RESUMEN
Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Although new technologies are being applied to tackle this problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in CRISPR/Cas9-mediated gene engineering have paved the way to accelerate plant breeding to meet this increasing demand. However, many traits are governed by multiple small-effect genes operating in complex interactive networks. Here, we present the gene discovery pipeline BREEDIT, which combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought tolerance. We induced gene knockouts in 48 growth-related genes into maize (Zea mays) using CRISPR/Cas9 and generated a collection of over 1,000 gene-edited plants. The edited populations displayed (on average) 5%-10% increases in leaf length and up to 20% increases in leaf width compared with the controls. For each gene family, edits in subsets of genes could be associated with enhanced traits, allowing us to reduce the gene space to be considered for trait improvement. BREEDIT could be rapidly applied to generate a diverse collection of mutants to identify promising gene modifications for later use in breeding programs.
Asunto(s)
Edición Génica , Zea mays , Zea mays/genética , Sistemas CRISPR-Cas/genética , Plantas Modificadas Genéticamente/genética , Herencia Multifactorial , Fitomejoramiento , Genoma de Planta/genéticaRESUMEN
Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium-mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene-editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1-VIR2 virulence helper plasmid to improve T-DNA delivery, and/or expressing a fusion protein between a GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1-VIR2 helper vector with no effect on event quality regarding T-DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5- to 6.5-fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR-/Cas9-mediated gene editing. Our results demonstrate how a GRF-GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene-editing applications and molecular biology studies in maize.
Asunto(s)
Vectores Genéticos , Plantas Modificadas Genéticamente , Transformación Genética , Zea mays , Zea mays/genética , Zea mays/crecimiento & desarrollo , Edición Génica/métodos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ADN Bacteriano/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Agrobacterium tumefaciens/genética , Plásmidos/genéticaRESUMEN
Plant flowers have a functional life span during which pollination and fertilization occur to ensure seed and fruit development. Once flower senescence is initiated, the potential to set seed or fruit is irrevocably lost. In maize, silk strands are the elongated floral stigmas that emerge from the husk-enveloped inflorescence to intercept airborne pollen. Here we show that KIRA1-LIKE1 (KIL1), an ortholog of the Arabidopsis NAC (NAM (NO APICAL MERISTEM), ATAF1/2 (Arabidopsis thaliana Activation Factor1 and 2) and CUC (CUP-SHAPED COTYLEDON 2)) transcription factor KIRA1, promotes senescence and programmed cell death (PCD) in the silk strand base, ending the window of accessibility for fertilization of the ovary. Loss of KIL1 function extends silk receptivity and thus strongly increases kernel yield following late pollination. This phenotype offers new opportunities for possibly improving yield stability in cereal crops. Moreover, despite diverging flower morphologies and the substantial evolutionary distance between Arabidopsis and maize, our data indicate remarkably similar principles in terminating floral receptivity by PCD, whose modulation offers the potential to be widely used in agriculture.
Asunto(s)
Arabidopsis , Arabidopsis/fisiología , Fertilidad/genética , Flores/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Seda/genética , Seda/metabolismo , Zea mays/genética , Zea mays/metabolismoRESUMEN
The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases coordinate the DNA damage response. The roles described for Arabidopsis thaliana ATR and ATM are assumed to be conserved over other plant species, but molecular evidence is scarce. Here, we demonstrate that the functions of ATR and ATM are only partially conserved between Arabidopsis and maize (Zea mays). In both species, ATR and ATM play a key role in DNA repair and cell cycle checkpoint activation, but whereas Arabidopsis plants do not suffer from the absence of ATR under control growth conditions, maize mutant plants accumulate replication defects, likely due to their large genome size. Moreover, contrarily to Arabidopsis, maize ATM deficiency does not trigger meiotic defects, whereas the ATR kinase appears to be crucial for the maternal fertility. Strikingly, ATR is required to repress premature endocycle onset and cell death in the maize endosperm. Its absence results in a reduction of kernel size, protein and starch content, and a stochastic death of kernels, a process being counteracted by ATM. Additionally, while Arabidopsis atr atm double mutants are viable, no such mutants could be obtained for maize. Therefore, our data highlight that the mechanisms maintaining genome integrity may be more important for vegetative and reproductive development than previously anticipated.
Asunto(s)
Reparación del ADN/genética , Endospermo/genética , Proteínas de Plantas/genética , Zea mays/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Sistemas CRISPR-Cas , Muerte Celular/genética , Roturas del ADN de Doble Cadena , Replicación del ADN/genética , Endospermo/citología , Inestabilidad Genómica , Mutación , Células Vegetales , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Semillas/citología , Semillas/genética , Semillas/crecimiento & desarrollo , Zea mays/citología , Zea mays/crecimiento & desarrolloRESUMEN
Agrobacterium spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although Agrobacterium spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of Agrobacterium has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agrobacterium rhizogenes As an example, we generated EHA105 strains with loss-of-function mutations in recA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development by A. rhizogenes K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of "engineering the engineer," leading to improved Agrobacterium strains for more efficient plant transformation and gene editing.
Asunto(s)
Agrobacterium/genética , Proteínas Asociadas a CRISPR/genética , Edición Génica/métodos , Agrobacterium tumefaciens/genética , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN de Plantas/genética , Genes de Plantas/genética , Genoma de Planta/genética , Mutagénesis/genética , Mutación/genética , Zea mays/genéticaRESUMEN
A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.
Asunto(s)
Edición Génica , Zea mays , Zea mays/genética , Sistemas CRISPR-Cas/genética , Genoma de Planta , Haploidia , Plantas Modificadas GenéticamenteRESUMEN
Plant organ size and shape are major agronomic traits that depend on cell division and expansion, which are both regulated by complex gene networks. In several eudicot species belonging to the rosid clade, organ growth is controlled by a repressor complex consisting of PEAPOD (PPD) and KINASE-INDUCIBLE DOMAIN INTERACTING (KIX) proteins. The role of these proteins in asterids, which together with the rosids constitute most of the core eudicot species, is unknown. We used Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 genome editing to target SlKIX8 and SlKIX9 in the asterid model species tomato (Solanum lycopersicum) and analyzed loss-of-function phenotypes. Loss-of-function of SlKIX8 and SlKIX9 led to the production of enlarged, dome-shaped leaves and these leaves exhibited increased expression of putative Solanum lycopersicum PPD (SlPPD target genes. Unexpectedly, kix8 kix9 mutants carried enlarged fruits with increased pericarp thickness due to cell expansion. At the molecular level, protein interaction assays indicated that SlKIX8 and SlKIX9 act as adaptors between the SlPPD and SlTOPLESS co-repressor proteins. Our results show that KIX8 and KIX9 are regulators of organ growth in asterids and can be used in strategies to improve important traits in produce such as thickness of the fruit flesh.
Asunto(s)
Frutas/crecimiento & desarrollo , Frutas/genética , Reguladores del Crecimiento de las Plantas/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/genética , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/genética , Productos Agrícolas/genética , Productos Agrícolas/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , FenotipoRESUMEN
SAMBA has been identified as a plant-specific regulator of the anaphase-promoting complex/cyclosome (APC/C) that controls unidirectional cell cycle progression in Arabidopsis (Arabidopsis thaliana), but so far its role has not been studied in monocots. Here, we show the association of SAMBA with the APC/C is conserved in maize (Zea mays). Two samba genome edited mutants showed growth defects, such as reduced internode length, shortened upper leaves with erect leaf architecture, and reduced leaf size due to an altered cell division rate and cell expansion, which aggravated with plant age. The two mutants differed in the severity and developmental onset of the phenotypes, because samba-1 represented a knockout allele, while translation re-initiation in samba-3 resulted in a truncated protein that was still able to interact with the APC/C and regulate its function, albeit with altered APC/C activity and efficiency. Our data are consistent with a dosage-dependent role for SAMBA to control developmental processes for which a change in growth rate is pivotal.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/genética , Zea mays/crecimiento & desarrollo , Zea mays/genética , Productos Agrícolas/genética , Productos Agrícolas/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Variación Genética , Genotipo , FenotipoRESUMEN
The plant shoot apex houses the shoot apical meristem, a highly organized and active stem-cell tissue where molecular signaling in discrete cells determines when and where leaves are initiated. We optimized a spatial transcriptomics approach, in situ sequencing (ISS), to colocalize the transcripts of 90 genes simultaneously on the same section of tissue from the maize (Zea mays) shoot apex. The RNA ISS technology reported expression profiles that were highly comparable with those obtained by in situ hybridizations (ISHs) and allowed the discrimination between tissue domains. Furthermore, the application of spatial transcriptomics to the shoot apex, which inherently comprised phytomers that are in gradual developmental stages, provided a spatiotemporal sequence of transcriptional events. We illustrate the power of the technology through PLASTOCHRON1 (PLA1), which was specifically expressed at the boundary between indeterminate and determinate cells and partially overlapped with ROUGH SHEATH1 and OUTER CELL LAYER4 transcripts. Also, in the inflorescence, PLA1 transcripts localized in cells subtending the lateral primordia or bordering the newly established meristematic region, suggesting a more general role of PLA1 in signaling between indeterminate and determinate cells during the formation of lateral organs. Spatial transcriptomics builds on RNA ISH, which assays relatively few transcripts at a time and provides a powerful complement to single-cell transcriptomics that inherently removes cells from their native spatial context. Further improvements in resolution and sensitivity will greatly advance research in plant developmental biology.
Asunto(s)
Células Vegetales , Proteínas de Plantas/química , Análisis de Secuencia de ARN/métodos , Zea mays/química , Expresión Génica , Proteínas de Plantas/genética , Análisis de Secuencia de ARN/instrumentación , Zea mays/genéticaRESUMEN
The HD-ZIP III transcription factor REVOLUTA (REV) is involved in early leaf development, as well as in leaf senescence. REV directly binds to the promoters of senescence-associated genes, including the central regulator WRKY53. As this direct regulation appears to be restricted to senescence, we aimed to characterize protein-interaction partners of REV which could mediate this senescence-specificity. The interaction between REV and the TIFY family member TIFY8 was confirmed by yeast two-hybrid assays, as well as by bimolecular fluorescence complementation in planta. This interaction inhibited REV's function as an activator of WRKY53 expression. Mutation or overexpression of TIFY8 accelerated or delayed senescence, respectively, but did not significantly alter early leaf development. Jasmonic acid (JA) had only a limited effect on TIFY8 expression or function; however, REV appears to be under the control of JA signaling. Accordingly, REV also interacted with many other members of the TIFY family, namely the PEAPODs and several JAZ proteins in the yeast system, which could potentially mediate the JA-response. Therefore, REV appears to be under the control of the TIFY family in two different ways: a JA-independent way through TIFY8, which controls REV function in senescence, and a JA-dependent way through PEAPODs and JAZ proteins.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ciclopentanos/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Oxilipinas/metabolismo , Hojas de la Planta/metabolismo , Senescencia de la Planta , Factores de Transcripción/metabolismoRESUMEN
Recent advances in genome editing have enormously enhanced the effort to develop biotechnology crops for more sustainable food production. CRISPR/Cas, the most versatile genome-editing tool, has shown the potential to create genome modifications that range from gene knockout and gene expression pattern modulations to allele-specific changes in order to design superior genotypes harboring multiple improved agronomic traits. However, a frequent bottleneck is the delivery of CRISPR/Cas to crops that are less amenable to transformation and regeneration. Several technologies have recently been proposed to overcome transformation recalcitrance, including HI-Edit/IMGE and ectopic/transient expression of genes encoding morphogenic regulators. These technologies allow the eroding of the barriers that make crops inaccessible for genome editing. In this review, we discuss the advances in genome editing in crops with a particular focus on the use of technologies to improve complex traits such as water use efficiency, drought stress, and yield in maize.
RESUMEN
Plants produce specialized metabolites to protect themselves from biotic enemies. Members of the Solanaceae family accumulate phenylpropanoid-polyamine conjugates (PPCs) in response to attackers while also maintaining a chemical barrier of steroidal glycoalkaloids (SGAs). Across the plant kingdom, biosynthesis of such defense compounds is promoted by jasmonate signaling in which clade IIIe basic helix-loop-helix (bHLH) transcription factors play a central role. By characterizing hairy root mutants obtained through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (CRISPR-Cas9) genome editing, we show that the tomato clade IIIe bHLH transcription factors, MYC1 and MYC2, redundantly control jasmonate-inducible PPC and SGA production, and are also essential for constitutive SGA biosynthesis. Double myc1 myc2 loss-of-function tomato hairy roots displayed suppressed constitutive expression of SGA biosynthesis genes, and severely reduced levels of the main tomato SGAs α-tomatine and dehydrotomatine. In contrast, basal expression of genes involved in PPC biosynthesis was not affected. CRISPR-Cas9(VQR) genome editing of a specific cis-regulatory element, targeted by MYC1/2, in the promoter of a SGA precursor biosynthesis gene led to decreased constitutive expression of this gene, but did not affect its jasmonate inducibility. Our results demonstrate that clade IIIe bHLH transcriptional regulators have evolved under the control of distinct regulatory cues to specifically steer constitutive and stress-inducible specialized metabolism.
Asunto(s)
Solanum lycopersicum , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Ciclopentanos/metabolismo , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Oxilipinas/metabolismo , Poliaminas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The plant hormone auxin, a master coordinator of development, regulates hypocotyl elongation during seedling growth. We previously identified the synthetic molecule RubNeddin 1 (RN1), which induces degradation of the AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors INDOLE-3-ACETIC ACID-INDUCIBLE3 (IAA3) and IAA7 in planta and strongly promotes hypocotyl elongation. In the present study, we show that despite the structural similarity of RN1 to the synthetic auxin 2,4-dichlorophenoxyacetic-acid (2,4-D), direct treatments with these compounds in Arabidopsis (Arabidopsis thaliana) result in distinct effects, possibly due to enhanced uptake of RN1 and low-level, chronic release of 2,4-D from RN1 in planta. We confirm RN1-induced hypocotyl elongation occurs via specific TRANSPORT INHIBITOR RESISTANT1 (TIR1)/AUXIN SIGNALING F-BOX (AFB) receptor-mediated auxin signaling involving TIR1, AFB2, and AFB5. Using a transcriptome profiling strategy and candidate gene approach, we identify the genes ZINC FINGER OF ARABIDOPSIS THALIANA10 (ZAT10), ARABIDOPSIS TOXICOS EN LEVADURA31 (ATL31), and WRKY DNA-BINDING PROTEIN33 (WRKY33) as being rapidly upregulated by RN1, despite being downregulated by 2,4-D treatment. RN1-induced expression of these genes also occurs via TIR1/AFB-mediated auxin signaling. Our results suggest both hypocotyl elongation and transcription of these genes are induced by RN1 via the promoted degradation of the AUX/IAA transcriptional repressor IAA7. Moreover, these three genes, which are known to be stress-related, act in an inter-dependent transcriptional regulatory network controlling hypocotyl elongation. Together, our results suggest ZAT10, ATL31, and WRKY33 take part in a common gene network regulating hypocotyl elongation in Arabidopsis downstream of a selective auxin perception module likely involving TIR1, AFB2, and AFB5 and inducing the degradation of IAA7.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Hipocótilo/crecimiento & desarrollo , Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Hipocótilo/genéticaRESUMEN
Auxin phytohormones control most aspects of plant development through a complex and interconnected signaling network. In the presence of auxin, AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors are targeted for degradation by the SKP1-CULLIN1-F-BOX (SCF) ubiquitin-protein ligases containing TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB). CULLIN1-neddylation is required for SCFTIR1/AFB functionality, as exemplified by mutants deficient in the NEDD8-activating enzyme subunit AUXIN-RESISTANT 1 (AXR1). Here, we report a chemical biology screen that identifies small molecules requiring AXR1 to modulate plant development. We selected four molecules of interest, RubNeddin 1 to 4 (RN1 to -4), among which RN3 and RN4 trigger selective auxin responses at transcriptional, biochemical, and morphological levels. This selective activity is explained by their ability to consistently promote the interaction between TIR1 and a specific subset of AUX/IAA proteins, stimulating the degradation of particular AUX/IAA combinations. Finally, we performed a genetic screen using RN4, the RN with the greatest potential for dissecting auxin perception, which revealed that the chromatin remodeling ATPase BRAHMA is implicated in auxin-mediated apical hook development. These results demonstrate the power of selective auxin agonists to dissect auxin perception for plant developmental functions, as well as offering opportunities to discover new molecular players involved in auxin responses.
Asunto(s)
Ácidos Indolacéticos/metabolismo , Desarrollo de la Planta/fisiología , Proteolisis , Factores de Transcripción/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas F-Box/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteína NEDD8/genética , Desarrollo de la Planta/genética , Reguladores del Crecimiento de las Plantas/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Receptores de Superficie Celular/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Plantones/metabolismo , Transducción de Señal , Transcripción Genética/efectos de los fármacosRESUMEN
Plant roots are specialized belowground organs that spatiotemporally shape their development in function of varying soil conditions. This root plasticity relies on intricate molecular networks driven by phytohormones, such as auxin and jasmonate (JA). Loss-of-function of the NOVEL INTERACTOR OF JAZ (NINJA), a core component of the JA signaling pathway, leads to enhanced triterpene biosynthesis, in particular of the thalianol gene cluster, in Arabidopsis thaliana roots. We have investigated the biological role of thalianol and its derivatives by focusing on Thalianol Synthase (THAS) and Thalianol Acyltransferase 2 (THAA2), two thalianol cluster genes that are upregulated in the roots of ninja mutant plants. THAS and THAA2 activity was investigated in yeast, and metabolite and phenotype profiling of thas and thaa2 loss-of-function plants was carried out. THAA2 was shown to be responsible for the acetylation of thalianol and its derivatives, both in yeast and in planta. In addition, THAS and THAA2 activity was shown to modulate root development. Our results indicate that the thalianol pathway is not only controlled by phytohormonal cues, but also may modulate phytohormonal action itself, thereby affecting root development and interaction with the environment.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Triterpenos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclopentanos , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos , Oxilipinas , Raíces de Plantas/metabolismo , Transducción de SeñalRESUMEN
Organ size control is of particular importance for developmental biology and agriculture, but the mechanisms underlying organ size regulation remain elusive in plants. Meristemoids, which possess stem cell-like properties, have been recognized to play important roles in leaf growth. We have recently reported that the Arabidopsis F-box protein STERILE APETALA (SAP)/SUPPRESSOR OF DA1 (SOD3) promotes meristemoid proliferation and regulates organ size by influencing the stability of the transcriptional regulators PEAPODs (PPDs). Here we demonstrate that KIX8 and KIX9, which function as adaptors for the corepressor TOPLESS and PPD, are novel substrates of SAP. SAP interacts with KIX8/9 and modulates their protein stability. Further results show that SAP acts in a common pathway with KIX8/9 and PPD to control organ growth by regulating meristemoid cell proliferation. Thus, these findings reveal a molecular mechanism by which SAP targets the KIX-PPD repressor complex for degradation to regulate meristemoid cell proliferation and organ size.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Arabidopsis , Complejos Multiproteicos/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/fisiología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proliferación Celular/genética , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Meristema/genética , Meristema/crecimiento & desarrollo , Tamaño de los Órganos/genética , Plantas Modificadas Genéticamente , Estabilidad Proteica , Proteolisis , Factores de Transcripción/genéticaRESUMEN
The sessile lifestyle of plants requires accurate physiology adjustments to be able to thrive in a changing environment. Plants integrate environmental timing signals to control developmental and stress responses. Here, we identified Far1 Related Sequence (FRS) 7 and FRS12, two transcriptional repressors that accumulate in short-day conditions, as regulators of Arabidopsis glucosinolate (GSL) biosynthesis. Loss of function of FRS7 and FRS12 results in plants with increased amplitudes of diurnal expression of GSL pathway genes. Protein interaction analyses revealed that FRS7 and FRS12 recruit the NOVEL INTERACTOR OF JAZ (NINJA) to assemble a transcriptional repressor complex. Genetic and molecular evidence demonstrated that FRS7, FRS12 and NINJA jointly regulate the expression of GSL biosynthetic genes, and thus constitute a molecular mechanism that modulates specialized metabolite accumulation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclopentanos , Regulación de la Expresión Génica de las Plantas , Glucosinolatos , Proteínas Nucleares , Oxilipinas , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In Arabidopsis (Arabidopsis thaliana), reduced expression of the transcriptional regulator PEAPOD2 (PPD2) results in propeller-like rosettes with enlarged and dome-shaped leaves. However, the molecular and cellular processes underlying this peculiar phenotype remain elusive. Here, we studied the interaction between PPD2 and NOVEL INTERACTOR OF JAZ (NINJA) and demonstrated that ninja loss-of-function plants produce rosettes with dome-shaped leaves similar to those of ppd mutants but without the increase in size. We showed that ninja mutants have a convex-shaped primary cell cycle arrest front, putatively leading to excessive cell division in the central leaf blade region. Furthermore, ppd and ninja mutants have a similar increase in the expression of CYCLIN D3;2 (CYCD3;2), and ectopic overexpression of CYCD3;2 phenocopies the ppd and ninja rosette and leaf shape phenotypes without affecting the size. Our results reveal a pivotal contribution of NINJA in leaf development, in addition to its well-studied function in jasmonate signaling, and imply a new function for D3-type cyclins in, at least partially, uncoupling the size and shape phenotypes of ppd leaves.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ciclina D3/genética , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Arabidopsis/anatomía & histología , Arabidopsis/citología , Proteínas de Arabidopsis/metabolismo , Puntos de Control del Ciclo Celular/genética , División Celular/genética , Ciclina D3/metabolismo , Mutación , Fenotipo , Hojas de la Planta/anatomía & histología , Hojas de la Planta/citología , Plantas Modificadas Genéticamente , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Phytohormones regulate the plasticity of plant growth and development, and responses to biotic and abiotic stresses. Many hormone signal transduction cascades involve ubiquitination and subsequent degradation of proteins by the 26S proteasome. The conjugation of ubiquitin to a substrate is facilitated by the E1 activating, E2 conjugating, and the substrate-specifying E3 ligating enzymes. The most prevalent type of E3 ligase in plants is the Cullin-RING ligase (CRL)-type, with F-box proteins (FBPs) as the substrate recognition component. The activity of these SKP-Cullin-F-box (SCF) complexes needs to be tightly regulated in time and place. Here, we review the regulation of SCF function in plants on multiple levels, with a focus on the auxin and jasmonate SCF-type receptor complexes. We discuss in particular the relevance of protein-protein interactions and post-translational modifications as mechanisms to keep SCF functioning under control. Additionally, we highlight the unique property of SCFTIR1/AFB and SCFCOI1 to recognize substrates by forming co-receptor complexes. Finally, we explore how engineered selective agonists can be used to study and uncouple the outcomes of the complex auxin and jasmonate signaling networks that are governed by these FBPs.