RESUMO
Xenia, the phenomenon in which the pollen genotype directly affects the phenotypic characteristics of maternal tissues (i.e., fruit ripening), has applications in crop production and breeding. However, the underlying molecular mechanism has yet to be elucidated. Here, we investigated whether mobile mRNAs from the pollen affect the ripening and quality-related characteristics of the fruit using cross-pollination between distinct Malus domestica (apple) cultivars. We demonstrated that hundreds of mobile mRNAs originating from the seeds are delivered to the fruit. We found that the movement of one of these mRNAs, ACC oxidase 3 (MdACO3), is coordinated with fruit ripening. Salicylic acid treatment, which can cause plasmodesmal closure, blocks MdACO3 movement, indicating that MdACO3 transcripts may move through the plasmodesmata. To assess the role of mobile MdACO3 transcripts in apple fruit, we created MdACO3-GFP-expressing apple seeds using MdACO3-GFP-overexpressing pollen for pollination and showed that MdACO3 transcripts in the transgenic seeds move to the flesh, where they promote fruit ripening. Furthermore, we demonstrated that MdACO3 can be transported from the seeds to fruit in the fleshy-fruited species tomato and strawberry. These results underscore the potential of mobile mRNAs from seeds to influence fruit characteristics, providing an explanation for the xenia phenomenon. Notably, our findings highlight the feasibility of leveraging diverse pollen genomic resources, without resorting to genome editing, to improve fruit quality.
Assuntos
Aminoácido Oxirredutases , Frutas , Malus , RNA Mensageiro , Sementes , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Malus/genética , Malus/crescimento & desenvolvimento , Malus/metabolismo , Malus/enzimologia , Frutas/genética , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , PolinizaçãoRESUMO
Flavonols are widely synthesized throughout the plant kingdom, playing essential roles in plant physiology and providing unique health benefits for humans. Their glycosylation plays significant role in improving their stability and solubility, thus their accumulation and function. However, the genes encoding the enzymes catalyze this glycosylation remain largely unknown in apple. This study utilized a combination of methods to identify genes encoding such enzymes. Initially, candidate genes were selected based on their potential to encode UDP-dependent glycosyltransferases (UGTs) and their expression patterns in response to light induction. Subsequently, through testing the in vitro enzyme activity of the proteins produced in Escherichia coli cells, four candidates were confirmed to encode a flavonol 3-O-galactosyltransferase (UGT78T6), flavonol 3-O-glucosyltransferase (UGT78S1), flavonol 3-O-xylosyltransferase/arabinosyltransferase (UGT78T5), and flavonol 3-O-rhamnosyltransferase (UGT76AE22), respectively. Further validation of these genes' functions was conducted by modulating their expression levels in stably transformed apple plants. As anticipated, a positive correlation was observed between the expression levels of these genes and the content of specific flavonol glycosides corresponding to each gene. Moreover, overexpression of a flavonol synthase gene, MdFLS, resulted in increased flavonol glycoside content in apple roots and leaves. These findings provide valuable insights for breeding programs aimed at enriching apple flesh with flavonols and for identifying flavonol 3-O-glycosyltransferases of other plant species.
Assuntos
Flavonóis , Glicosídeos , Glicosiltransferases , Malus , Proteínas de Plantas , Malus/genética , Malus/metabolismo , Malus/enzimologia , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Flavonóis/metabolismo , Flavonóis/biossíntese , Glicosídeos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , GlicosilaçãoRESUMO
Wall-associated kinases (WAKs) have been determined to recognize pathogenic signals and initiate plant immune responses. However, the roles of the family members in host resistance against Valsa canker, a serious fungal disease of apples and pears, are largely unknown. Here, we identified MbWAK1 in Malus baccata, a resistant germplasm differentially expressed during infection by Valsa mali (Vm). Over-expression of MbWAK1 enhanced the Valsa canker resistance of apple and pear fruits and 'Duli-G03' (Pyrus betulifolia) suspension cells. A large number of phloem, cell wall, and lipid metabolic process-related genes were differentially expressed in overexpressed suspension cell lines in response to Valsa pyri (Vp) signals. Among these, the expression of xyloglucan endotransglucosylase/hydrolase (XTH) gene PbeXTH1 and sieve element occlusion B-like (SEOB) gene PbeSEOB1 were significantly inhibited. Transient expression of PbeXTH1 or PbeSEOB1 compromised the expressional induction of MbWAK1 and the resistance contributed by MbWAK1. In addition, PbeXTH1 and PbeSEOB1 suppressed the immune response induced by MbWAK1. Our results enriched the molecular mechanisms for MbWAK1 against Valsa canker and resistant breeding.
Assuntos
Resistência à Doença , Regulação da Expressão Gênica de Plantas , Malus , Doenças das Plantas , Proteínas de Plantas , Pyrus , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Resistência à Doença/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pyrus/genética , Pyrus/microbiologia , Malus/genética , Malus/microbiologia , Malus/imunologia , Malus/enzimologia , Parede Celular/metabolismoRESUMO
The utilization of xylanase in juice clarification is contingent upon its stability within acidic environments. We generated a mutant xynA-1 by substituting the N-terminal segment of the recombinant xylanase xynA to investigate the correlation between the N-terminal region of xylanase and its acid stability. The enzymatic activity of xynA-1 was found to be superior under acidic conditions (pH 5.0). It exhibited enhanced acid stability, surpassing the residual enzyme activity values of xynA at pH 4.0 (53.07 %), pH 4.5 (69.8 %), and pH 5.0 (82.4 %), with values of 60.16 %, 77.74 %, and 87.3 %, respectively. Additionally, the catalytic efficiency of xynA was concurrently improved. Through molecular dynamics simulation, we observed that N-terminal shortening induced a reduction in motility across most regions of the protein structure while enhancing its stability, particularly Lys131-Phe146 and Leu176-Gly206. Furthermore, the application of treated xynA-1 in the process of apple juice clarification led to a significant increase in clarity within a short duration of 20 min at 35 °C while ensuring the quality of the apple juice. This study not only enhances the understanding of the N-terminal region of xylanase but also establishes a theoretical basis for augmenting xylanase resources employed in fruit juice clarification.
Assuntos
Endo-1,4-beta-Xilanases , Estabilidade Enzimática , Sucos de Frutas e Vegetais , Malus , Proteínas Recombinantes , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Concentração de Íons de Hidrogênio , Malus/química , Malus/enzimologia , Simulação de Dinâmica MolecularRESUMO
Protein S-acyltransferases (PATs) are a category of eukaryotic transmembrane proteins that mediate the S-acylation of their target proteins. S-acylation, commonly known as palmitoylation, is a reversible protein modification that regulates the membrane association and function of target proteins. However, the functions and mechanisms of PATs in apple (Malus domestica) remain poorly understood. In this study, an MdPAT family member, MdPAT16, was identified and shown to have palmitoyltransferase activity. We demonstrated that this gene responds to salt stress and that its expression improves plant salt stress resistance. In addition, its overexpression significantly promotes the accumulation of soluble sugars. The same phenotypes were observed in transgenic tissue culture seedlings, transgenic roots, and Arabidopsis thaliana that ectopically expressed MdPAT16. MdPAT16 was shown to interact with MdCBL1 and stabilize MdCBL1 protein levels through palmitoylation. The N-terminal sequence of MdCBL1 contains a palmitoylation site, and its N-terminal deletion led to changes in MdCBL1 protein stability and subcellular localization. The phenotypes of MdCBL1 transgenic roots and transiently injected apple fruits were fully consistent with the sugar accumulation phenotype of MdPAT16. Mutation of the palmitoylation site interfered with this phenotype. These findings suggest that MdPAT16 palmitoylates its downstream target proteins, improving their stability. This may be a missing link in the plant salt stress response pathway and have an important impact on fruit quality.
Assuntos
Aciltransferases/metabolismo , Frutas/metabolismo , Malus/enzimologia , Proteínas de Plantas/metabolismo , Açúcares/metabolismo , Frutas/enzimologia , Malus/metabolismo , Redes e Vias Metabólicas , Proteínas de Plantas/fisiologia , Tolerância ao SalRESUMO
A plant pathway that initiates with the formation of citramalate from pyruvate and acetyl-CoA by citramalate synthase (CMS) is shown to contribute to the synthesis of α-ketoacids and important odor-active esters in apple (Malus × domestica) fruit. Microarray screening led to the discovery of a gene with high amino acid similarity to 2-isopropylmalate synthase (IPMS). However, functional analysis of recombinant protein revealed its substrate preference differed substantially from IPMS and was more typical of CMS. MdCMS also lacked the regulatory region present in MdIPMS and was not sensitive to feedback inhibition. 13C-acetate feeding of apple tissue labeled citramalate and α-ketoacids in a manner consistent with the presence of the citramalate pathway, labeling both straight- and branched-chain esters. Analysis of genomic DNA (gDNA) revealed the presence of two nearly identical alleles in "Jonagold" fruit (MdCMS_1 and MdCMS_2), differing by two nonsynonymous single-nucleotide polymorphisms (SNPs). The mature proteins differed only at amino acid 387, possessing either glutamine387 (MdCMS_1) or glutamate387 (MdCMS_2). Glutamate387 was associated with near complete loss of activity. MdCMS expression was fruit-specific, increasing severalfold during ripening. The translated protein product was detected in ripe fruit. Transient expression of MdCMS_1 in Nicotiana benthamiana induced the accumulation of high levels of citramalate, whereas MdCMS_2 did not. Domesticated apple lines with MdCMS isozymes containing only glutamate387 produced a very low proportion of 2-methylbutanol- and 2-methylbutanoate (2MB) and 1-propanol and propanoate (PROP) esters. The citramalate pathway, previously only described in microorganisms, is shown to function in ripening apple and contribute to isoleucine and 2MB and PROP ester biosynthesis without feedback regulation.
Assuntos
Vias Biossintéticas/genética , Ésteres/metabolismo , Malatos/metabolismo , Proteínas de Plantas/metabolismo , Aminoácidos/metabolismo , Frutas/enzimologia , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Isoleucina/metabolismo , Malus/enzimologia , Malus/metabolismo , Nicotiana/genéticaRESUMO
In this study we describe the crystal structures of the apoform, the binary and the ternary complexes of a double bond reductase from Malus domestica L. (MdDBR) and explore a range of potential substrates. The overall fold of MdDBR is similar to that of the medium chain reductase/dehydrogenase/zinc-dependent alcohol dehydrogenase-like family. Structural comparison of MdDBR with Arabidopsis thaliana DBR (AtDBR), Nicotiana tabacum DBR (NtDBR) and Rubus idaeus DBR (RiDBR) allowed the identification of key amino acids involved in cofactor and ligands binding and shed light on how these residues may guide the orientation of the substrates. The enzyme kinetic for the substrate trans-4-phenylbuten-2-one has been analyzed, and MdDBR activity towards a variety of substrates was tested. This enzyme has been reported to be involved in the phenylpropanoid pathway where it would catalyze the NADPH-dependent reduction of the α, ß-unsaturated double bond of carbonyl metabolites. Our study provides new data towards the identification of MdDBR natural substrate and the biosynthetic pathway where it belongs. Furthermore, the originally proposed involvement in dihydrochalcone biosynthesis in apple must be questioned.
Assuntos
Apoproteínas/química , Butanonas/química , Malus/química , NADP/química , Oxirredutases/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Arabidopsis/química , Arabidopsis/enzimologia , Sítios de Ligação , Butanonas/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Malus/enzimologia , Modelos Moleculares , NADP/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rubus/química , Rubus/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica , Nicotiana/química , Nicotiana/enzimologiaRESUMO
The effects of treatment with melatonin on ripening of 'Fuji' apples during storage at 1 °C for 56 d were investigated. The apples were harvested at the commercial ripening stage and treated with 1 mmol L-1 melatonin. Compared with the control, melatonin treated apples had significant reduced ethylene production (28 d-56 d) and weight loss (14 d-56 d) during storage (p < 0.05). Also, the melatonin treatment maintained better apple skin structure throughout storage. The reduced ethylene production was regulated by the decreased expressions of MdACO1, MdACS1, MdAP2.4 and MdERF109, based on RNA-Seq analysis, which was validated using qRT-PCR analysis. Moreover, the activity of 3 enzymes, including peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), were significantly increased in melatonin treated fruit (p < 0.05). Taken together, this study highlights the inhibitory effects of melatonin in ethylene biosynthesis and factors influencing postharvest quality in apple.
Assuntos
Etilenos/biossíntese , Qualidade dos Alimentos , Armazenamento de Alimentos/métodos , Frutas/efeitos dos fármacos , Malus/efeitos dos fármacos , Malus/metabolismo , Melatonina/farmacologia , Malus/enzimologiaRESUMO
Selenium is an essential trace element that improves fruit quality and nutritional value. However, the effect of sodium selenite on apple quality and its relative sucrose metabolism activity remains unclear. In this study, we investigated the roles of selenite spraying, in improving Fuji apple quality and sucrose metabolism-related enzyme activity. Results showed that foliar spraying of sodium selenite significantly (P < 0.05) increased apple fruit yield and internal quality, but no significant effects on external quality. The apple yield, vitamin C content, sugar-acid ratio and total soluble sugar increased 4.4% to 11.7%, 4.68% to 20.86%, 3.07% to 31.57%, and 4.53% to 18.89%, respectively. Se content is 9.5-fold compared to the control. Significant correlations were observed between neutral invertase, sucrose synthase activity and sucrose phosphate synthase enzymes, and sucrose phosphate synthase enzyme was most crucial. Spraying sodium selenite of 100-150 mg/L could be appropriate for improving Fuji apple yield and quality.
Assuntos
Malus/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Selenito de Sódio/farmacologia , Sacarose/metabolismo , Ácido Ascórbico/metabolismo , Qualidade dos Alimentos , Frutas/efeitos dos fármacos , Frutas/enzimologia , Frutas/metabolismo , Glucosiltransferases/metabolismo , Malus/enzimologia , Malus/metabolismo , beta-Frutofuranosidase/metabolismoRESUMO
We investigated the inhibitory effect and binding mechanism of four selected compounds (ascorbic acid, l-cysteine, glutathione, and citric acid) on membrane-bound polyphenol oxidases (mPPO) using spectroscopic and molecular docking techniques. Kinetic analysis demonstrated that these inhibitors reversibly inhibited the mPPO activity. Fluorescence spectroscopy revealed that the intrinsic fluorescence intensity of mPPO was quenched by inhibitors with a single class of the inhibition site on mPPO. Amino acid residues His 180, His 201, His 366, Cys 184, Glu 328, and Asn 333 were the important binding sites in the active center. These sites were identified using molecular docking techniques. Our findings suggested that the inhibitors were allosterically bound to the active center of mPPO through hydrogen bonds and ion contacts. This study provides new insights into the active site residues responsible for catalyzing mPPO and provides applicable information about the design of mPPO inhibitors.
Assuntos
Catecol Oxidase/metabolismo , Malus/enzimologia , Simulação de Acoplamento Molecular , Proteínas de Plantas/metabolismo , Regulação Alostérica , Ácido Ascórbico/química , Ácido Ascórbico/metabolismo , Sítios de Ligação , Catecol Oxidase/antagonistas & inibidores , Cisteína/química , Cisteína/metabolismo , Glutationa/química , Glutationa/metabolismo , Cinética , Proteínas de Plantas/antagonistas & inibidores , Espectrometria de FluorescênciaRESUMO
Calcium-dependent protein kinases (CDPKs) are important calcium receptors, which play a crucial part in the process of sensing and decoding intracellular calcium signals during plant development and adaptation to various environmental stresses. In this study, a CDPK gene MdCPK1a, was isolated from apple (Malus×domestica) which contains 1701bp nucleotide and encodes a protein of 566 amino acid residues, and contains the conserved domain of CDPKs. The transient expression and western blot experiment showed that MdCPK1a protein was localized in the nucleus and cell plasma membrane. Ectopic expression of MdCPK1a in Nicotiana benthamiana increased the resistance of the tobacco plants to salt and cold stresses. The mechanism of MdCPK1a regulating cold resistance was further investigated. The overexpressed MdCPK1a tobacco plants had higher survival rates and longer root length than wild type (WT) plants under cold stress, and the electrolyte leakages (EL), the content of malondialdehyde (MDA) and reactive oxygen species (ROS) were lower, and accordingly, antioxidant enzyme activities, such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were higher, suggesting the transgenic plants suffered less chilling injury than WT plants. Moreover, the transcript levels of ROS-scavenging and stress-related genes were higher in the transgenic plants than those in WT plants whether under normal conditions or cold stress. The above results suggest that the improvement of cold tolerance in MdCPK1a-overexpressed plants was due to scavenging ROS accumulation and modulating the expression of stress-related genes.
Assuntos
Regulação da Expressão Gênica de Plantas , Malus/enzimologia , Malus/genética , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Adaptação Fisiológica/genética , Antioxidantes/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Catalase/metabolismo , Membrana Celular/metabolismo , Temperatura Baixa , Biologia Computacional , Fases de Leitura Aberta , Peroxidase/metabolismo , Fenótipo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Domínios Proteicos , Proteínas Quinases/genética , Estresse Fisiológico/genética , Superóxido Dismutase/metabolismo , Nicotiana/metabolismoRESUMO
COP1, an important RING ubiquitin ligase E3, is a molecular switch for light regulation in plant development. As an interacting protein of COP1, CIP8 contains a RING-H2 domain, but its biological function is unclear. Here, the apple MdCIP8 was identified based on its homology with AtCIP8 in Arabidopsis. MdCIP8 was constitutively expressed at different levels in various apple tissues, and the expression level of MdCIP8 was not affected by light and dark conditions. MdCIP8 reversed the short hypocotyl phenotype of the cip8 mutant under light conditions. Furthermore, the yeast two-hybrid experiment showed that MdCIP8 interacted with the RING domain of MdCOP1 through its RING-H2 domain. MdCIP8-OX/cop1-4 exhibited the phenotype of the cop1-4 mutant, indicating that CIP8 acts upstream of COP1. In addition, an apple transient injection experiment showed that MdCIP8 inhibited anthocyanin accumulation in an MdCOP1-dependent pathway. Overall, our findings reveal that CIP8 plays an inhibitory role in the light-regulation responses of plants.
Assuntos
Antocianinas/metabolismo , Malus/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Hipocótilo/enzimologia , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/efeitos da radiação , Luz , Malus/enzimologia , Malus/crescimento & desenvolvimento , Malus/efeitos da radiação , Mutação , Proteínas de Plantas/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Enzymatic browning is one of the main problems faced by the food industry due to the enzyme polyphenol oxidase (PPO) provoking an undesirable color change in the presence of oxygen. Here, we report the evaluation of 10 different azamacrocyclic compounds with diverse morphologies as potential inhibitors against the activity of PPO, both in model and real systems. An initial screening of 10 ligands shows that all azamacrocyclic compounds inhibit to some extent the enzymatic browning, but the molecular structure plays a crucial role on the power of inhibition. Kinetic studies of the most active ligand (L2) reveal a S-parabolic I-parabolic noncompetitive inhibition mechanism and a remarkable inhibition at micromolar concentration (IC50 = 10 µM). Furthermore, L2 action has been proven on apple juice to significantly reduce the enzymatic browning.
Assuntos
Catecol Oxidase/antagonistas & inibidores , Inibidores Enzimáticos/química , Malus/enzimologia , Proteínas de Plantas/antagonistas & inibidores , Catecol Oxidase/química , Frutas/química , Frutas/enzimologia , Sucos de Frutas e Vegetais/análise , Cinética , Ligantes , Malus/química , Proteínas de Plantas/químicaRESUMO
The aerial parts of apple are protected against environmental stress by cuticular wax. Although it has been suggested that several long-chain acyl-CoA synthetases are involved in wax biosynthesis, the molecular pathway of apple cuticular wax biosynthesis remains unclear. In this study, an MdLACS4 protein with long-chain acyl-CoA synthetase activity was isolated from apple. The MdLACS4 gene was highly expressed in pericarp, stem, and mature leaf tissues. Ectopic expression of MdLACS4 in Arabidopsis induced early flowering. Compared with wild-type plants, MdLACS4 transgenic Arabidopsis exhibited lower water loss rates, reduced epidermal permeability, increased cuticular wax in stems and leaves, and altered cuticular ultrastructure. Furthermore, the accumulation of cuticular wax enhanced the resistance of MdLACS4 transgenic plants to drought and salt stress. Finally, predicted protein functional interaction networks for LACS4 suggested that the molecular regulation pathway of MdLACS4 mediates wax biosynthesis in apple.
Assuntos
Coenzima A Ligases/fisiologia , Flores/crescimento & desenvolvimento , Malus/enzimologia , Proteínas de Plantas/fisiologia , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Coenzima A Ligases/genética , Sequência Conservada/genética , Flores/enzimologia , Cromatografia Gasosa-Espectrometria de Massas , Genes de Plantas/genética , Genes de Plantas/fisiologia , Liases/genética , Liases/fisiologia , Malus/genética , Microscopia Eletrônica de Varredura , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Estresse FisiológicoRESUMO
Apple fruit is covered by cuticle wax, which plays important roles protecting fruits from adverse environmental conditions. ß-Ketoacyl-CoA synthase (KCS) is the key rate-limiting enzyme in plant wax synthesis. In this study, we identified 28 KCS gene family members from apple (Malus × domestica Borkh.) by homology analysis. Multi-sequence alignment and phylogenetic analyses revealed that the 28 MdKCS genes were divided into four subgroups, including KCS1-like, FAE1-like, FDH-like, and CER6. A chromosomal localization analysis revealed that 27 apple KCS genes were located on 11 chromosomes, while MdKCS28 was localized to the unassembled genomic scaffold. Most of the MdKCS proteins were hydrophilic proteins and they had similar secondary and tertiary structures. The prediction of cis-acting elements of the MdKCS gene promoters suggested that the MdKCS genes may be widely involved in hormone signaling and the stress response. Furthermore, the quantitative real-time polymerase chain reaction results showed that eight MdKCS genes were highly expressed in the apple pericarp, and were significantly induced by drought, abscisic acid (ABA), and NaCl treatments. We transformed the MdKCS21 gene into apple calli, and found the MdKCS21 overexpressing transgenic apple calli exhibited higher tolerance to ABA treatment. Finally, the MdKCS proteins were localized to the endoplasmic reticulum and vacuolar membrane by confocal laser microscopy. This study established a foundation to further analyze the function of KCS genes and provided candidate genes for molecular improvement of wax content in apple.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase , Genoma de Planta , Malus , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Regulação da Expressão Gênica de Plantas , Estudo de Associação Genômica Ampla , Malus/classificação , Malus/enzimologia , Malus/genética , Filogenia , Proteínas de Plantas/genéticaRESUMO
Phytohormonal interactions are crucial for plant development. Auxin and cytokinin (CK) both play critical roles in regulating plant growth and development; however, the interaction between these two phytohormones is complex and not fully understood. Here, we isolated a wild apple (Malus sieversii Roem) GRETCHEN HAGEN3 (GH3) gene, MsGH3.5, encoding an indole-3-acetic acid (IAA)-amido synthetase. Overexpression of MsGH3.5 significantly reduced the free IAA content and increased the content of some IAA-amino acid conjugates, and MsGH3.5-overexpressing lines were dwarfed and produced fewer adventitious roots (ARs) than the control. This phenotype is consistent with the role of GH3 in conjugating excess free active IAA to amino acids in auxin homeostasis. Surprisingly, overexpression of MsGH3.5 significantly increased CK concentrations in the whole plant, and altered the expression of genes involved in CK biosynthesis, metabolism and signaling. Furthermore, exogenous CK application induced MsGH3.5 expression through the activity of the CK type-B response regulator, MsRR1a, which mediates the CK primary response. MsRR1a activated MsGH3.5 expression by directly binding to its promoter, linking auxin and CK signaling. Plants overexpressing MsRR1a also displayed fewer ARs, in agreement with the regulation of MsGH3.5 expression by MsRR1a. Taken together, we reveal that MsGH3.5 affects apple growth and development by modulating auxin and CK levels and signaling pathways. These findings provide insight into the interaction between the auxin and CK pathways, and might have substantial implications for efforts to improve apple architecture.
Assuntos
Citocininas/metabolismo , Ácidos Indolacéticos/metabolismo , Ligases/fisiologia , Malus/genética , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Ligases/metabolismo , Malus/enzimologia , Malus/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Plantas Geneticamente ModificadasRESUMO
Arbuscular mycorrhizal fungi (AMF) can form a symbiotic relationships with most terrestrial plants and play an important role in plant growth and adaptation to various stresses. To study the role of AMF in regulating drought resistance in apple, the effects of drought stress on Malus hupehensis inoculated with AMF were investigated. Inoculation of AMF enhanced apple plants growth. Mycorrhizal plants had higher total chlorophyll concentrations but lower relative electrolyte leakage under drought stress. Mycorrhizal plants increased net photosynthetic rate, stomatal conductance, and transpiration rate under drought stress, however, they showed lower inhibition in the quantum yield of PSII photochemistry. Mycorrhizal plants also had higher superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) enzyme activities under drought conditions. Thus, mycorrhizal plants had lower accumulated MDA, H2O2, and O2- than non-mycorrhizal seedlings. Total sugar and proline concentrations also significantly increased, helping maintain the osmotic balance. Furthermore, mitogen-activated protein kinase (MAPK) cascades, which participate in the regulation of responses of plants and microorganisms to biotic and abiotic stress, were up-regulated in apple plants and AMF during drought. We saw that there were at least two motifs that were identical in MAPK proteins and many elements that responded to hormones and stress from these MAPK genes. In summary, our results showed that mycorrhizal colonization enhanced apple drought tolerance by improving gas exchange capacity, increasing chlorophyll fluorescence parameters, creating a greater osmotic adjustment capacity, increasing scavenging of reactive oxygen species (ROS), and using MAPK signals for interactions between AMF and their apple plant hosts.
Assuntos
Secas , Regulação da Expressão Gênica de Plantas , Malus , Proteínas Quinases Ativadas por Mitógeno , Micorrizas , Estresse Fisiológico , Malus/enzimologia , Malus/genética , Malus/microbiologia , Proteínas Quinases Ativadas por Mitógeno/genética , Micorrizas/fisiologia , Estresse Fisiológico/genéticaRESUMO
Apple exocarp was used to investigate the effect of acibenzolar-S-methyl (ASM) and dehydroepiandrosterone (DHEA) treatments on reaction oxygen species (ROS) metabolism. The results indicated that ASM enhanced the hydrogen peroxide (H2O2) content, the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH). ASM also increased the contents of ascorbic acid (AsA), reduced glutathione (GSH) and nicotinamide ademine dinucleotidephosphate (NADPH), MdSOD and MdAPX expression, but decreased MdMDHAR and dehydroascorbate reductase (MdDHAR) expression. DHEA suppressed H2O2 accumulation and POD, APX, MDHAR, G6PDH activities, but increased SOD, CAT and GR activities compared to the control. ASM and DHEA treatments suppressed the contents of AsA, GSH and NADPH, and expression of MdSOD, MdAPX and MdMDHAR. These results suggest that DHEA treatment prevented ROS metabolism induced by ASM which showed the important role of G6PDH in maintaining redox homeostasis in apple exocarp.
Assuntos
Glucosefosfato Desidrogenase/metabolismo , Malus/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Ascorbato Peroxidases/metabolismo , Ácido Ascórbico/metabolismo , Catalase/metabolismo , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Peróxido de Hidrogênio/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Oxirredutases/metabolismo , Superóxido Dismutase/metabolismo , Tiadiazóis/metabolismoRESUMO
MYB transcription factors (TFs) have been demonstrated to play diverse roles in plant growth and development through interaction with basic helix-loop-helix (bHLH) TFs. MdbHLH33, an apple bHLH TF, has been identified as a positive regulator in cold tolerance and anthocyanin accumulation by activating the expressions of MdCBF2 and MdDFR. In the present study, a MYB TF MdMYB308L was found to also positively regulate cold tolerance and anthocyanin accumulation in apple. We found that MdMYB308L interacted with MdbHLH33 and enhanced its binding to the promoters of MdCBF2 and MdDFR. In addition, an apple RING E3 ubiquitin ligase MYB30-INTERACTING E3 LIGASE 1 (MdMIEL1) was identified to be an MdMYB308L-interacting protein and promoted the ubiquitination degradation of MdMYB308L, thus negatively regulated cold tolerance and anthocyanin accumulation in apple. These results suggest that MdMYB308L acts as a positive regulator in cold tolerance and anthocyanin accumulation in apple by interacting with MdbHLH33 and undergoes MdMIEL1-mediated protein degradation. The dynamic change in MYB-bHLH protein complex seems to play a key role in the regulation of plant growth and development.
Assuntos
Adaptação Fisiológica , Antocianinas , Malus , Proteínas de Plantas , Fatores de Transcrição , Adaptação Fisiológica/genética , Antocianinas/metabolismo , Regulação da Expressão Gênica de Plantas , Malus/enzimologia , Malus/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
MAIN CONCLUSION: Certain apple cultivars accumulate to high levels in their nectar and stigma exudate an acidic chitinase III protein that can protect against pathogens including fire blight disease causing Erwinia amylovora. To prevent microbial infections, flower nectars and stigma exudates contain various antimicrobial compounds. Erwinia amylovora, the causing bacterium of the devastating fire blight apple disease, is the model pathogen that multiplies in flower secretions and infects through the nectaries. Although Erwinia-resistant apples are not available, certain cultivars are tolerant. It was reported that in flower infection assay, the 'Freedom' cultivar was Erwinia tolerant, while the 'Jonagold' cultivar was susceptible. We hypothesized that differences in the nectar protein compositions lead to different susceptibility. Indeed, we found that an acidic chitinase III protein (Machi3-1) selectively accumulates to very high levels in the nectar and the stigma exudate of the 'Freedom' cultivar. We show that three different Machi3-1 alleles exist in apple cultivars and that only the 5B-Machi3-1 allele expresses the Machi3-1 protein in the nectar and the stigma exudate. We demonstrate that the 5B-Machi3-1 allele was introgressed from the Malus floribunda 821 clone into different apple cultivars including the 'Freedom'. Our data suggest that MYB-binding site containing repeats of the 5B-Machi3-1 promoter is responsible for the strong nectar- and stigma exudate-specific expression. As we found that in vitro, the Machi3-1 protein impairs growth and biofilm formation of Erwinia at physiological concentration, we propose that the Machi3-1 protein could partially protect 5B-Machi3-1 allele containing cultivars against Erwinia by inhibiting the multiplication and biofilm formation of the pathogen in the stigma exudate and in the nectar.