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1.
BMC Plant Biol ; 21(1): 376, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399701

RESUMO

BACKGROUND: Glycolytic pathway is common in all plant organs, especially in oxygen-deficient tissues. Phosphofructokinase (PFK) is a rate-limiting enzyme in the glycolytic pathway and catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. Cassava (M. esculenta) root is a huge storage organ with low amount of oxygen. However, less is known about the functions of PFK from M. esculenta (MePFK). We conducted a systematic analysis of MePFK genes to explore the function of the MePFK gene family under hypoxic stress. RESULTS: We identified 13 MePFK genes and characterised their sequence structure. The phylogenetic tree divided the 13 genes into two groups: nine were MePFKs and four were pyrophosphate-fructose-6-phosphate phosphotransferase (MePFPs). We confirmed by green fluorescent protein fusion protein expression that MePFK03 and MePFPA1 were localised in the chloroplast and cytoplasm, respectively. The expression profiles of the 13 MePFKs detected by quantitative reverse transcription polymerase chain reaction revealed that MePFK02, MePFK03, MePFPA1, MePFPB1 displayed higher expression in leaves, root and flower. The expression of MePFK03, MePFPA1 and MePFPB1 in tuber root increased gradually with plant growth. We confirmed that hypoxia occurred in the cassava root, and the concentration of oxygen was sharply decreasing from the outside to the inside root. The expression of MePFK03, MePFPA1 and MePFPB1 decreased with the decrease in the oxygen concentration in cassava root. Waterlogging stress treatment showed that the transcript level of PPi-dependent MePFP and MeSuSy were up-regulated remarkably and PPi-dependent glycolysis bypass was promoted. CONCLUSION: A systematic survey of phylogenetic relation, molecular characterisation, chromosomal and subcellular localisation and cis-element prediction of MePFKs were performed in cassava. The expression profiles of MePFKs in different development stages, organs and under waterlogging stress showed that MePFPA1 plays an important role during the growth and development of cassava. Combined with the transcriptional level of MeSuSy, we found that pyrophosphate (PPi)-dependent glycolysis bypass was promoted when cassava was under waterlogging stress. The results would provide insights for further studying the function of MePFKs under hypoxic stress.


Assuntos
Genoma de Planta , Manihot/enzimologia , Manihot/genética , Fosfofrutoquinases/genética , Fosfofrutoquinases/metabolismo , Cloroplastos/enzimologia , Mapeamento Cromossômico , Cromossomos de Plantas , Sequência Conservada , Citoplasma/enzimologia , Éxons , Flores/enzimologia , Íntrons , Família Multigênica , Oxigênio/metabolismo , Filogenia , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Regiões Promotoras Genéticas , Estresse Fisiológico/genética , Transcriptoma
2.
Gene ; 769: 145191, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33007377

RESUMO

Sucrose synthase (SUS), a key enzyme of the sucrose metabolism pathway, is encoded by a multi-gene family in plants. To date, dozens of SUS gene families have been characterized in various plant genomes. However, only a few studies have performed comprehensive analyses in tropical crops like cassava (Manihot esculenta Crantz). In the present study, seven non-redundant members of the SUS gene family (MeSUS1-7) were identified and characterized from the cassava genome. The MeSUS genes were distributed on five chromosomes (Chr1, Chr2, Chr3, Chr14, and Chr16) and the encoded proteins could be classified into three major groups with other SUS proteins from both dicot and monocot species (SUS I, SUS II, and SUS III). The spatio-temporal expression profiles of MeSUS genes showed a developmental stage-dependent, partially overlapping pattern, mainly expressed in the source and sink tissues. Cold and drought treatments significantly induced the expressions of MeSUS2, MeSUS4, MeSUS6, and MeSUS7 and the activities of the encoded enzymes, indicating that these genes may play crucial roles in resistance against abiotic stresses. These results provide new insights into the multifaceted role of the SUS gene family members in various physiological processes, especially sucrose transport and starch accumulation in cassava roots.


Assuntos
Glucosiltransferases/genética , Manihot/enzimologia , Proteínas de Plantas/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Temperatura Baixa , Secas , Éxons , Perfilação da Expressão Gênica , Genoma de Planta , Glucosiltransferases/fisiologia , Íntrons , Manihot/genética , Manihot/crescimento & desenvolvimento , Família Multigênica , Filogenia , Desenvolvimento Vegetal/genética , Sacarose/metabolismo
3.
J Integr Plant Biol ; 62(6): 832-846, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180179

RESUMO

High amylose starch can be produced by plants deficient in the function of branching enzymes (BEs). Here we report the production of transgenic cassava (Manihot esculenta Crantz) with starches containing up to 50% amylose due to the constitutive expression of hair-pin dsRNAs targeting the BE1 or BE2 genes. All BE1-RNAi plant lines (BE1i) and BE2-RNAi plant lines (BE2i) were grown up in the field, but with reduced total biomass production. Considerably high amylose content in the storage roots of BE2i plant lines was achieved. Storage starch granules of BE1i and BE2i plants had similar morphology as wild type (WT), however, the size of BE1i starch granules were bigger than that of WT. Comparisons of amylograms and thermograms of all three sources of storage starches revealed dramatic changes to the pasting properties and a higher melting temperature for BE2i starches. Glucan chain length distribution analysis showed a slight increase in chains of DP>36 in BE1i lines and a dramatic increase in glucan chains between DP 10-20 and DP>40 in BE2i lines. Furthermore, BE2i starches displayed a B-type X-ray diffraction pattern instead of the A-type pattern found in BE1i and WT starches. Therefore, cassava BE1 and BE2 function differently in storage root starch biosynthesis.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Amilose/biossíntese , Inativação Gênica , Genes de Plantas , Manihot/enzimologia , Manihot/genética , Transcrição Genética , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilopectina/química , Cristalização , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Manihot/crescimento & desenvolvimento , Fenótipo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Amido/metabolismo , Amido/ultraestrutura , Temperatura , Viscosidade , Difração de Raios X
4.
Phytochemistry ; 169: 112164, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31622858

RESUMO

Previous studies have shown that several d-amino acids are widely present in plants, and serine racemase (SerR), which synthesizes d-serine in vivo, has already been identified from three plant species. However, the full picture of the d-amino acid synthesis pathway in plants is not well understood. To clarify the distribution of amino acid racemases in plants, we have cloned, expressed and characterized eight SerR homologous genes from five plant species, including green alga. These SerR homologs exhibited racemase activity towards serine or aspartate and were identified on the basis of their maximum activity as SerR or aspartate racemase (AspR). The plant AspR gene is identified for the first time from Medicago truncatula, Manihot esculenta, Solanum lycopersicum, Sphagnum girgensohnii and Spirogyra pratensis. In addition to the AspR gene, three SerR genes are identified in the former three species. Phylogenetic tree analysis showed that SerR and AspR are widely distributed in plants and form a serine/aspartate racemase family cluster. The catalytic efficiency (kcat/Km) of plant AspRs was more than 100 times higher than that of plant SerRs, suggesting that d-aspartate, as well as d-serine, can be synthesized in vivo by AspR. The amino acid sequence alignment and comparison of the chromosomal gene arrangement have revealed that plant AspR genes independently evolved from SerR in each ancestral lineage of plant species by gene duplication and acquisition of two serine residues at position 150 to 152.


Assuntos
Isomerases de Aminoácido/metabolismo , Racemases e Epimerases/metabolismo , Isomerases de Aminoácido/genética , Biocatálise , Regulação Enzimológica da Expressão Gênica/genética , Lycopersicon esculentum/enzimologia , Manihot/enzimologia , Medicago truncatula/enzimologia , Filogenia , Racemases e Epimerases/genética , Sphagnopsida/enzimologia , Spirogyra/enzimologia
5.
J Mol Model ; 25(9): 295, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31478108

RESUMO

Cyanogenesis is an enzyme-promoted cleavage of ß-cyanoglucosides; the release of hydrogen cyanide is believed to produce food poisoning by consumption of certain crops as Cassava (Manihot esculenta Crantz). The production of hydrogen cyanide by some disruption of the plant wall is related to the content of two ß-cyanoglucosides (linamarin and lotaustralin) which are stored within the tuber. Some features about the mechanistic bases of these transformations have been published; nevertheless, there are still questions about the exact mechanism, such as the feasibility of a difference in the kinetics of cyanogenesis between both cyanoglucosides. In this work, we have performed a theoretical analysis using DFT and QTAIM theoretical frameworks to propose a feasible mechanism of the observed first step of the enzyme-catalyzed rupture of these glucosides; our results led us to explain the observed difference between linamarin and lotaustralin. Meanwhile, DFT studies suggest that there are no differences between local reactivity indexes of both glucosides; QTAIM topological analysis suggests two important intramolecular interactions which we found to fix the glucoside in such a way that suggests the linamarin as a more reactive system towards a nucleophilic attack, thus explaining the readiness to liberate hydrogen cyanide.


Assuntos
Glucosídeos/química , Cianeto de Hidrogênio/química , Manihot/química , Nitrilas/química , Biocatálise , Biotransformação , Glucosídeos/metabolismo , Cianeto de Hidrogênio/metabolismo , Cinética , Manihot/enzimologia , Estrutura Molecular , Nitrilas/metabolismo , Tubérculos/química , Tubérculos/enzimologia , Teoria Quântica , Termodinâmica
6.
Planta ; 250(5): 1621-1635, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31399791

RESUMO

MAIN CONCLUSION: Cassava AGPase and AGPase genes have some unique characteristics. ADP-glucose pyrophosphorylase (AGPase) is a rate-limiting enzyme for starch synthesis. In this study, cassava AGPase genes (MeAGP) were analyzed based on six cultivars and one wild species. A total of seven MeAGPs was identified, including four encoding AGPase large subunits (MeAGPLs 1, 2, 3 and 4) and three encoding AGPase small subunits (MeAGPSs 1, 2 and 3). The copy number of MeAGPs varied in cassava germplasm materials. There were 14 introns for MeAGPLs 1, 2 and 3, 13 introns for MeAGPL4, and 8 introns for other three MeAGPSs. Multiple conservative amino acid sequence motifs were found in the MeAGPs. There were differences in amino acids at binding sites of substrates and regulators among different MeAGP subunits and between MeAGPs and a potato AGPase small subunit (1YP2:B). MeAGPs were all located in chloroplasts. MeAGP expression was not only associated with gene copy number and types/combinations, regions and levels of the DNA methylation but was also affected by environmental factors with the involvement of various transcription factors in multiple regulation networks and in various cis-elements in the gene promoter regions. The MeAGP activity also changed with environmental conditions and had potential differences among the subunits. Taken together, MeAGPs differ in number from those of Arabidopsis, potato, maize, banana, sweet potato, and tomato.


Assuntos
Variações do Número de Cópias de DNA , Genoma de Planta/genética , Glucose-1-Fosfato Adenililtransferase/genética , Manihot/enzimologia , Motivos de Aminoácidos , Sítios de Ligação , Cloroplastos/metabolismo , Evolução Molecular , Manihot/genética , Proteínas de Plantas/genética , Subunidades Proteicas , Especificidade da Espécie , Amido/metabolismo
7.
J Food Biochem ; 43(5): e12840, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31353528

RESUMO

The aim of this study was to examine oxidative protection and enzymatic browning in the storage of minimally processed cassava and their relationship with population density and harvest age. Population densities were 1.0, 1.25, 1.5, and 1.75 plants m-2 . After being harvested at 300, 360, or 420 days after planting, cassava were minimally processed and stored at 5 ± 2°C. It was observed that superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) play key roles in the tolerance of young roots to browning. Planting density, however, does not appear to be a key factor modulating the activity of the enzymes studied. PRACTICAL APPLICATIONS: Younger harvested cassava roots, harvested at 300 days, are more tolerant to enzymatic browning. This appears to be in part due to enzymatic activity modulation of the SOD, CAT, and POD enzymes. In addition, it has been demonstrated that agronomic techniques aimed at increasing productivity, such as increasing the planting density of cassava, do not alter the biomarkers of postharvest quality. In summary, evidence that field management may be an efficient approach to improving the conservation of minimally processed cassava is provided. We believe that the findings of this paper will be of great interest regarding the influence of field management on the postharvest quality of freshly cut cassava and will also provide applicable results relating to its production chain.


Assuntos
Manipulação de Alimentos/métodos , Manihot/química , Proteínas de Plantas/metabolismo , Catalase/metabolismo , Cor , Manihot/enzimologia , Oxirredução/efeitos dos fármacos , Peroxidase/metabolismo , Raízes de Plantas/química , Raízes de Plantas/enzimologia , Superóxido Dismutase/metabolismo
8.
Plant Physiol Biochem ; 132: 281-286, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30240990

RESUMO

Isoamylase (EC.3.2.1.68), an essential enzyme in starch metabolism, catalyses the cleavage of α-1,6 glucosidic linkages of branched α-polyglucans such as beta-limit dextrin and amylopectin, but not pullulan. Three different isoamylase isoforms have been reported in plants and algae. We herein report on the first success in preparation of full-length isoamylase3 gene (MeISA3) of cassava Manihot esculenta Crantz 'KU50' from 5' Rapid Amplification of cDNA Ends (5' RACE). The MeISA3 was cloned to pET21b and expressed in E. coli. The HistrapTM-purified rMeISA3 appeared as a single band protein with approximate molecular size of 75 kDa on SDS-PAGE and Western blot, while 80 kDa was shown by gel filtration chromatography. This indicated the existence of a monomeric enzyme. Biochemical characterisation of rMeISA3 showed that the enzyme was specific towards beta-limit dextrin, with optimal activity at 37 °C pH 6.0. Activity of rMeISA3 could be significantly promoted by Mg2+ and Co2+. rMeISA3 debranched glucan chains of amylopectin were confirmed by HPAEC-PAD analysis.


Assuntos
Escherichia coli/genética , Expressão Gênica , Genes de Plantas , Isoamilase/genética , Manihot/enzimologia , Manihot/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Clonagem Molecular , Sequência Conservada , Isoamilase/química , Modelos Moleculares , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
9.
Biochem Biophys Res Commun ; 504(4): 647-653, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30205957

RESUMO

Dalcochinase from Dalbergia cochinchinensis Pierre and linamarase from Manihot esculenta Crantz are ß-glucosidases which share 47% sequence identity, but show distinct substrate specificities in hydrolysis and transglucosylation. Previously, three amino acid residues of dalcochinase, namely I185, N189 and V255, were identified as being important for determining substrate specificity. In this study, kinetic analysis of the ensuing double and triple mutants of dalcochinase showed that only those containing the 185A mutation could appreciably hydrolyze linamarin as well as transfer glucose to 2-methyl-2-propanol. So, the space provided by the I185A mutation appeared to be a prerequisite for accommodation of the aglycone moiety containing three substituents at the carbinol carbon. However, quantitative analysis of the energy parameters revealed mostly antagonistic interactions between these mutations. In addition, the N189F mutant showed a potential for use in enzymatic synthesis of alkyl glucosides via transglucosylation and reverse hydrolysis reactions. Thus, substitution of only 2-3 key residues in the aglycone binding pocket of dalcochinase could convert its specificities to that of linamarase, as well as to be suitable for any chosen hydrolytic or synthetic applications.


Assuntos
Glicosídeos/metabolismo , Mutação , Proteínas de Plantas/genética , beta-Glucosidase/genética , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Sítios de Ligação/genética , Dalbergia/enzimologia , Dalbergia/genética , Glicosídeos/química , Hidrólise , Cinética , Manihot/enzimologia , Manihot/genética , Modelos Moleculares , Estrutura Molecular , Nitrilas/química , Nitrilas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Domínios Proteicos , Especificidade por Substrato , beta-Glucosidase/química , beta-Glucosidase/metabolismo
10.
Plant Mol Biol ; 97(3): 201-214, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29679263

RESUMO

KEY MESSAGE: MeGAPCs were identified as negative regulators of plant disease resistance, and the interaction of MeGAPCs and MeATG8s was highlighted in plant defense response. As an important enzyme of glycolysis metabolic pathway, glyceraldehyde-3-P dehydrogenase (GAPDH) plays important roles in plant development, abiotic stress and immune responses. Cassava (Manihot esculenta) is most important tropical crop and one of the major food crops, however, no information is available about GAPDH gene family in cassava. In this study, 14 MeGAPDHs including 6 cytosol GAPDHs (MeGAPCs) were identified from cassava, and the transcripts of 14 MeGAPDHs in response to Xanthomonas axonopodis pv manihotis (Xam) indicated their possible involvement in immune responses. Further investigation showed that MeGAPCs are negative regulators of disease resistance against Xam. Through transient expression in Nicotiana benthamiana, we found that overexpression of MeGAPCs led to decreased disease resistance against Xam. On the contrary, MeGAPCs-silenced cassava plants through virus-induced gene silencing (VIGS) conferred improved disease resistance. Notably, MeGAPCs physically interacted with autophagy-related protein 8b (MeATG8b) and MeATG8e and inhibited autophagic activity. Moreover, MeATG8b and MeATG8e negatively regulated the activities of NAD-dependent MeGAPDHs, and are involved in MeGAPCs-mediated disease resistance. Taken together, this study highlights the involvement of MeGAPCs in plant disease resistance, through interacting with MeATG8b and MeATG8e.


Assuntos
Resistência à Doença/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/genética , Manihot/fisiologia , Doenças das Plantas/microbiologia , Xanthomonas axonopodis , Gliceraldeído-3-Fosfato Desidrogenases/fisiologia , Manihot/enzimologia , Manihot/genética , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Técnicas do Sistema de Duplo-Híbrido
11.
Plant Mol Biol ; 96(4-5): 417-427, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29380100

RESUMO

KEY MESSAGE: Cloning of two isoamylase genes, MeISA1 and MeISA2, from cassava (Manihot esculenta Crantz) tubers, accompanied by their co-expression in E. coli demonstrates a requirement for heteromeric complex formation to achieve debranching activity. Starch debranching enzyme (DBE) or isoamylase (ISA) (EC.3.2.1.68), an important enzyme in starch metabolism, catalyses the hydrolysis of α-1,6 glycosidic linkages of amylopectin. Isoforms of ISAs have been reported in higher plants and algae (Fujita et al. in Planta 208:283-293, 1999; Hussain et al. in Plant Cell 15:133-149, 2003; Ishizaki et al. in Agric Biol Chem 47:771-779, 1983; Mouille et al. in Plant Cell 8:1353-1366, 1996). In the current work, cassava ISA genes were isolated from cDNA generated from total RNA from tubers of Manihot esculanta Crantz cultivar KU50. MeISA1 and MeISA2 were successfully amplified and cloned into a pETDuet1 vector. The putative MeISA1 and MeISA2 proteins comprised 763 and 882 amino acids, with substantial similarity to StISA1 and StISA2 from potato (84.4% and 68.9%, respectively). Recombinant MeISA1 and MeISA2 were co-expressed in Escherichia coli SoluBL21 (DE3). HistrapTM-Purified rMeISA1 and rMeISA2 showed approximate molecular weights of 87 and 99 kDa, respectively, by SDS-PAGE. Debranching activity was only detectable in the column fractions where both recombinant ISA isoforms were present. The heteromeric DBE from crude extracts of 4-5 h induced cultures analysed by gel filtration chromatography and western blot showed combinations of rMeISA1 and rMeISA2 at ratios of 1:1 to 4:1. Pooled fractions with DBE activity were used for enzyme characterisation, which showed that the enzyme was specific for amylopectin, with optimum activity at 37 °C and pH 7.0. Enzyme activity was enhanced by Co2+, Mg2+ and Ca2+, but was strongly inhibited by Cu2+. Debranched amylopectin products showed chain length distributions typical of plant DBE.


Assuntos
Escherichia coli/metabolismo , Genes de Plantas , Isoamilase/genética , Manihot/enzimologia , Manihot/genética , Multimerização Proteica , Sequência de Aminoácidos , Clonagem Molecular , Isoamilase/química , Isoamilase/metabolismo , Peso Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Recombinação Genética/genética , Especificidade por Substrato
12.
Int J Mol Sci ; 18(11)2017 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-29137155

RESUMO

Fructokinase (FRK) proteins play important roles in catalyzing fructose phosphorylation and participate in the carbohydrate metabolism of storage organs in plants. To investigate the roles of FRKs in cassava tuber root development, seven FRK genes (MeFRK1-7) were identified, and MeFRK1-6 were isolated. Phylogenetic analysis revealed that the MeFRK family genes can be divided into α (MeFRK1, 2, 6, 7) and ß (MeFRK3, 4, 5) groups. All the MeFRK proteins have typical conserved regions and substrate binding residues similar to those of the FRKs. The overall predicted three-dimensional structures of MeFRK1-6 were similar, folding into a catalytic domain and a ß-sheet ''lid" region, forming a substrate binding cleft, which contains many residues involved in the binding to fructose. The gene and the predicted three-dimensional structures of MeFRK3 and MeFRK4 were the most similar. MeFRK1-6 displayed different expression patterns across different tissues, including leaves, stems, tuber roots, flowers, and fruits. In tuber roots, the expressions of MeFRK3 and MeFRK4 were much higher compared to those of the other genes. Notably, the expression of MeFRK3 and MeFRK4 as well as the enzymatic activity of FRK were higher at the initial and early expanding tuber stages and were lower at the later expanding and mature tuber stages. The FRK activity of MeFRK3 and MeFRK4 was identified by the functional complementation of triple mutant yeast cells that were unable to phosphorylate either glucose or fructose. The gene expression and enzymatic activity of MeFRK3 and MeFRK4 suggest that they might be the main enzymes in fructose phosphorylation for regulating the formation of tuber roots and starch accumulation at the tuber root initial and expanding stages.


Assuntos
Frutoquinases/genética , Genes de Plantas , Manihot/enzimologia , Manihot/genética , Família Multigênica , Motivos de Aminoácidos , Sequência de Aminoácidos , Cromossomos de Plantas/genética , Clonagem Molecular , Sequência Conservada , DNA Complementar/genética , Éxons/genética , Frutoquinases/química , Frutoquinases/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Íntrons/genética , Filogenia , Raízes de Plantas/genética , Tubérculos/genética , Domínios Proteicos , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por Substrato
13.
Sci Rep ; 7(1): 10016, 2017 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-28855620

RESUMO

The TCP transcription factors usually act as integrators of multiple growth regulatory and environmental stimuli. However, little is known about this gene family in the important tropical crop cassava (Manihot esculenta). In this study, 36 TCP genes were identified and renamed based on cassava whole-genome sequence and their sequence similarity with Arabidopsis TCPs. Typical TCP domains were detected in these proteins by multiple sequence alignment analysis. Evolutionary analysis indicated that MeTCPs could be divided into 8 subgroups, which was further supported by gene structure and conserved motif analyses. qRT-PCR analysis revealed tissue-specific and hormone-responsive expression patterns of MeTCP genes. Moreover, with global expression and promoter analysis, we found that MeTCPs showed similar or distinct expression patterns under cold and/or drought stress, suggesting that they might participate in distinct signaling pathways. Our study provides the first comprehensive analysis of TCP gene family in the cassava genome. The data will be useful for uncovering the potential functions of MeTCP genes, and their possible roles in mediating hormone and abiotic stress responses in cassava.


Assuntos
Perfilação da Expressão Gênica , Variação Genética , Manihot/enzimologia , Filogenia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Temperatura Baixa , Secas , Reação em Cadeia da Polimerase em Tempo Real , Plântula/enzimologia , Alinhamento de Sequência , Transdução de Sinais , Estresse Fisiológico , Fatores de Transcrição/classificação
14.
Int J Mol Sci ; 18(5)2017 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-28498327

RESUMO

Hexokinase (HXK) proteins play important roles in catalyzing hexose phosphorylation and sugar sensing and signaling. To investigate the roles of HXKs in cassava tuber root development, seven HXK genes (MeHXK1-7) were isolated and analyzed. A phylogenetic analysis revealed that the MeHXK family can be divided into five subfamilies of plant HXKs. MeHXKs were clearly divided into type A (MeHXK1) and type B (MeHXK2-7) based on their N-terminal sequences. MeHXK1-5 all had typical conserved regions and similar protein structures to the HXKs of other plants; while MeHXK6-7 lacked some of the conserved regions. An expression analysis of the MeHXK genes in cassava organs or tissues demonstrated that MeHXK2 is the dominant HXK in all the examined tissues (leaves, stems, fruits, tuber phloems, and tuber xylems). Notably, the expression of MeHXK2 and the enzymatic activity of HXK were higher at the initial and expanding tuber stages, and lower at the mature tuber stage. Furthermore, the HXK activity of MeHXK2 was identified by functional complementation of the HXK-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). The gene expression and enzymatic activity of MeHXK2 suggest that it might be the main enzyme for hexose phosphorylation during cassava tuber root development, which is involved in sucrose metabolism to regulate the accumulation of starch.


Assuntos
Hexoquinase/genética , Manihot/genética , Proteínas de Plantas/genética , Sequência Conservada , Hexoquinase/química , Hexoquinase/metabolismo , Manihot/enzimologia , Família Multigênica , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Domínios Proteicos
15.
Plant Mol Biol ; 94(1-2): 185-195, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28315989

RESUMO

Cassava (Manihot esculenta Crantz) is a major world crop, whose storage roots provide food for over 800 million throughout the humid tropics. Despite many advantages as a crop, the development of cassava is seriously constrained by the rapid post-harvest physiological deterioration (PPD) of its roots that occurs within 24-72 h of harvest, rendering the roots unpalatable and unmarketable. PPD limits cassava's marketing possibilities in countries that are undergoing increased development and urbanisation due to growing distances between farms and consumers. The inevitable wounding of the roots caused by harvesting triggers an oxidative burst that spreads throughout the cassava root, together with the accumulation of secondary metabolites including phenolic compounds, of which the coumarin scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) is the most abundant. Scopoletin oxidation yields a blue-black colour, which suggests its involvement in the discoloration observed during PPD. Feruloyl CoA 6'-hydroxylase is a controlling enzyme in the biosynthesis of scopoletin. The cassava genome contains a seven membered family of feruloyl CoA 6'-hydroxylase genes, four of which are expressed in the storage root and, of these, three were capable of functionally complementing Arabidopsis T-DNA insertion mutants in this gene. A RNA interference construct, designed to a highly conserved region of these genes, was used to transform cassava, where it significantly reduced feruloyl CoA 6'-hydroxylase gene expression, scopoletin accumulation and PPD symptom development. Collectively, our results provide evidence that scopoletin plays a major functional role in the development of PPD symptoms, rather than merely paralleling symptom development in the cassava storage root.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Manihot/enzimologia , Oxigenases de Função Mista/metabolismo , Proteínas de Plantas/metabolismo , Escopoletina/metabolismo , Arabidopsis , Clonagem Molecular , Armazenamento de Alimentos , Manihot/genética , Manihot/metabolismo , Oxigenases de Função Mista/genética , Filogenia , Proteínas de Plantas/genética , Raízes de Plantas , Plantas Geneticamente Modificadas , Interferência de RNA
16.
Sci Rep ; 7: 40179, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-28054665

RESUMO

To explore the role of protective enzymes in cassava (Manihot esculenta Crantz) resistance to mites, transgenic cassava lines overproducing copper/zinc superoxide dismutase (MeCu/ZnSOD) and catalase (MeCAT1) were used to evaluate and molecularly confirm cassava resistance to Tetranychus cinnabarinus. Laboratory evaluation demonstrated that, compared with the control cultivar TMS60444 (wild type, WT), the survival, reproduction, development and activities of SOD and CAT in T. cinnabarinus feeding on transgenic cassava lines SC2, SC4, and SC11 significantly inhibited. Furthermore, the activities of SOD and CAT in transgenic cassava lines SC2, SC4, and SC11 damaged by T. cinnabarinus significantly increased. These findings were similar to the results in the mite-resistant cassava cultivars. Besides, field evaluation indicated that the transgenic cassava lines SC2, SC4, and SC11 were slightly damaged as the highly mite-resistant control C1115, while the highly mite-susceptible WT was severely damaged by T. cinnabarinus. Laboratory and field evaluation demonstrated that transgenic cassava lines were resistant to T. cinnabarinus, which directly confirmed that the increase in SOD and CAT activities was positively related to cassava resistance to T. cinnabarinus. These results will help in understanding the antioxidant defense responses in the cassava-mite interaction and molecular breeding of mite-resistant cassava for effective pest control.


Assuntos
Catalase/metabolismo , Resistência à Doença , Manihot/enzimologia , Manihot/parasitologia , Doenças das Plantas/parasitologia , Superóxido Dismutase/metabolismo , Tetranychidae/crescimento & desenvolvimento , Animais , Antioxidantes/metabolismo , Sequestradores de Radicais Livres/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/parasitologia
17.
J Plant Physiol ; 204: 66-73, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27518222

RESUMO

Adenylate kinase (ADK) is a phosphotransferase that plays an important role in cellular energy homeostasis. Many isozymes located in different subcellular compartments have been reported. In this study, we focus on the characterization of cassava (Manihot esculenta) ADKs. We found 15 ADKs that are publicly available in the African cassava genome database. We cloned two ADKs, namely MeADK1 and MeADK2, which are phylogenetically grouped together with the plastidial ADK in potato. Both MeADK1 and MeADK2 showed 66% identity in the amino acid sequences with plastidial ADK in potato. However, we demonstrated that they are localized to mitochondria using GFP fusions of MeADK1 and MeADK2. The Escherichia coli-produced recombinant MeADK1 and MeADK2 preferred forward reactions that produce ATP. They exhibited similar specific activities. The semi-quantitative RT-PCR analysis showed that MeADK1 and MeADK2 in 2-month-old leaves have similar expression patterns under a diurnal light-dark cycle. However, MeADK2 transcripts were expressed at much higher levels than MeADK1 in 5-month-old leaves and roots. Thus, we conclude that MeADK2 might play a vital role in energy homeostasis in cassava mitochondria.


Assuntos
Adenilato Quinase/genética , Manihot/enzimologia , Manihot/genética , Clonagem Molecular , Simulação por Computador , Fluorescência , Regulação da Expressão Gênica de Plantas , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/isolamento & purificação , Frações Subcelulares/enzimologia
18.
Plant Physiol Biochem ; 102: 92-6, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26919397

RESUMO

Soluble starch synthase is a key enzyme in the starch biosynthesis pathway, and its enzyme activity significantly influences starch components in cassava storage root. However, studies on the regulation mechanism of soluble starch synthase gene are rare. In this study, we cloned the 5' flanking sequence of the MeSSIIb gene and predicted the distribution of cis-elements. The region from -453 to -1 was considered the primary core promoter by the quantitative detection of GUS activity in transgenic tobacco plants containing 5' truncated promoters fused with the GUS gene. Analysis results clarified that the region from -531 to -454 significantly repressed promoter activity. The region from -453 to -388 was a repressive domain of ethylene, and some unknown drought responsive cis-elements were located in the region from -387 to -1. These findings will provide useful information on the functional assay and transcriptional regulation mechanisms of the MeSSIIb gene.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Manihot/genética , Proteínas de Plantas , Plantas Geneticamente Modificadas , Sintase do Amido , Tabaco , Manihot/enzimologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Sintase do Amido/biossíntese , Sintase do Amido/genética , Tabaco/enzimologia , Tabaco/genética
19.
Food Chem ; 197(Pt A): 737-46, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26617011

RESUMO

This study aimed to investigate the role of ascorbate peroxidase (APX), guaiacol peroxidase (GPX), polysaccharides, and protein contents associated with the early events of postharvest physiological deterioration (PPD) in cassava roots. Increases in APX and GPX activity, as well as total protein contents occurred from 3 to 5 days of storage and were correlated with the delay of PPD. Cassava samples stained with Periodic Acid-Schiff (PAS) highlighted the presence of starch and cellulose. Degradation of starch granules during PPD was also detected. Slight metachromatic reaction with toluidine blue is indicative of increasing of acidic polysaccharides and may play an important role in PPD delay. Principal component analysis (PCA) classified samples according to their levels of enzymatic activity based on the decision tree model which showed GPX and total protein amounts to be correlated with PPD. The Oriental (ORI) cultivar was more susceptible to PPD.


Assuntos
Antioxidantes/análise , Ascorbato Peroxidases/análise , Manihot/química , Manihot/fisiologia , Peroxidase/análise , Amido/análise , Conservação de Alimentos , Armazenamento de Alimentos , Manihot/enzimologia , Fenômenos Fisiológicos , Raízes de Plantas/química , Raízes de Plantas/enzimologia , Análise de Componente Principal
20.
Mol Cell Biochem ; 406(1-2): 273-84, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25981533

RESUMO

Starch branching enzyme (SBE) is one of the key enzymes involved in starch biosynthetic metabolism. In this study, six SBE family genes were identified from the cassava genome. Phylogenetic analysis divided the MeSBE family genes into dicot family A, B, C, and the new group. Tissue-specific analysis showed that MeSBE2.2 was strongly expressed in leaves, stems cortex, and root stele, and MeSBE3 had high expression levels in stem cortex and root stele of plants in the rapid growth stage under field condition, whereas the expression levels of MeSBE2.1, MeSBE4, and MeSBE5 were low except for in stems cortex. The transcriptional activity of MeSBE2.2 and MeSBE3 was higher compared with other members and gradually increased in the storage roots during root growth process, while the other MeSBE members normally remained low expression levels. Expression of MeSBE2.2 could be induced by salt, drought, exogenous abscisic acid, jasmonic acid, and salicylic acid signals, while MeSBE3 had positive response to drought, salt, exogenous abscisic acid, and salicylic acid in leaves but not in storage root, indicating that they might be more important in starch biosynthesis pathway under diverse environments.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Manihot/genética , Proteínas de Plantas/genética , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Manihot/enzimologia , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas , Estresse Fisiológico
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