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1.
Plant Commun ; 5(1): 100681, 2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-37660253

RESUMO

Bananas (Musa spp.) are monocotyledonous plants with high genetic diversity in the Musaceae family that are cultivated mainly in tropical and subtropical countries. The fruits are a popular food, and the plants themselves have diverse uses. Four genetic groups (genomes) are thought to have contributed to current banana cultivars: Musa acuminata (A genome), Musa balbisiana (B genome), Musa schizocarpa (S genome), and species of the Australimusa section (T genome). However, the T genome has not been effectively explored. Here, we present the high-quality TT genomes of two representative accessions, Abaca (Musa textilis), with high-quality natural fiber, and Utafun (Musa troglodytarum, Fe'i group), with abundant ß-carotene. Both the Abaca and Utafun assemblies comprise 10 pseudochromosomes, and their total genome sizes are 613 Mb and 619 Mb, respectively. Comparative genome analysis revealed that the larger size of the T genome is likely attributable to rapid expansion and slow removal of transposons. Compared with those of Musa AA or BB accessions or sisal (Agava sisalana), Abaca fibers exhibit superior mechanical properties, mainly because of their thicker cell walls with a higher content of cellulose, lignin, and hemicellulose. Expression of MusaCesA cellulose synthesis genes peaks earlier in Abaca than in AA or BB accessions during plant development, potentially leading to earlier cellulose accumulation during secondary cell wall formation. The Abaca-specific expressed gene MusaMYB26, which is directly regulated by MusaMYB61, may be an important regulator that promotes precocious expression of secondary cell wall MusaCesAs. Furthermore, MusaWRKY2 and MusaNAC68, which appear to be involved in regulating expression of MusaLAC and MusaCAD, may at least partially explain the high accumulation of lignin in Abaca. This work contributes to a better understanding of banana domestication and the diverse genetic resources in the Musaceae family, thus providing resources for Musa genetic improvement.


Assuntos
Musa , Musa/genética , Genoma de Planta , Lignina
2.
Plant Commun ; 5(2): 100766, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-37974402

RESUMO

Bananas (Musa spp.) are one of the world's most important fruit crops and play a vital role in food security for many developing countries. Most banana cultivars are triploids derived from inter- and intraspecific hybridizations between the wild diploid ancestor species Musa acuminate (AA) and M. balbisiana (BB). We report two haplotype-resolved genome assemblies of the representative AAB-cultivated types, Plantain and Silk, and precisely characterize ancestral contributions by examining ancestry mosaics across the genome. Widespread asymmetric evolution is observed in their subgenomes, which can be linked to frequent homologous exchange events. We reveal the genetic makeup of triploid banana cultivars and verify that subgenome B is a rich source of disease resistance genes. Only 58.5% and 59.4% of Plantain and Silk genes, respectively, are present in all three haplotypes, with >50% of genes being differentially expressed alleles in different subgenomes. We observed that the number of upregulated genes in Plantain is significantly higher than that in Silk at one-week post-inoculation with Fusarium wilt tropical race 4 (Foc TR4), which confirms that Plantain can initiate defense responses faster than Silk. Additionally, we compared genomic and transcriptomic differences among the genes related to carotenoid synthesis and starch metabolism between Plantain and Silk. Our study provides resources for better understanding the genomic architecture of cultivated bananas and has important implications for Musa genetics and breeding.


Assuntos
Fusarium , Musa , Musa/genética , Fusarium/genética , Haplótipos , Perfilação da Expressão Gênica , Transcriptoma
4.
Front Plant Sci ; 14: 1125375, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36866367

RESUMO

Introduction: Polyphenol oxidases (PPOs), which are widely present in plants, play an important role in the growth, development, and stress responses. They can catalyze the oxidization of polyphenols and result in the browning of damaged or cut fruit, which seriously affects fruit quality and compromises the sale of fruit. In banana (Musa acuminata, AAA group), 10 PPO genes were determined based on the availability of a high-quality genome sequence, but the role of PPO genes in fruit browning remains unclear. Methods: In this study, we analyzed the physicochemical properties, gene structure, conserved structural domains, and evolutionary relationship of the PPO gene family of banana. The expression patterns were analyzed based on omics data and verified by qRT-PCR analysis. Transient expression assay in tobacco leaves was used to identify the subcellular localization of selected MaPPOs, and we analyzed the polyphenol oxidase activity using recombinant MaPPOs and transient expression assay. Results and discussion: We found that more than two-thirds of the MaPPO genes had one intron, and all contained three conserved structural domains of PPO, except MaPPO4. Phylogenetic tree analysis revealed that MaPPO genes were categorized into five groups. MaPPOs did not cluster with Rosaceae and Solanaceae, indicating distant affinities, and MaPPO6/7/8/9/10 clustered into an individual group. Transcriptome, proteome, and expression analyses showed that MaPPO1 exhibits preferential expression in fruit tissue and is highly expressed at respiratory climacteric during fruit ripening. Other examined MaPPO genes were detectable in at least five different tissues. In mature green fruit tissue, MaPPO1 and MaPPO6 were the most abundant. Furthermore, MaPPO1 and MaPPO7 localized in chloroplasts, and MaPPO6 was a chloroplast- and Endoplasmic Reticulum (ER)-localized protein, whereas MaPPO10 only localized in the ER. In addition, the enzyme activity in vivo and in vitro of the selected MaPPO protein showed that MaPPO1 had the highest PPO activity, followed by MaPPO6. These results imply that MaPPO1 and MaPPO6 are the main contributors to banana fruit browning and lay the foundation for the development of banana varieties with low fruit browning.

5.
Plant Physiol Biochem ; 194: 643-650, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36535104

RESUMO

Fruit ripening is the last phase of fruit growth and development. The initiation and progression of fruit ripening are highly modulated by a plethora of key genes, such as transcription factor (TF) genes. The WRKY gene family is a large group of TFs that play important roles in various cellular processes; nevertheless, the role of WRKY TF on fruit ripening remains enigmatic. Here, we report that a banana WRKY TF, MaWRKY49 functions in ethylene-induced fruit ripening by modulating the expression of fruit softening-related genes. We found that the expression of MaWRKY49 is highly induced by ethephon and inhibited by 1-methylcyclopropene, which is synchronous with the ripening process. Moreover, based on transcriptome data on fruit ripening, two pectate lyase (PL) genes that are involved in fruit softening were determined, and their expression pattern is also consistent with the fruit ripening process. Yeast one-hybrid and dual-luciferase assay confirmed that MaWRKY49 activated the transcription of two PL genes. In addition, transient overexpression of MaWRKY49 in banana fruits can apparently accelerate fruit ripening processs. Taken together, our findings indicate that MaWRKY49 acts as a potential modulator of fruit ripening by direct regulation of PL expression. This work contributes to developing the technology for improving the shelf-life of banana fruit.


Assuntos
Musa , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Musa/genética , Musa/metabolismo , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Etilenos/metabolismo , Proteínas de Plantas/metabolismo
6.
Front Nutr ; 9: 849385, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35369102

RESUMO

Litchi fruit contains abundant polyphenols and is susceptible to browning after harvest. Herein the combined treatments of malic acid (MA) and lycopene (LYC) to delay the development of browning in litchi fruit stored at room temperature (25°C) and low temperature (4°C) was investigated. The results showed that the pericarp browning could be alleviated, and the increase of malondialdehyde (MDA) content and relative leakage rate was retarded by the combined MA and LYC during storage. As compared to control, the content of pericarp anthocyanins, flavonoids, and the total phenols maintained higher levels; and the decrease of antioxidant activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity and reducing power were slowed down in treated fruit. The enzyme activity of polyphenol oxidase (PPO) and peroxidase (POD) related to oxidation of polyphenols were depressed by the combined treatments. Furthermore, correlation analysis revealed that the content of phenols in the pericarp negatively affected the changes in the browning index, and was positively related to the DPPH radical scavenging capacity. Taken together, the combined treatments of MA and LYC exhibited potential effects in delaying the pericarp browning of litchi fruit by maintaining the content of polyphenols, antioxidant activity, and membrane integrity.

7.
PeerJ ; 10: e12664, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35036088

RESUMO

The CRISPR/Cas9-mediated genome editing system has been used extensively to engineer targeted mutations in a wide variety of species. Its application in banana, however, has been hindered because of the species' triploid nature and low genome editing efficiency. This has delayed the development of a DNA-free genome editing approach. In this study, we reported that the endogenous U6 promoter and banana codon-optimized Cas9 apparently increased mutation frequency in banana, and we generated a method to validate the mutation efficiency of the CRISPR/Cas9-mediated genome editing system based on transient expression in protoplasts. The activity of the MaU6c promoter was approximately four times higher than that of the OsU6a promoter in banana protoplasts. The application of this promoter and banana codon-optimized Cas9 in CRISPR/Cas9 cassette resulted in a fourfold increase in mutation efficiency compared with the previous CRISPR/Cas9 cassette for banana. Our results indicated that the optimized CRISPR/Cas9 system was effective for mutating targeted genes in banana and thus will improve the applications for basic functional genomics. These findings are relevant to future germplasm improvement and provide a foundation for developing DNA-free genome editing technology in banana.


Assuntos
Edição de Genes , Musa , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Musa/genética , Mutação , Mutagênese Sítio-Dirigida
8.
Front Plant Sci ; 12: 792384, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34956291

RESUMO

The plant cuticle covers almost all the outermost surface of aerial plant organs, which play a primary function in limiting water loss and responding to the environmental interactions. Banana fruit is susceptible to thermal changes with chilling injury below 13°C and green ripening over 25°C. Herein, the changes of surface morphology, chemical compositions of cuticle, and the relative expression of cuticle biosynthesis genes in banana fruit under low-temperature storage were investigated. Banana fruit exhibited chilling injury rapidly with browned peel appearance stored at 4°C for 6 days. The surface altered apparently from the clear plateau with micro-crystals to smooth appearance. As compared to normal ones, the overall coverage of the main cuticle pattern of waxes and cutin monomers increased about 22% and 35%, respectively, in browned banana stored under low temperature at 6 days. Fatty acids (C16-C18) and ω-OH, mid-chain-epoxy fatty acids (C18) dominated cutin monomers. The monomers of fatty acids, the low abundant ω, mid-chain-diOH fatty acids, and 2-hydroxy fatty acids increased remarkably under low temperature. The cuticular waxes were dominated by fatty acids (> C19), n-alkanes, and triterpenoids; and the fatty acids and aldehydes were shifted to increase accompanied by the chilling injury. Furthermore, RNA-seq highlighted 111 cuticle-related genes involved in fatty acid elongation, biosynthesis of very-long-chain (VLC) aliphatics, triterpenoids, and cutin monomers, and lipid-transfer proteins were significantly differentially regulated by low temperature in banana. Results obtained indicate that the cuticle covering on the fruit surface was also involved to respond to the chilling injury of banana fruit after harvest. These findings provide useful insights to link the cuticle on the basis of morphology, chemical composition changes, and their biosynthesis regulations in response to the thermal stress of fruit during storage.

9.
BMC Plant Biol ; 21(1): 125, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33648452

RESUMO

BACKGROUND: Banana plant height is an important trait for horticultural practices and semi-dwarf cultivars show better resistance to damages by wind and rain. However, the molecular mechanisms controlling the pseudostem height remain poorly understood. Herein, we studied the molecular changes in the pseudostem of a semi-dwarf banana mutant Aifen No. 1 (Musa spp. Pisang Awak sub-group ABB) as compared to its wild-type dwarf cultivar using a combined transcriptome and metabolome approach. RESULTS: A total of 127 differentially expressed genes and 48 differentially accumulated metabolites were detected between the mutant and its wild type. Metabolites belonging to amino acid and its derivatives, flavonoids, lignans, coumarins, organic acids, and phenolic acids were up-regulated in the mutant. The transcriptome analysis showed the differential regulation of genes related to the gibberellin pathway, auxin transport, cell elongation, and cell wall modification. Based on the regulation of gibberellin and associated pathway-related genes, we discussed the involvement of gibberellins in pseudostem elongation in the mutant banana. Genes and metabolites associated with cell wall were explored and their involvement in cell extension is discussed. CONCLUSIONS: The results suggest that gibberellins and associated pathways are possibly developing the observed semi-dwarf pseudostem phenotype together with cell elongation and cell wall modification. The findings increase the understanding of the mechanisms underlying banana stem height and provide new clues for further dissection of specific gene functions.


Assuntos
Musa/crescimento & desenvolvimento , Musa/genética , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/genética , Parede Celular/genética , Parede Celular/metabolismo , Giberelinas/metabolismo , Metaboloma , Fenótipo , Reguladores de Crescimento de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma
10.
BMC Plant Biol ; 21(1): 97, 2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33596830

RESUMO

BACKGROUND: Banana is a tropical fruit with a high economic impact worldwide. Cold stress greatly affects the development and production of banana. RESULTS: In the present study, we investigated the functions of MaMAPK3 and MaICE1 involved in cold tolerance of banana. The effect of RNAi of MaMAPK3 on Dajiao (Musa spp. 'Dajiao'; ABB Group) cold tolerance was evaluated. The leaves of the MaMAPK3 RNAi transgenic plants showed wilting and severe necrotic symptoms, while the wide-type (WT) plants remained normal after cold exposure. RNAi of MaMAPK3 significantly changed the expressions of the cold-responsive genes, and the oxidoreductase activity was significantly changed in WT plants, while no changes in transgenic plants were observed. MaICE1 interacted with MaMAPK3, and the expression level of MaICE1 was significantly decreased in MaMAPK3 RNAi transgenic plants. Over-expression of MaICE1 in Cavendish banana (Musa spp. AAA group) indicated that the cold resistance of transgenic plants was superior to that of the WT plants. The POD P7 gene was significantly up-regulated in MaICE1-overexpressing transgenic plants compared with WT plants, and the POD P7 was proved to interact with MaICE1. CONCLUSIONS: Taken together, our work provided new and solid evidence that MaMAPK3-MaICE1-MaPOD P7 pathway positively improved the cold tolerance in monocotyledon banana, shedding light on molecular breeding for the cold-tolerant banana or other agricultural species.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Musa/fisiologia , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Temperatura Baixa , Resposta ao Choque Frio , Proteína Quinase 3 Ativada por Mitógeno/genética , Musa/genética , Musa/crescimento & desenvolvimento , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Fatores de Transcrição/genética
12.
BMC Plant Biol ; 20(1): 402, 2020 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-32867686

RESUMO

BACKGROUND: Pollen formation and development is important for crop fertility and is a key factor for hybrid development. Previous reports have indicated that Arabidopsis thaliana TAPETUM DETERMINANT1 (AtTPD1) and its rice (Oryza sativa) homolog, OsTPD1-like (OsTDL1A), are required for cell specialization and greatly affect pollen formation and development. Little is known about the role of the TPD1 homolog in banana pollen development. RESULTS: Here, we report the identification and characterization of TPD1 homologs in diploid banana (Musa itinerans) and examine their role in pollen development by overexpressing the closest homolog, MaTPD1A. MaTPD1A exhibits high expression in stamen and localizes in the plasma membrane. MaTPD1A-overexpressing plants produce no pollen grains and smaller and seedless fruit compared to wild-type plants. Transcriptome analysis showed that in plant hormone, starch and sucrose metabolism, and linolenic acid metabolism-related pathways were affected by overexpression of MaTPD1A, and the expression of several key regulators, such as PTC1 and MYB80, which are known to affect anther development, is affected in MaTPD1A-overexpressing lines. CONCLUSIONS: Our results indicate that MaTPD1A plays an important role in pollen formation and fruit development in diploid banana, possibly by affecting the expression of some key regulators of pollen development.


Assuntos
Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Musa/genética , Proteínas de Plantas/genética , Pólen/crescimento & desenvolvimento , Frutas/genética , Genes de Plantas , Musa/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Pólen/genética
13.
BMC Plant Biol ; 20(1): 425, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32933485

RESUMO

BACKGROUND: To date, CRISPR/Cas9 RNP editing tools have not been applied to the genetic modification of banana. Here, the establishment of a PEG-mediated banana protoplast transformation system makes it possible to build an efficient DNA-free method for a site-directed mutagenesis system. RESULTS: Protoplasts constitute a versatile platform for transient expression in plant science. In this study, we established a PEG-mediated banana protoplast transformation system. This system was further optimized for successfully delivering CRISPR/Cas9 and CRISPR/Cas12a plasmids and CRISPR/Cas9 ribonucleoproteins (RNPs) for targeted delivery of the PDS gene into banana protoplasts. Specific bands were observed in PCR-Restriction Enzyme Digestion (PCR-RE) assays, and Sanger sequencing of single clones further confirmed the occurrence of indels at target sites. Deep amplicon sequencing results showed that the editing efficiency of the CRISPR/Cas9 system was higher than that of the other two systems. CONCLUSIONS: The PEG-mediated banana protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in banana. The application of the CRISPR/Cas9 RNP system enables the generation of banana plants engineered by DNA-free gene editing.


Assuntos
Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Musa/genética , Musa/metabolismo , Polietilenoglicóis/metabolismo , Protoplastos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Regulação da Expressão Gênica de Plantas , Mutagênese Sítio-Dirigida/métodos , Melhoramento Vegetal/métodos
14.
Front Plant Sci ; 11: 145, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32161611

RESUMO

Ceramide sphingolipids are major components of membranes. C2 and C6 ceramides induce programmed cell death (PCD) in animals and plants, and we previously showed that C2 and C6 ceramides induce PCD in rice (Oryza sativa) protoplasts. However, the mechanistic link between sphingolipids and PCD in rice remains unclear. Here, we observed that calcium levels increased rapidly after ceramide treatment. Moreover, the calcium channel inhibitor LaCl3 and the intracellular calcium chelator acetoxymethyl-1, 2-bis (2-aminophenoxy) ethic acid (BAPTA-AM) inhibited this calcium increase and prevented ceramide-induced PCD. Moreover, caspase-3-like protease activity increased significantly in C6 ceramide-treated protoplasts, and a caspase-specific inhibitor prevented C6 ceramide-induced cell death. We also detected the other typical PCD events including ATP loss. DIDS (4, 49-diisothiocyanatostilbene- 2, 29-disulfonic acid), an inhibitor of voltage-dependent anion channels (VDACs), decreased C6 ceramide-induced cell death. Together, this evidence suggests that mitochondria played an important role in C6 ceramide-induced PCD.

15.
Int J Mol Sci ; 21(2)2020 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-31963632

RESUMO

Banana (Musa acuminata, AAA group) is a representative climacteric fruit with essential nutrients and pleasant flavors. Control of its ripening determines both the fruit quality and the shelf life. NAC (NAM, ATAF, CUC2) proteins, as one of the largest superfamilies of transcription factors, play crucial roles in various functions, especially developmental processes. Thus, it is important to conduct a comprehensive identification and characterization of the NAC transcription factor family at the genomic level in M. acuminata. In this article, a total of 181 banana NAC genes were identified. Phylogenetic analysis indicated that NAC genes in M. acuminata, Arabidopsis, and rice were clustered into 18 groups (S1-S18), and MCScanX analysis disclosed that the evolution of MaNAC genes was promoted by segmental duplication events. Expression patterns of NAC genes during banana fruit ripening induced by ethylene were investigated using RNA-Seq data, and 10 MaNAC genes were identified as related to fruit ripening. A subcellular localization assay of selected MaNACs revealed that they were all localized to the nucleus. These results lay a good foundation for the investigation of NAC genes in banana toward the biological functions and evolution.


Assuntos
Perfilação da Expressão Gênica/métodos , Musa/fisiologia , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Sequenciamento Completo do Genoma/métodos , Núcleo Celular/genética , Etilenos/farmacologia , Evolução Molecular , Armazenamento de Alimentos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Família Multigênica , Musa/efeitos dos fármacos , Musa/genética , Filogenia
16.
Front Plant Sci ; 11: 600704, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33488646

RESUMO

Anthocyanins spatiotemporally accumulate in certain tissues of particular species in the banana plant, and MYB transcription factors (TFs) serve as their primary regulators. However, the precise regulatory mechanism in banana remains to be determined. Here, we report the identification and characterization of MaMYB4, an R2R3-MYB repressor TF, characterized by the presence of EAR (ethylene-responsive element binding factor-associated amphiphilic repression) and TLLLFR motifs. MaMYB4 expression was induced by the accumulation of anthocyanins. Transgenic banana plants overexpressing MaMYB4 displayed a significant reduction in anthocyanin compared to wild type. Consistent with the above results, metabolome results showed that there was a decrease in all three identified cyanidins and one delphinidin, the main anthocyanins that determine the color of banana leaves, whereas both transcriptome and reverse transcription-quantitative polymerase chain reaction analysis showed that many key anthocyanin synthesis structural genes and TF regulators were downregulated in MaMYB4 overexpressors. Furthermore, dual-luciferase assays showed that MaMYB4 was able to bind to the CHS, ANS, DFR, and bHLH promoters, leading to inhibition of their expression. Yeast two-hybrid analysis verified that MaMYB4 did not interact with bHLH, which ruled out the possibility that MaMYB4 could be incorporated into the MYB-bHLH-WD40 complex. Our results indicated that MaMYB4 acts as a repressor of anthocyanin biosynthesis in banana, likely due to a two-level repression mechanism that consists of reduced expression of anthocyanin synthesis structural genes and the parallel downregulation of bHLH to interfere with the proper assembly of the MYB-bHLH-WD40 activation complex. To the best of our knowledge, this is the first MYB TF that regulates anthocyanin synthesis that was identified by genetic methods in bananas, which will be helpful for manipulating anthocyanin coloration in banana programs in the future.

20.
J Biol Chem ; 295(3): 717-728, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31819005

RESUMO

Cellular membranes contain many lipids, some of which, such as sphingolipids, have important structural and signaling functions. The common sphingolipid glucosylceramide (GlcCer) is present in plants, fungi, and animals. As a major plant sphingolipid, GlcCer is involved in the formation of lipid microdomains, and the regulation of GlcCer is key for acclimation to stress. Although the GlcCer biosynthetic pathway has been elucidated, little is known about GlcCer catabolism, and a plant GlcCer-degrading enzyme (glucosylceramidase (GCD)) has yet to be identified. Here, we identified AtGCD3, one of four Arabidopsis thaliana homologs of human nonlysosomal glucosylceramidase, as a plant GCD. We found that recombinant AtGCD3 has a low Km for the fluorescent lipid C6-NBD GlcCer and preferentially hydrolyzes long acyl-chain GlcCer purified from Arabidopsis leaves. Testing of inhibitors of mammalian glucosylceramidases revealed that a specific inhibitor of human ß-glucosidase 2, N-butyldeoxynojirimycin, inhibits AtGCD3 more effectively than does a specific inhibitor of human ß-glucosidase 1, conduritol ß-epoxide. We also found that Glu-499 and Asp-647 in AtGCD3 are vital for GCD activity. GFP-AtGCD3 fusion proteins mainly localized to the plasma membrane or the endoplasmic reticulum membrane. No obvious growth defects or changes in sphingolipid contents were observed in gcd3 mutants. Our results indicate that AtGCD3 is a plant glucosylceramidase that participates in GlcCer catabolism by preferentially hydrolyzing long-acyl-chain GlcCers.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Glucosilceramidase/genética , Glucosilceramidas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacologia , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/química , Vias Biossintéticas/efeitos dos fármacos , Glucosilceramidase/antagonistas & inibidores , Glucosilceramidase/química , Glucosilceramidas/genética , Humanos , Metabolismo/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/química , Folhas de Planta/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transdução de Sinais/efeitos dos fármacos , Esfingolipídeos/metabolismo
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