real-time PCR (qPCR) technology found that the expression of MbMYB4 was enriched in the young leaf and root, and it was highly affected by cold and drought treatments in M. baccata seedlings. When MbMYB4 was introduced into Arabidopsis thaliana, it greatly increased the cold and drought tolerance in the transgenic plant. Under cold and drought stresses, the proline and chlorophyll content, and peroxidase (POD) and catalase (CAT) activities of transgenic A. thaliana increased significantly, and the content of malondialdehyde (MDA) and the relative conductivity decreased significantly, indicating that the plasma membrane damage of transgenic A. thaliana was lesser. Therefore, the overexpression of the MbMYB4 gene in A. thaliana can enhance the tolerance of transgenic plants to cold and drought stresses.

Overexpression of a Malus baccata NAC Transcription Factor Gene MbNAC25 Increases Cold and Salinity Tolerance in Arabidopsis.

NAC (no apical meristem (NAM), Arabidopsis thaliana transcription activation factor (ATAF1/2) and cup shaped cotyledon (CUC2)) transcription factors play crucial roles in plant development and stress responses. Nevertheless, to date, only a few reports regarding stress-related NAC genes are available in Malus baccata (L.) Borkh. In this study, the transcription factor MbNAC25 in M. baccata was isolated as a member of the plant-specific NAC family that regulates stress responses. Expression of MbNAC25 was induced by abiotic stresses such as drought, cold, high salinity and heat. The ORF of MbNAC25 is 1122 bp, encodes 373 amino acids and subcellular localization showed that MbNAC25 protein was localized in the nucleus. In addition, MbNAC25 was highly expressed in new leaves and stems using real-time PCR. To analyze the function of MbNAC25 in plants, we generated transgenic Arabidopsis plants that overexpressed MbNAC25. Under low-temperature stress (4 °C) and high-salt stress (200 mM NaCl), plants overexpressing MbNAC25 enhanced tolerance against cold and drought salinity conferring a higher survival rate than that of wild-type (WT). Correspondingly, the chlorophyll content, proline content, the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were significantly increased, while malondialdehyde (MDA) content was lower. These results indicated that the overexpression of MbNAC25 in Arabidopsis plants improved the tolerance to cold and salinity stress via enhanced scavenging capability of reactive oxygen species (ROS).

Genome-based breeding approaches in major vegetable crops.

Vegetable crops are major nutrient sources for humanity and have been well-cultivated since thousands of years of domestication. With the rapid development of next-generation sequencing and high-throughput genotyping technologies, the reference genome of more than 20 vegetables have been well-assembled and published. Resequencing approaches on large-scale germplasm resources have clarified the domestication and improvement of vegetable crops by human selection; its application on genetic mapping and quantitative trait locus analysis has led to the discovery of key genes and molecular markers linked to important traits in vegetables. Moreover, genome-based breeding has been utilized in many vegetable crops, including Solanaceae, Cucurbitaceae, Cruciferae, and other families, thereby promoting molecular breeding at a single-nucleotide level. Thus, genome-wide SNP markers have been widely used, and high-throughput genotyping techniques have become one of the most essential methods in vegetable breeding. With the popularization of gene editing technology research on vegetable crops, breeding efficiency can be rapidly increased, especially by combining the genomic and variomic information of vegetable crops. This review outlines the present genome-based breeding approaches used for major vegetable crops to provide insights into next-generation molecular breeding for the increasing global population.

The MADS-Box Gene CsSHP Participates in Fruit Maturation and Floral Organ Development in Cucumber.

Cucumber is an important vegetable crop bearing fleshy pepo fruit harvested immature. Fruits left unpicked in time during summer production, as well as unfavorable environmental conditions during post-harvest shelf, will cause cucumber fruits to turn yellow and ripen, and thus impair the market value. Identification of maturity-related genes is of great agricultural and economic importance for cucumber production. Here, we isolated and characterized a MADS-box gene, Cucumis sativus SHATTERPROOF (CsSHP) in cucumber. Expression analysis indicated that CsSHP was specifically enriched in reproductive organs including stamens and carpels. Ectopic expression of CsSHP was unable to rescue the indehiscence silique phenotype of shp1 shp2 mutant plant in Arabidopsis. Instead, overexpression of CsSHP resulted in early flowering, precocious phenotypes, and capelloid organs in wild-type Arabidopsis. Biochemical analysis indicated that CsSHP directly interacted with cucumber SEPALLATA (SEP) proteins. CsSHP expression increased significantly during the yellowing stage of cucumber ripening, and was induced by exogenous application of abscisic acid (ABA). Therefore, CsSHP may participate in fruit maturation through the ABA pathway and floral organ specification via interaction with CsSEPs to form protein complex in cucumber.