Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 465
Filtrar
Mais filtros










Filtros aplicados

Base de dados
Intervalo de ano de publicação
1.
Carbohydr Polym ; 230: 115656, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31887861

RESUMO

How various isoforms of rice-starch biosynthesis enzymes interact during amylose and amylopectin synthesis is explored. The chain-length distributions of amylopectin and amylose from 95 varieties with different environmental and genetic backgrounds were obtained using size- exclusion chromatography, and fitted with biosynthesis-derived models based on isoforms of starch synthase (SSI-SSIV), starch branching enzyme (SBE, including SBEI and SBEII) and granule-bound starch synthase (GBSS) that are involved in amylose and amylopectin synthesis. It is usually thought that these are synthesized by separate enzymes. However, the amount of longer amylopectin chains correlated with that of shorter amylose chains, indicating that GBSS, SBE and SS affect both amylose and amylopectin synthesis. Further, the activity of GBSS in amylose correlated with that of SS in amylopectin. This new understanding of which enzymes are suggested by the statistics to be involved in both amylose and amylopectin synthesis could help rice breeders develop cereals with targeted properties.


Assuntos
Amilopectina/biossíntese , Amilose/biossíntese , Endosperma/metabolismo , Oryza/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/química , Grão Comestível/metabolismo , Proteínas de Plantas/química , Sintase do Amido/química
2.
PLoS One ; 14(10): e0223783, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31603940

RESUMO

Starch is synthesized from a series of reactions catalyzed by enzymes. ADP-glucose pyrophosphorylase (AGPase) initiates the synthesis pathway and synthesizes ADP-glucose, the substrate of starch synthase (SS), of which SSIV is an isoform. Mutations of the AGPase subunit and SSIV-coding genes affect starch content and cause variation in the number of granules. Here, we pyramided the functional mutation alleles of the AGPase subunit gene TaAGP.L-B1 and the SSIV-coding gene TaSSIVb-D to elucidate their synergistic effects on other key starch biosynthesis genes and their impact on starch content. Both the TaAGP.L-B1 and TaSSIVb-D genes were expressed in wheat grain development, and the expression level of TaAGP.L-B1 was higher than that of TaSSIVb-D. The TaAGP.L-B1 gene was downregulated in the agp.L-B1 single and agp.L-B1/ssIV-D double mutants at 12 to 18 days after flowering (DAF). TaSSIVb-D expression was significantly reduced at 6 DAF in both ssIV-D single and double mutants. In the agp.L-B1/ssIV-D double mutant, TaGBSSII was upregulated, while TaAGPSS, TaSSI, and TaSBEII were downregulated. Under the interaction of these genes, the total starch and amylopectin contents were significantly decreased in agp.L-B1 and agp.L-B1/ssIV-D mutants. The results suggested that the mutations of TaAGP.L-B1 and TaSSIVb-D genes resulted in variation in the expression patterns of the other four starch synthetic genes and led to a reduction in starch and amylopectin contents. These mutants could be used further as germplasm for resistant starch analysis.


Assuntos
Glucose-1-Fosfato Adenililtransferase/genética , Mutação , Sintase do Amido/genética , Amido/biossíntese , Triticum/crescimento & desenvolvimento , Amilopectina/metabolismo , Vias Biossintéticas , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Glucose-1-Fosfato Adenililtransferase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sintase do Amido/metabolismo , Triticum/genética , Triticum/metabolismo
3.
Theor Appl Genet ; 132(12): 3357-3374, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31624872

RESUMO

KEY MESSAGE: Seven novel alleles of SBEIIb and one allele of SSIIa co-segregated with the ASV phenotype and contributed to distinct starch quality traits important for food-processing applications. Sorghum is an important food crop for millions of people in Africa and Asia. Whole-genome re-sequencing of sorghum EMS mutants exhibiting an alkali spreading value (ASV) phenotype revealed candidate SNPs in Sobic.004G163700 and Sobic.010G093400. Comparative genomics identified Sobic.010G093400 as a starch synthase IIa and Sobic.004G163700 as a starch branching enzyme IIb. Segregation analyses showed that mutations in Sobic.010G093400 or Sobic.004G163700 co-segregated with the ASV phenotype. Mutants in SSIIa exhibited no change in amylose content but expressed lower final viscosity and lower starch gelatinization temperature (GT) than starches from non-mutant plants. The sbeIIb mutants exhibited significantly higher amylose levels and starch GT and lower viscosity compared to non-mutant starches and ssIIa mutants. Mutations in SBEIIb had a dosage-dependent effect on amylose content. Double mutants of sbeIIb and ssIIa resembled their sbeIIb parent in amylose content, starch thermal properties and viscosity profiles. These variants will provide opportunities to produce sorghum varieties with modified starch end-use qualities important for the beer brewing and baking industries and specialty foods for humans with diabetes.


Assuntos
Amilose/análise , Farinha/análise , Sorghum/genética , Amido/análise , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Álcalis , Alelos , Análise Mutacional de DNA , Dosagem de Genes , Mutação , Fenótipo , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único , Alinhamento de Sequência , Sintase do Amido/genética , Viscosidade
4.
BMC Plant Biol ; 19(1): 303, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31291892

RESUMO

BACKGROUND: Polyploidy and hybridization are ubiquitous in Rubus L., a large and taxonomically challenging genus. Chinese Rubus are mainly concentrated into two major sections, the diploid Idaeobatus and the polyploid Malachobatus. However, it remains unclear to be auto- or allo- polyploid origin of polyploids in Rubus. We investigated the homoeologs and the structure of the GBSSI-1 (granule-bound starch synthase I) gene in 140 Rubus individuals representing 102 taxa in 17 (out of the total 24) subsections of 7 (total of 12) sections at different ploidy levels. RESULTS: Based on the gene structure and sequence divergence, we defined three gene variants, GBSSI-1a, GBSSI-1b, and GBSSI-1c. When compared with GBSSI-1a, both GBSSI-1b and GBSSI-1c have a shorter fourth intron, and GBSSI-1c had an additional deletion in the fifth intron. For diploids, either GBSSI-1a or GBSSI-1b was detected in 56 taxa consisting of 82 individuals from sect. Idaeobatus, while both alleles existed in R. pentagonus and R. peltatus. Both homoeologs GBSSI-1a and GBSSI-1b were identified in 39 taxa (48 individuals) of Malachobatus polyploids. They were also observed in two sect. Dalibardastrum taxa, in one sect. Chamaebatus taxon, and in three taxa from sect. Cylactis. Interestingly, all three homoeologs were observed in the three tetraploid taxa. Phylogenetic trees and networks suggested two clades (I and II), corresponding to GBSSI-1a, and GBSSI-1b/1c sequences, respectively. GBSSI-1 homoeologs from the same polyploid individual were resolved in different well-supported clades, and some of these homoelogs were more closely related to homoelogs in other species than they were to each other. This implied that the homoeologs of these polyploids were donated by different ancestral taxa, indicating their allopolyploid origin. Two kinds of diploids hybridized to form most allotetraploid species. The early-divergent diploid species with GBSSI-1a or -1b emerged before polyploid formation in the evolutionary history of Rubus. CONCLUSION: This study provided new insights into allopolyploid origin and evolution from diploid to polyploid within the genus Rubus at the molecular phylogenetic level, consistent with the taxonomic treatment by Yü et al. and Lu.


Assuntos
Rubus/genética , Sintase do Amido/genética , Núcleo Celular/enzimologia , Diploide , Evolução Molecular , Hibridização Genética , Filogenia , Poliploidia , Rubus/enzimologia , Tetraploidia
5.
Food Chem ; 297: 124991, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253316

RESUMO

Plant species differ greatly in their ability to acclimatise to and survive, cold stress. Normally, potato tubers are stored at low temperatures (below 10 °C) to delay sprouting. In this research, combined transcriptomic and proteomic analysis was conducted on potato tubers stored at 15 °C, 4 °C and 0 °C to investigate the mechanism of cold responses during postharvest storage. Results showed that soluble sugars were accumulated under low temperatures, regulating by granule-bound starch synthase 1, beta-amylase, invertase inhibitor and fructokinase. In addition, fifteen heat shock proteins (Hsps), including three Hsp70s, two Hsp80s, one Hsp90, one Hsp100 and eight small Hsps, were induced by low temperatures, which may act individually or synergistically to prevent physiological or cellular damage from cold stress in postharvest potato tubers. This research provided general information of sugar accumulation and defense response in potato tuber under cold storage.


Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/metabolismo , Açúcares/metabolismo , Transcriptoma , Temperatura Baixa , Armazenamento de Alimentos , Tubérculos/genética , Tubérculos/metabolismo , Proteômica , Solanum tuberosum/genética , Sintase do Amido/metabolismo , beta-Amilase/metabolismo
6.
BMC Plant Biol ; 19(1): 275, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234778

RESUMO

BACKGROUND: Amylose content (AC) is a critical factor for the quality of rice. It is determined by the biosynthesis gene Waxy (Wx) and a variety of quantitative trait loci (QTLs). Although many QTLs have been reported to affect rice AC, few of them have been investigated under varying growth conditions, especially various temperatures, which are known to greatly influence the AC. RESULTS: We analyzed the AC at different temperatures and planting seasons in a set of chromosome segment substitution lines (CSSLs) which were derived from a cross between the indica variety 9311 and the japonica variety Nipponbare carrying the same Wxb allele. A joint analysis detected a single locus, qSAC3, with a high logarithm of odds (LOD) score in four different conditions. The qSAC3 from indica 9311 (qSAC3ind) substantially increased the AC in japonica Nipponbare under all tested growth conditions. Furthermore, introducing the qSAC3ind into the soft rice variety Nangeng9108 with Wxmq, a mutant allele of Wxb, also moderately increased its AC and improved its appearance quality significantly by reducing the chalkiness of the polished rice. CONCLUSIONS: Our results indicate that the qSAC3ind could increase the AC of japonica rice in different environments as well as in the background of different Wx alleles and that qSAC3 is a valuable locus for fine-tuning the rice AC and ameliorating the dull endosperm in rice varieties with the Wxmq allele.


Assuntos
Amilose/metabolismo , Genes de Plantas , Oryza/genética , Oryza/química , Locos de Características Quantitativas , Sintase do Amido/genética
8.
Plant Cell Rep ; 38(9): 1065-1080, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31101972

RESUMO

KEY MESSAGE: The StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base-editing strategies, leading to plants with impaired amylose biosynthesis. Genome editing has recently become a method of choice for basic research and functional genomics, and holds great potential for molecular plant-breeding applications. The powerful CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to generate knockout mutants. Recently, the development of base editors has broadened the scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knock out the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective screening strategy based on high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but unwanted insertions of plasmid DNA were also observed. We successfully created tetra-allelic mutants with impaired amylose biosynthesis, confirming the loss of function of the StGBSSI protein. The second main objective of this work was to demonstrate the proof of concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete variation in the amino acid sequence and generating a loss-of-function allele. The successful application of base editing in the tetraploid potato opens up new avenues for genome engineering in this species.


Assuntos
Edição de Genes , Solanum tuberosum/genética , Sintase do Amido/genética , Alelos , Sistemas CRISPR-Cas , Tetraploidia
9.
New Phytol ; 224(2): 761-774, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31111487

RESUMO

Root gravitropism is one of the most important processes allowing plant adaptation to the land environment. Auxin plays a central role in mediating root gravitropism, but how auxin contributes to gravitational perception and the subsequent response are still unclear. Here, we showed that the local auxin maximum/gradient within the root apex, which is generated by the PIN directional auxin transporters, regulates the expression of three key starch granule synthesis genes, SS4, PGM and ADG1, which in turn influence the accumulation of starch granules that serve as a statolith perceiving gravity. Moreover, using the cvxIAA-ccvTIR1 system, we also showed that TIR1-mediated auxin signaling is required for starch granule formation and gravitropic response within root tips. In addition, axr3 mutants showed reduced auxin-mediated starch granule accumulation and disruption of gravitropism within the root apex. Our results indicate that auxin-mediated statolith production relies on the TIR1/AFB-AXR3-mediated auxin signaling pathway. In summary, we propose a dual role for auxin in gravitropism: the regulation of both gravity perception and response.


Assuntos
Arabidopsis/fisiologia , Gravitropismo/fisiologia , Ácidos Indolacéticos/farmacologia , Raízes de Plantas/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Cinurenina/farmacologia , Amido/genética , Amido/metabolismo , Sintase do Amido/genética , Sintase do Amido/metabolismo
10.
Theor Appl Genet ; 132(7): 2097-2109, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30993362

RESUMO

KEY MESSAGE: An EMS-induced single-base mutation at a splice site caused abnormal RNA splicing and resulted in the gene inactivation and the lack of Wx-A1 protein in a wheat EMS mutant line. An EMS-mutagenized population was generated using common wheat cv. SM126 consisting of 10,600 M2 plants. One Wx-A1 null mutant was identified through analyses of 390 grains produced from 130 M2 plants using electrophoresis analyses. The Wx-A1 sequences of parental line SM126 and M2-31 mutant were determined as 2781 bp, and there was only one SNP mutation between them. The SNP was a mutation from G to A at nucleotide sequence position 2168 bp (G2168A) downstream of the start codon which was located at the splicing site within the eighth intron. All 52 cDNA transcripts were found to be incorrectly spliced and can be summarized as five types of variations. The deletion of the exon and the exclusion of intron were structural features in abnormal splicing RNA. Together with the prediction of potential splice regulatory motifs, the mutation G2168A happened within the 5' splice site of the eighth intron and destroyed the splice donor site from GU to AU, which may have brought about a barrier against correct RNA splice, and generated abnormal mRNA, which was the mechanism of the inactivation of Wx-A1 in M2-31. The lack of Wx-A1 has resulted in changes in starch properties in the M2-31 mutant, with the reduction in amylose and starch contents. The increased grains hardness was observed in M2-31, which may be related to the lower expression level of Pinb-D1 gene. As the waxy wheat foods have a lot of advantages, the null waxy genes will be widely applied in breeding waxy wheat for varied amylose contents.


Assuntos
Inativação Gênica , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único , Processamento de RNA , Sintase do Amido/genética , Triticum/genética , Amilose/análise , Sequência de Bases , Éxons , Íntrons , Mutação , RNA Mensageiro/genética , Deleção de Sequência , Amido/análise
11.
Genes (Basel) ; 10(4)2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31003564

RESUMO

Wheat is a staple food commodity grown worldwide, and wheat starch is a valuable source of energy and carbon that constitutes 80% of the grain weight. Manipulation of genes involved in starch synthesis significantly affects wheat grain weight and yield. TaSSIV plays an important role in starch synthesis and its main function is granule formation. To mine and stack more favorable alleles, single nucleotide polymorphisms (SNPs) of TaSSIV-A, B, and D were investigated across 362 wheat accessions by Ecotype-Targeting Induced Local Lesions IN Genome (EcoTILLING). As a result, a total of 38 SNPs in the amplified regions of three TaSSIV genes were identified, of which 10, 15, and 13 were in TaSSIV-A, B, and D, respectively. These 38 SNPs were evaluated by using KASP and six SNPs showed an allele frequency >5% whereas the rest were <5%, i.e., considered to be minor alleles. In the Chinese mini core collection, three haplotypes were detected for TaSSIV-A and three for TaSSIV-B. The results of an association study in the Chinese mini core collection with thousand grain weight (TGW) and spike length (SPL) showed that Hap-2-1A was significantly associated with TGW and Hap-3-1B with SPL. Allelic frequency and geographic distribution indicated that the favored haplotype (Hap-2-1A) has been positively selected in Chinese wheat breeding. These results suggested that the Kompetitive Allele Specific PCR (KASP) markers can be applied in starch improvement to ultimately improve wheat yield by marker assisted selection in wheat breeding.


Assuntos
Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Sintase do Amido/genética , Triticum/crescimento & desenvolvimento , China , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Frequência do Gene , Haplótipos , Tamanho do Órgão , Proteínas de Plantas/genética , Característica Quantitativa Herdável , Amido/biossíntese , Triticum/genética , Triticum/metabolismo
12.
J Agric Food Chem ; 67(22): 6302-6312, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30925057

RESUMO

Granule-bound starch synthase I (GBSSI) and starch-branching enzymes I and II (SBEI and SBEII) are crucial enzymes that biosynthesize starches with varied apparent amylose contents and amylopectin branching structure. With a sweet potato ( Ipomoea batatas [L.] Lam. cv. Xushu22), this work shows that downregulating GBSSI (for waxy starch) or SBE (for high-amylose starch) activity allowed the formation of new semicrystalline lamellae (named Type II) in sweet potato starch in addition to the widely reported Type I lamellae. Small-angle X-ray scattering (SAXS) results show that, compared with Type I lamellae, Type II lamellae displayed increased average thickness and thickness-distribution width, with thickened amorphous and crystalline components. The size-exclusion-chromatography (SEC) data revealed mainly two enzyme sets, (i) and (ii), synthesizing amylopectin chains. Reducing the GBSSI or SBE activity increased the amounts of amylopectin long chains (degree of polymerization (DP) ≥ 33). Combined SAXS and SEC analyses indicate that parts of these long chains from enzyme set (i) could be confined to Type II lamellae, followed by DP ≤ 32 short chains in Type I lamellae and the rest of the long chains from enzyme sets (i) and (ii) spanning more than a single lamella.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Ipomoea batatas/enzimologia , Proteínas de Plantas/genética , Sintase do Amido/genética , Amido/biossíntese , Amido/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Regulação para Baixo , Ipomoea batatas/genética , Ipomoea batatas/metabolismo , Proteínas de Plantas/metabolismo , Espalhamento a Baixo Ângulo , Sintase do Amido/metabolismo
13.
Plant Cell Rep ; 38(3): 417-433, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30715580

RESUMO

KEY MESSAGE: Induced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery. The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway. Surprisingly this aspect of global starch metabolism has received little attention. We used CRISPR/Cas9 to introduce mutations affecting the Waxy (Wx) locus encoding granule-bound starch synthase I (GBSSI) in rice endosperm. Our specific objective was to develop a mechanistic understanding of how the endogenous starch biosynthetic machinery might be affected at the transcriptional level following the targeted knock out of GBSSI in the endosperm. We found that the mutations reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the upregulation of GBSSII. The GBSS activity in the mutants was 61-71% of wild-type levels, similarly to two irradiation mutants, but the amylose content declined to 8-12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Expression of many other starch biosynthetic genes was modulated in seeds and leaves. This modulation of gene expression resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars.


Assuntos
Oryza/metabolismo , Sintase do Amido/metabolismo , Alelos , Sistemas CRISPR-Cas/genética , Endosperma/metabolismo , Mutação/genética , Oryza/genética , Sintase do Amido/genética , Ceras/metabolismo
14.
Plant Physiol ; 179(4): 1457-1478, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30670603

RESUMO

Diel starch turnover responds rapidly to changes in the light regime. We investigated if these responses require changes in the temporal dynamics of the circadian clock. Arabidopsis (Arabidopsis thaliana) was grown in a 12-h photoperiod for 19 d, shifted to three different reduced light levels or to low CO2 for one light period, and returned to growth conditions. The treatments produced widespread changes in clock transcript abundance. However, almost all of the changes were restricted to extreme treatments that led to carbon starvation and were small compared to the magnitude of the circadian oscillation. Changes included repression of EARLY FLOWERNG 4, slower decay of dusk components, and a slight phase delay at the next dawn, possibly due to abrogated Evening Complex function and sustained expression of PHYTOCHROME INTERACTING FACTORs and REVEILLEs during the night. Mobilization of starch in the night occurred in a linear manner and was paced to dawn, both in moderate treatments that did not alter clock transcripts and in extreme treatments that led to severe carbon starvation. We conclude that pacing of starch mobilization to dawn does not require retrograde carbon signaling to the transcriptional clock. On the following day, growth decreased, sugars rose, and starch accumulation was stimulated in low-light-treated plants compared to controls. This adaptive response was marked after moderate treatments and occurred independently of changes in the transcriptional clock. It is probably a time-delayed response to low-C signaling in the preceding 24-h cycle, possibly including changes in PHYTOCHROME INTERACTING FACTOR and REVEILLE expression.


Assuntos
Arabidopsis/efeitos da radiação , Dióxido de Carbono/metabolismo , Relógios Circadianos , Amido/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ritmo Circadiano , Luz , Sintase do Amido/metabolismo , Fatores de Transcrição/metabolismo
15.
Plant Cell Physiol ; 60(5): 961-972, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30690625

RESUMO

CO2-responsive CCT protein (CRCT) is suggested to be a positive regulator of starch biosynthesis in the leaf sheaths of rice, regulating the expression levels of starch biosynthesis-related genes. In this study, the effects of CRCT expression levels on the expression of starch biosynthesis-related enzymes and the quality of starch were studied. Using native-PAGE/activity staining and immunoblotting, we found that the protein levels of starch synthase I, branching enzyme I, branching enzyme IIa, isoamylase 1 and phosphorylase 1 were largely correlated with the CRCT expression levels in the leaf sheaths of CRCT transgenic lines. In contrast, the CRCT expression levels largely did not affect the expression levels and/or activities of starch biosynthesis-related enzymes in the leaf blades and endosperm tissues. The analysis of the chain-length distribution of starch in the leaf sheaths showed that short chains with a degree of polymerization from 5 to 14 were increased in the overexpression lines but decreased in the knockdown lines. The amylose content of starch in the leaf sheath was greatly increased in the overexpression lines. In contrast, the molecular weight of the amylopectin of starch in the leaf sheath of overexpression lines did not change compared with those of the non-transgenic rice. These results suggest that CRCT can control the quality and the quantity of starch in the leaf sheath by regulating the expression of particular starch biosynthesis-related enzymes.


Assuntos
Dióxido de Carbono/metabolismo , Oryza/metabolismo , Folhas de Planta/metabolismo , Amido/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilose/metabolismo , Isoamilase/metabolismo , Sintase do Amido/metabolismo
16.
New Phytol ; 221(1): 356-370, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30055112

RESUMO

The initiation of starch granule formation is still poorly understood. However, the soluble starch synthase 4 (SS4) appears to be a major component of this process since it is required to synthesize the correct number of starch granules in the chloroplasts of Arabidopsis thaliana plants. A yeast two-hybrid screen allowed the identification of several putative SS4 interacting partners. We identified the product of At4g32190 locus as a chloroplast-targeted PROTEIN INVOLVED IN STARCH INITIATION (named PII1). Arabidopsis mutants devoid of PII1 display an alteration of the starch initiation process and accumulate, on average, one starch granule per plastid instead of the five to seven granules found in plastids of wild-type plants. These granules are larger than in wild-type, and they remain flat and lenticular. pii1 mutants display wild-type growth rates and accumulate standard starch amounts. Moreover, starch characteristics, such as amylopectin chain length distribution, remain unchanged. Our results reveal the involvement of PII1 in the starch priming process in Arabidopsis leaves through interaction with SS4.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Amido/metabolismo , Amilopectina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Cloroplastos/genética , Cloroplastos/genética , Microscopia Eletrônica de Varredura , Mutação , Cadeias Pesadas de Miosina/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plastídeos/genética , Plastídeos/metabolismo , Amido/genética , Amido/ultraestrutura , Sintase do Amido/genética , Sintase do Amido/metabolismo
17.
Plant J ; 97(6): 1073-1088, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30523657

RESUMO

The CS8 transgenic rice (Oryza sativa L.) lines expressing an up-regulated glgC gene produced higher levels of ADPglucose (ADPglc), the substrate for starch synthases. However, the increase in grain weight was much less than the increase in ADPglc levels suggesting one or more downstream rate-limiting steps. Endosperm starch levels were not further enhanced in double transgenic plants expressing both glgC and the maize brittle-1 gene, the latter responsible for transport of ADPglc into the amyloplast. These studies demonstrate that critical processes within the amyloplast stroma restrict maximum carbon flow into starch. RNA-seq analysis showed extensive re-programming of gene expression in the CS8 with 2073 genes up-regulated and 140 down-regulated. One conspicuous gene, up-regulated ~15-fold, coded for a biochemically uncharacterized starch binding domain-containing protein (SBDCP1) possessing a plastid transit peptide. Confocal microscopy and transmission electron microscopy analysis confirmed that SBDCP1 was located in the amyloplasts. Reciprocal immunoprecipitation and pull-down assays indicated an interaction between SBDCP1 and starch synthase IIIa (SSIIIa), which was down-regulated at the protein level in the CS8 line. Furthermore, binding by SBDCP1 inhibited SSIIIa starch polymerization activity in a non-competitive manner. Surprisingly, artificial microRNA gene suppression of SBDCP1 restored protein expression levels of SSIIIa in the CS8 line resulting in starch with lower amylose content and increased amylopectin chains with a higher degree of polymerization. Collectively, our results support the involvement of additional non-enzymatic factors such as SBDCP in starch biosynthesis.


Assuntos
Metabolismo dos Carboidratos , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Amido/biossíntese , Zea mays/genética , Regulação para Baixo , Endosperma/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Oryza/genética , Oryza/fisiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Plastídeos/metabolismo , Sintase do Amido/genética , Sintase do Amido/metabolismo , Regulação para Cima
18.
Plant Biotechnol J ; 17(7): 1222-1235, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30552799

RESUMO

Identification of seed development regulatory genes is the key for the genetic improvement in rice grain quality. NF-Ys are the important transcription factors, but their roles in rice grain quality control and the underlying molecular mechanism remain largely unknown. Here, we report the functional characterization a rice NF-Y heterotrimer complex NF-YB1-YC12-bHLH144, which is formed by the binding of NF-YB1 to NF-YC12 and then bHLH144 in a sequential order. Knock-out of each of the complex genes resulted in alteration of grain qualities in all the mutants as well as reduced grain size in crnf-yb1 and crnf-yc12. RNA-seq analysis identified 1496 genes that were commonly regulated by NF-YB1 and NF-YC12, including the key granule-bound starch synthase gene Wx. NF-YC12 and bHLH144 maintain NF-YB1 stability from the degradation mediated by ubiquitin/26S proteasome, while NF-YB1 directly binds to the 'G-box' domain of Wx promoter and activates Wx transcription, hence to regulate rice grain quality. Finally, we revealed a novel grain quality regulatory pathway controlled by NF-YB1-YC12-bHLH144 complex, which has great potential for rice genetic improvement.


Assuntos
Regulação da Expressão Gênica de Plantas , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Sintase do Amido/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Grão Comestível , Oryza/genética , Proteínas de Plantas/genética , Sementes , Sintase do Amido/genética
19.
Sci Rep ; 8(1): 13753, 2018 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-30214055

RESUMO

CRISPR/Cas9 is a programmable nuclease composed of the Cas9 protein and a guide RNA (gRNA) molecule. To create a mutant potato, a powerful genome-editing system was required because potato has a tetraploid genome. The translational enhancer dMac3, consisting of a portion of the OsMac3 mRNA 5'-untranslated region, greatly enhanced the production of the protein encoded in the downstream ORF. To enrich the amount of Cas9, we applied the dMac3 translational enhancer to the Cas9 expression system with multiple gRNA genes. CRISPR/Cas9 systems targeting the potato granule-bound starch synthase I (GBSSI) gene examined the frequency of mutant alleles in transgenic potato plants. The efficiency of the targeted mutagenesis strongly increased when the dMac3-installed Cas9 was used. In this case, the ratio of transformants containing four mutant alleles reached approximately 25% when estimated by CAPS analysis. The mutants that exhibited targeted mutagenesis in the GBSSI gene showed characteristics of low amylose starch in their tubers. This result suggests that our system may facilitate genome-editing events in polyploid plants.


Assuntos
Plantas Geneticamente Modificadas/genética , RNA Guia/genética , Solanum tuberosum/genética , Sintase do Amido/genética , Alelos , Sistemas CRISPR-Cas/genética , Edição de Genes , Vetores Genéticos/genética , Mutagênese/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Sequências Reguladoras de Ácido Nucleico/genética , Solanum tuberosum/crescimento & desenvolvimento
20.
Sci Adv ; 4(9): eaat6086, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30191180

RESUMO

Crop diversification required to meet demands for food security and industrial use is often challenged by breeding time and amenability of varieties to genome modification. Cassava is one such crop. Grown for its large starch-rich storage roots, it serves as a staple food and a commodity in the multibillion-dollar starch industry. Starch is composed of the glucose polymers amylopectin and amylose, with the latter strongly influencing the physicochemical properties of starch during cooking and processing. We demonstrate that CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated targeted mutagenesis of two genes involved in amylose biosynthesis, PROTEIN TARGETING TO STARCH (PTST1) or GRANULE BOUND STARCH SYNTHASE (GBSS), can reduce or eliminate amylose content in root starch. Integration of the Arabidopsis FLOWERING LOCUS T gene in the genome-editing cassette allowed us to accelerate flowering-an event seldom seen under glasshouse conditions. Germinated seeds yielded S1, a transgene-free progeny that inherited edited genes. This attractive new plant breeding technique for modified cassava could be extended to other crops to provide a suite of novel varieties with useful traits for food and industrial applications.


Assuntos
Manihot/genética , Melhoramento Vegetal/métodos , Proteínas de Plantas/genética , Sintase do Amido/genética , Amido/genética , Proteínas de Arabidopsis/genética , Sistemas CRISPR-Cas , Produtos Agrícolas/genética , Edição de Genes , Germinação , Manihot/química , Mutagênese , Plantas Geneticamente Modificadas/genética , Amido/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA