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1.
J Integr Plant Biol ; 2020 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-32149461

RESUMO

Male sterility is a prerequisite for hybrid seed production. The phytohormone gibberellin (GA) are involved in regulating the male reproductive development, but the mechanism underlying GA homeostasis in anther development remains less understood. Here, we report the isolation and characterization of a new positive regulator of GA homeostasis, Swollen Anther Wall 1 (SAW1), for anther development in rice (Oryza sativa). Rice plants carrying the recessive mutant allele saw1 produces abnormal anthers with a swollen anther wall and aborted pollen. CRISPR/Cas9-mediated knockout of SAW1 in rice generated similar male sterile plants. SAW1 encodes a novel nucleus-localizing CCCH-tandem zinc finger protein, and this protein could directly bind to the promoter region of the GA synthesis gene OsGA20ox3 to induce its anther-specific expression. In the saw1 anther, the significantly decreased OsGA20ox3 expression resulted in lower bioactive GA content, which in turn caused the lower expression of the GA-inducible anther-regulator gene OsGAMYB. Thus, our results disclose the mechanism of the SAW1-GA20ox3-GAMYB pathway in controlling rice anther development, and provide a new target gene for the rapid generation of male sterile lines by genome editing for hybrid breeding. This article is protected by copyright. All rights reserved.

6.
Mol Plant ; 11(12): 1440-1448, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30296601

RESUMO

Carotenoids are important phytonutrients with antioxidant properties, and are widely used in foods and feedstuffs as supplements. Astaxanthin, a red-colored ketocarotenoid, has strong antioxidant activity and thus can benefit human health. However, astaxanthin is not produced in most higher plants. Here we report the bioengineering of astaxanthin biosynthesis in rice endosperm by introducing four synthetic genes, sZmPSY1, sPaCrtI, sCrBKT, and sHpBHY, which encode the enzymes phytoene synthase, phytoene desaturase, ß-carotene ketolase, and ß-carotene hydroxylase, respectively. Transgneic overexpression of two (sZmPSY1 and sPaCrtI), three (sZmPSY1, sPaCrtI and sCrBKT), and all these four genes driven by rice endosperm-specific promoters established the carotenoid/ketocarotenoid/astaxanthin biosynthetic pathways in the endosperm and thus resulted in various types of germplasm, from the yellow-grained ß-carotene-enriched Golden Rice to orange-red-grained Canthaxanthin Rice and Astaxanthin Rice, respectively. Grains of Astaxanthin Rice were enriched with astaxanthin in the endosperm and had higher antioxidant activity. These results proved that introduction of a minimal set of four transgenes enables de novo biosynthesis of astaxanthin in the rice endosperm. This work provides a successful example for synthetic biology in plants and biofortification in crops; the biofortified rice products generated by this study could be consumed as health-promoting foods and processed to produce dietary supplements.


Assuntos
Endosperma/metabolismo , Engenharia Genética , Oryza/genética , Oryza/metabolismo , Antioxidantes/metabolismo , Biofortificação , Cantaxantina/biossíntese , Cantaxantina/metabolismo , Plantas Geneticamente Modificadas , Xantofilas/biossíntese , Xantofilas/metabolismo , beta Caroteno/biossíntese , beta Caroteno/metabolismo
7.
Mol Plant ; 11(5): 720-735, 2018 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-29524649

RESUMO

Plant height has a major effect on grain yield in crops such as rice (Oryza sativa), and the hormone gibberellic acid (GA) regulates many developmental processes that feed into plant height. Rice ELONGATED UPPERMOST INTERNODE1 (Eui1) encodes a GA-deactivating enzyme governing elongation of the uppermost internode. The expression of Eui1 is finely tuned, thereby maintaining homeostasis of endogenous bioactive GA and producing plants of normal plant height. Here, we identified a dominant dwarf mutant, dEui1, caused by the deletion of an RY motif-containing cis-silencing element (SE1) in the intron of Eui1. Detailed genetic and molecular analysis of SE1 revealed that this intronic cis element recruits at least one trans-acting repressor complex, containing the B3 repressors OsVAL2 and OsGD1, the SAP18 co-repressor, and the histone deacetylase OsHDA710, to negatively regulate the expression of Eui1. This complex generates closed chromatin at Eui1, suppressing Eui1 expression and modulating GA homeostasis. Loss of SE1 or dysfunction of the complex components impairs histone deacetylation and H3K27me3 methylation of Eui1 chromatin, thereby increasing Eui1 transcription and decreasing bioactive GA, producing dwarfism in rice. Together, our results reveal a novel silencing mechanism in which the intronic cis element SE1 negatively regulates Eui1 expression via repressor complexes that modulate histone deacetylation and/or methylation.


Assuntos
Regulação da Expressão Gênica de Plantas , Íntrons , Oryza/genética , Proteínas de Plantas/metabolismo , Proteínas Repressoras/metabolismo , Acetilação , Inativação Gênica , Histona Desmetilases/metabolismo , Histonas/metabolismo , Mutagênese , Oryza/metabolismo
8.
Nat Commun ; 8(1): 1310, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-29101356

RESUMO

Hybrids between divergent populations commonly show hybrid sterility; this reproductive barrier hinders hybrid breeding of the japonica and indica rice (Oryza sativa L.) subspecies. Here we show that structural changes and copy number variation at the Sc locus confer japonica-indica hybrid male sterility. The japonica allele, Sc-j, contains a pollen-essential gene encoding a DUF1618-domain protein; the indica allele, Sc-i, contains two or three tandem-duplicated ~ 28-kb segments, each carrying an Sc-j-homolog with a distinct promoter. In Sc-j/Sc-i hybrids, the high-expression of Sc-i in sporophytic cells causes suppression of Sc-j expression in pollen and selective abortion of Sc-j-pollen, leading to transmission ratio distortion. Knocking out one or two of the three Sc-i copies by CRISPR/Cas9 rescues Sc-j expression and male fertility. Our results reveal the gene dosage-dependent allelic suppression as a mechanism of hybrid incompatibility, and provide an effective approach to overcome the reproductive barrier for hybrid breeding.


Assuntos
Oryza/genética , Infertilidade das Plantas/genética , Alelos , Variações do Número de Cópias de DNA , DNA de Plantas/genética , Dosagem de Genes , Técnicas de Inativação de Genes , Genes de Plantas , Variação Estrutural do Genoma , Hibridização Genética , Modelos Genéticos , Oryza/classificação , Oryza/fisiologia , Melhoramento Vegetal , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento , Especificidade da Espécie
10.
Front Plant Sci ; 8: 1071, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28676815

RESUMO

Chimeric fluorescent fusion proteins have been employed as a powerful tool to reveal the subcellular localizations and dynamics of proteins in living cells. Co-expression of a fluorescent fusion protein with well-known organelle markers in the same cell is especially useful in revealing its spatial and temporal functions of the protein in question. However, the conventional methods for co-expressing multiple fluorescent tagged proteins in plants have the drawbacks of low expression efficiency, variations in the expression level and time-consuming genetic crossing. Here, we have developed a novel robust system that allows for high-efficient co-expression of multiple chimeric fluorescent fusion proteins in plants in a time-saving fashion. This system takes advantage of employing a single expression vector which consists of multiple semi-independent expressing cassettes for the protein co-expression thereby overcoming the limitations of using multiple independent expressing plasmids. In addition, it is a highly manipulable DNA assembly system, in which modification and recombination of DNA molecules are easily achieved through an optimized one-step assembly reaction. By employing this effective system, we demonstrated that co-expression of two chimeric fluorescent fusion reporter proteins of vacuolar sorting receptor and secretory carrier membrane protein gave rise to their perspective subcellular localizations in plants via both transient expression and stable transformation. Thus, we believed that this technical advance represents a promising approach for multi-color-protein co-expression in plant cells.

11.
Mol Plant ; 10(7): 918-929, 2017 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-28666688

RESUMO

Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice (Oryza sativa L.), could provide health-promoting foods for improving human health. However, engineering metabolic pathways for biofortification remains difficult, and previous attempts to engineer anthocyanin production in rice endosperm failed because of the sophisticated genetic regulatory network of its biosynthetic pathway. In this study, we developed a high-efficiency vector system for transgene stacking and used it to engineer anthocyanin biosynthesis in rice endosperm. We made a construct containing eight anthocyanin-related genes (two regulatory genes from maize and six structural genes from Coleus) driven by the endosperm-specific promoters,plus a selectable marker and a gene for marker excision. Transformation of rice with this construct generated a novel biofortified germplasm "Purple Endosperm Rice" (called "Zijingmi" in Chinese), which has high anthocyanin contents and antioxidant activity in the endosperm. This anthocyanin production results from expression of the transgenes and the resulting activation (or enhancement) of expression of 13 endogenous anthocyanin biosynthesis genes that are silenced or expressed at low levels in wild-type rice endosperm. This study provides an efficient, versatile toolkit for transgene stacking and demonstrates its use for successful engineering of a sophisticated biological pathway, suggesting the potential utility of this toolkit for synthetic biology and improvement of agronomic traits in plants.


Assuntos
Antocianinas/biossíntese , Endosperma/metabolismo , Oryza/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Endosperma/genética , Engenharia Genética , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética
13.
Front Plant Sci ; 8: 206, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28261256

RESUMO

Yield is one of the most important yet complex crop traits. To improve our understanding of the genetic basis of yield establishment, and to identify candidate genes responsible for yield improvement in Brassica napus, we performed genome-wide association studies (GWAS) for seven yield-determining traits [main inflorescence pod number (MIPN), branch pod number (BPN), pod number per plant (PNP), seed number per pod (SPP), thousand seed weight, main inflorescence yield (MIY), and branch yield], using data from 520 diverse B. napus accessions from two different yield environments. In total, we detected 128 significant single nucleotide polymorphisms (SNPs), 93 of which were revealed as novel by integrative analysis. A combination of GWAS and transcriptome sequencing on 21 haplotype blocks from samples pooled by four extremely high-yielding or low-yielding accessions revealed the differential expression of 14 crucial candiate genes (such as Bna.MYB83, Bna.SPL5, and Bna.ROP3) associated with multiple traits or containing multiple SNPs associated with the same trait. Functional annotation and expression pattern analyses further demonstrated that these 14 candiate genes might be important in developmental processes and biomass accumulation, thus affecting the yield establishment of B. napus. These results provide valuable information for understanding the genetic mechanisms underlying the establishment of high yield in B. napus, and lay the foundation for developing high-yielding B. napus varieties.

14.
Mol Plant ; 9(7): 961-74, 2016 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-27108381

RESUMO

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) genome editing system (CRISPR/Cas9) is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adaption of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, factors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement.


Assuntos
Sistemas CRISPR-Cas/fisiologia , Edição de RNA/genética , Sistemas CRISPR-Cas/genética , Edição de Genes , Engenharia Genética , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética
15.
PLoS One ; 10(10): e0139392, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26448466

RESUMO

Proanthocyanidins (PAs) play an important role in plant disease defense and have beneficial effects on human health. We isolated and characterized a novel R2R3 MYB-type PA-regulator SsMYB3 from a well-known ornamental plant, coleus (Solenostemon scutellarioides), to study the molecular regulation of PAs and to engineer PAs biosynthesis. The expression level of SsMYB3 was correlated with condensed tannins contents in various coleus tissues and was induced by wounding and light. A complementation test in the Arabidopsis tt2 mutant showed that SsMYB3 could restore the PA-deficient seed coat phenotype and activated expression of the PA-specific gene ANR and two related genes, DFR and ANS. In yeast two-hybrid assays, SsMYB3 interacted with the Arabidopsis AtTT8 and AtTTG1 to reform the ternary transcriptional complex, and also interacted with two tobacco bHLH proteins (NtAn1a and NtJAF13-1) and a WD40 protein, NtAn11-1. Ectopic overexpression of SsMYB3 in transgenic tobacco led to almost-white flowers by greatly reducing anthocyanin levels and enhancing accumulation of condensed tannins. This overexpression of SsMYB3 upregulated the key PA genes (NtLAR and NtANR) and late anthocyanin structural genes (NtDFR and NtANS), but downregulated the expression of the final anthocyanin gene NtUFGT. The formative SsMYB3-complex represses anthocyanin accumulation by directly suppressing the expression of the final anthocyanin structural gene NtUFGT, through competitive inhibition or destabilization of the endogenous NtAn2-complex formation. These results suggested that SsMYB3 may form a transcription activation complex to regulate PA biosynthesis in the Arabidopsis tt2 mutant and transgenic tobacco. Our findings suggest that SsMYB3 is involved in the regulation of PA biosynthesis in coleus and has the potential as a molecular tool for manipulating biosynthesis of PAs in fruits and other crops using metabolic engineering.


Assuntos
Coleus/genética , Expressão Ectópica do Gene , Pigmentação/genética , Proteínas de Plantas/genética , Proantocianidinas/metabolismo , Tabaco/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Clonagem Molecular , Flores/genética , Dados de Sequência Molecular , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Proantocianidinas/análise , Alinhamento de Sequência , Espectrofotometria Ultravioleta
16.
Molecules ; 20(9): 16833-51, 2015 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-26389875

RESUMO

Phenylalanine ammonia-lyase (PAL) is the first enzyme involved in the phenylpropanoid pathway and plays important roles in the secondary metabolisms, development and defense of plants. To study the molecular function of PAL in anthocyanin synthesis of Coleus (Solenostemon scutellarioides (L.) Codd), a Coleus PAL gene designated as SsPAL1 was cloned and characterized using a degenerate oligonucleotide primer PCR and RACE method. The full-length SsPAL1 was 2450 bp in size and consisted of one intron and two exons encoding a polypeptide of 711 amino acids. The deduced SsPAL1 protein showed high identities and structural similarities with other functional plant PAL proteins. A series of putative cis-acting elements involved in transcriptional regulation, light and stress responsiveness were found in the upstream regulatory sequence of SsPAL1. Transcription pattern analysis indicated that SsPAL1 was constitutively expressed in all tissues examined and was enhanced by light and different abiotic factors. The recombinant SsPAL1 protein exhibited high PAL activity, at optimal conditions of 60 °C and pH 8.2. Although the levels of total PAL activity and total anthocyanin concentration have a similar variation trend in different Coleus cultivars, there was no significant correlation between them (r = 0.7529, p > 0.1), suggesting that PAL was not the rate-limiting enzyme for the downstream anthocyanin biosynthetic branch in Coleus. This study enables us to further understand the role of SsPAL1 in the phenylpropanoid (flavonoids, anthocyanins) biosynthesis in Coleus at the molecular level.


Assuntos
Coleus/enzimologia , Fenilalanina Amônia-Liase/isolamento & purificação , Fenilalanina Amônia-Liase/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Dados de Sequência Molecular , Fenilalanina Amônia-Liase/química , Fenilalanina Amônia-Liase/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Reação em Cadeia da Polimerase , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos
17.
Mol Plant ; 8(8): 1274-84, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25917172

RESUMO

CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.


Assuntos
Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Genoma de Planta , Oryza/genética , Edição de RNA/genética , Alelos , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Vetores Genéticos/metabolismo , Padrões de Herança/genética , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Plantas Geneticamente Modificadas , RNA Guia/genética
19.
Mol Plant ; 8(2): 290-302, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25616388

RESUMO

The majority of plant disease resistance (R) genes encode proteins that share common structural features. However, the transcription activator-like effector (TALE)-associated executor type R genes show no considerable sequence homology to any known R genes. We adopted a map-based cloning approach and TALE-based technology to isolate and characterize Xa23, a new executor R gene derived from wild rice (Oryza rufipogon) that confers an extremely broad spectrum of resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xa23 encodes a 113 amino acid protein that shares 50% identity with the known executor R protein XA10. The predicted transmembrane helices in XA23 also overlap with those of XA10. Unlike Xa10, however, Xa23 transcription is specifically activated by AvrXa23, a TALE present in all examined Xoo field isolates. Moreover, the susceptible xa23 allele has an identical open reading frame of Xa23 but differs in promoter region by lacking the TALE binding element (EBE) for AvrXa23. XA23 can trigger a strong hypersensitive response in rice, tobacco, and tomato. Our results provide the first evidence that plant genomes have an executor R gene family of which members execute their function and spectrum of disease resistance by recognizing the cognate TALEs in the pathogen.


Assuntos
Oryza/metabolismo , Oryza/microbiologia , Proteínas de Plantas/metabolismo , Resistência à Doença , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Xanthomonas/fisiologia
20.
Mol Plant ; 2014 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-25385701

RESUMO

The majority of plant disease resistance (R) genes encode proteins that share common structural features. However, the transcription activator-like effector (TALE) associated executor type R genes show no considerable sequence homology to any known R genes. We adopted a map-based cloning approach and TALE-based technology to isolate and characterize Xa23, a new executor R gene derived from the wild rice (Oryza rufipogon) that confers an extremely broad spectrum of resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xa23 encodes a 113-amino acid protein that shares 50% identity to the known executor R protein XA10. The predicted transmembrane helices in XA23 also overlap with those of XA10. Unlike Xa10, however, Xa23 transcription is specifically activated by AvrXa23, a TALE present in all examined Xoo field isolates. Moreover, the susceptible xa23 allele has an identical open reading frame of Xa23, but differs in promoter region by lacking the TALE binding-element (EBE) for AvrXa23. XA23 can trigger strong hypersensitive response in rice, tobacco and tomato. Our results provide the first evidence that plant genomes have an executor R gene family in which members execute their function and spectrum of disease resistance by recognizing the cognate TALEs in pathogen.

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