RESUMEN
Drought stress is one of the dominating challenges to the growth and productivity in crop plants. Elucidating the molecular mechanisms of plants responses to drought stress is fundamental to improve fruit quality. However, such molecular mechanisms are poorly understood in apple (Malus domestica Borkh.). In this study, we explored that the BTB-BACK-TAZ protein, MdBT2, negatively modulates the drought tolerance of apple plantlets. Moreover, we identified a novel Homeodomain-leucine zipper (HD-Zip) transcription factor, MdHDZ27, using a yeast two-hybrid (Y2H) screen with MdBT2 as the bait. Overexpression of MdHDZ27 in apple plantlets, calli, and tomato plantlets enhanced their drought tolerance by promoting the expression of drought tolerance-related genes [responsive to dehydration 29A (MdRD29A) and MdRD29B]. Biochemical analyses demonstrated that MdHDZ27 directly binds to and activates the promoters of MdRD29A and MdRD29B. Furthermore, in vitro and in vivo assays indicate that MdBT2 interacts with and ubiquitinates MdHDZ27, via the ubiquitin/26S proteasome pathway. This ubiquitination results in the degradation of MdHDZ27 and weakens the transcriptional activation of MdHDZ27 on MdRD29A and MdRD29B. Finally, a series of transgenic analyses in apple plantlets further clarified the role of the relationship between MdBT2 and MdHDZ27, as well as the effect of their interaction on drought resistance in apple plantlets. Collectively, our findings reveal a novel mechanism by which the MdBT2-MdHDZ27 regulatory module controls drought tolerance, which is of great significance for enhancing the drought resistance of apple and other plants.
Asunto(s)
Resistencia a la Sequía , Malus , Proteínas de Plantas , Factores de Transcripción , Ubiquitinación , Resistencia a la Sequía/genética , Regulación de la Expresión Génica de las Plantas , Malus/genética , Malus/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Estrés Fisiológico , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Malic acid accumulation in the vacuole largely determines acidity and perception of sweetness of apple. It has long been observed that reduction in malate level is associated with increase in ethylene production during the ripening process of climacteric fruits, but the molecular mechanism linking ethylene to malate reduction is unclear. Here, we show that ethylene-modulated WRKY transcription factor 31 (WRKY31)-Ethylene Response Factor 72 (ERF72)-ALUMINUM ACTIVATED MALATE TRANSPORTER 9 (Ma1) network regulates malate accumulation in apple fruit. ERF72 binds to the promoter of ALMT9, a key tonoplast transporter for malate accumulation of apple, transcriptionally repressing ALMT9 expression in response to ethylene. WRKY31 interacts with ERF72, suppressing its transcriptional inhibition activity on ALMT9. In addition, WRKY31 directly binds to the promoters of ERF72 and ALMT9, transcriptionally repressing and activating ERF72 and ALMT9, respectively. The expression of WRKY31 decreases in response to ethylene, lowering the transcription of ALMT9 directly and via its interactions with ERF72. These findings reveal that the regulatory complex WRKY31 forms with ERF72 responds to ethylene, linking the ethylene signal to ALMT9 expression in reducing malate transport into the vacuole during fruit ripening.
Asunto(s)
Malus , Malus/genética , Malus/metabolismo , Malatos/metabolismo , Aluminio/metabolismo , Frutas/genética , Frutas/metabolismo , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Sugars are involved in plant growth, fruit quality, and signaling perception. Therefore, understanding the mechanisms involved in soluble sugar accumulation is essential to understand fruit development. Here, we report that MdPFPß, a pyrophosphate-dependent phosphofructokinase gene, regulates soluble sugar accumulation by enhancing the photosynthetic performance and sugar-metabolizing enzyme activities in apple (Malus domestica Borkh.). Biochemical analysis revealed that a basic helix-loop-helix (bHLH) transcription factor, MdbHLH3, binds to the MdPFPß promoter and activates its expression, thus promoting soluble sugar accumulation in apple fruit. In addition, MdPFPß overexpression in tomato influenced photosynthesis and carbon metabolism in the plant. Furthermore, we determined that MdbHLH3 increases photosynthetic rates and soluble sugar accumulation in apple by activating MdPFPß expression. Our results thus shed light on the mechanism of soluble sugar accumulation in apple leaves and fruit: MdbHLH3 regulates soluble sugar accumulation by activating MdPFPß gene expression and coordinating carbohydrate allocation.
Asunto(s)
Malus , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carbohidratos , Frutas/genética , Frutas/metabolismo , Expresión Génica , Regulación de la Expresión Génica de las Plantas/genética , Malus/genética , Malus/metabolismo , Fosfofructoquinasas/genética , Fosfofructoquinasas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Azúcares/metabolismoRESUMEN
Changes in carbohydrates and organic acids largely determine the palatability of edible tissues of horticulture crops. Elucidating the potential molecular mechanisms involved in the change in carbohydrates and organic acids, and their temporal and spatial crosstalk are key steps in understanding fruit developmental processes. Here, we used apple (Malus domestica Borkh.) as research materials and found that MdbHLH3, a basic helix-loop-helix transcription factor (bHLH TF), modulates the accumulation of malate and carbohydrates. Biochemical analyses demonstrated that MdbHLH3 directly binds to the promoter of MdcyMDH that encodes an apple cytosolic NAD-dependent malate dehydrogenase, activating its transcriptional expression, thereby promoting malate accumulation in apple fruits. Additionally, MdbHLH3 overexpression increased the photosynthetic capacity and carbohydrate levels in apple leaves and also enhanced the carbohydrate accumulation in fruits by adjusting carbohydrate allocation from sources to sinks. Overall, our findings provide new insights into the mechanism of how the bHLH TF MdbHLH3 modulates the fruit quality. It directly regulates the expression of cytosolic malate dehydrogenase MdcyMDH to coordinate carbohydrate allocation and malate accumulation in apple.
Asunto(s)
Malus , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Fructosa , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas , Malatos , Malus/genética , Malus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Excessive application of nitrate, an essential macronutrient and a signal regulating diverse physiological processes, decreases malate accumulation in apple (Malus domestica) fruit, but the underlying mechanism remains poorly understood. Here, we show that an apple BTB/TAZ protein, MdBT2, is involved in regulating malate accumulation and vacuolar pH in response to nitrate. In vitro and in vivo assays indicate that MdBT2 interacts directly with and ubiquitinates a bHLH transcription factor, MdCIbHLH1, via the ubiquitin/26S proteasome pathway in response to nitrate. This ubiquitination results in the degradation of MdCIbHLH1 protein and reduces the transcription of MdCIbHLH1-targeted genes involved in malate accumulation and vacuolar acidification, including MdVHA-A, which encodes a vacuolar H+-ATPase, and MdVHP1, which encodes a vacuolar H+-pyrophosphatase, as well as MdALMT9, which encodes an aluminum-activated malate transporter. A series of transgenic analyses in apple materials including fruits, plantlets, and calli demonstrate that MdBT2 controls nitrate-mediated malate accumulation and vacuolar pH at least partially, if not completely, via regulating the MdCIbHLH1 protein level. Taken together, these findings reveal that MdBT2 regulates the stability of MdCIbHLH1 via ubiquitination in response to nitrate, which in succession transcriptionally reduces the expression of malate-associated genes, thereby controlling malate accumulation and vacuolar acidification in apples under high nitrate supply.
Asunto(s)
Malatos/metabolismo , Nitratos/farmacología , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Pirofosfatasa Inorgánica/genética , Pirofosfatasa Inorgánica/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Ubiquitinación/efectos de los fármacos , Ubiquitinación/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
The plant hormone ethylene is critical for climacteric fruit ripening, while glucose and anthocyanins determine the fruit quality of climacteric fruits such as apple. Understanding the exact molecular mechanism for this process is important for elucidating the interconnection of ethylene and fruit quality. Overexpression of apple MdbHLH3 gene, an anthocyanin-related basic helix-loop-helix transcription factor (bHLH TF) gene, promotes ethylene production, and transgenic apple plantlets and trees exhibit ethylene-related root developmental abnormalities, premature leaf senescence, and fruit ripening. Biochemical analyses demonstrate that MdbHLH3 binds to the promoters of three genes that are involved in ethylene biosynthesis, including MdACO1, MdACS1, and MdACS5A, activating their transcriptional expression, thereby promoting ethylene biosynthesis. High glucose-inhibited U-box-type E3 ubiquitin ligase MdPUB29, the ortholog of Arabidopsis AtPUB29 in apple, influences the expression of ethylene biosynthetic genes and ethylene production by direct ubiquitination of the MdbHLH3 protein. Our findings provide new insights into the ubiquitination of MdbHLH3 by glucose-inhibited ubiquitin E3 ligase MdPUB29 in the regulation of ethylene biosynthesis as well as indicate that the regulatory module MdPUB29-MdbHLH3 connects ethylene biosynthesis with fruit quality in apple.
Asunto(s)
Vías Biosintéticas/genética , Etilenos/biosíntesis , Frutas/genética , Redes Reguladoras de Genes , Malus/genética , Vías Biosintéticas/efectos de los fármacos , Frutas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Genes de Plantas , Glucosa/farmacología , Malus/efectos de los fármacos , Modelos Biológicos , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Transducción de Señal/genética , Transcripción Genética/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
Glucose induces anthocyanin accumulation in many plant species; however, the molecular mechanism involved in this process remains largely unknown. Here, we found that apple hexokinase MdHXK1, a glucose sensor, was involved in sensing exogenous glucose and regulating anthocyanin biosynthesis. In vitro and in vivo assays suggested that MdHXK1 interacted directly with and phosphorylated an anthocyanin-associated bHLH transcription factor (TF) MdbHLH3 at its Ser361 site in response to glucose. Furthermore, both the hexokinase_2 domain and signal peptide are crucial for the MdHXK1-mediated phosphorylation of MdbHLH3. Moreover, phosphorylation modification stabilized MdbHLH3 protein and enhanced its transcription of the anthocyanin biosynthesis genes, thereby increasing anthocyanin biosynthesis. Finally, a series of transgenic analyses in apple calli and fruits demonstrated that MdHXK1 controlled glucose-induced anthocyanin accumulation at least partially, if not completely, via regulating MdbHLH3. Overall, our findings provide new insights into the mechanism of the glucose sensor HXK1 modulation of anthocyanin accumulation, which occur by directly regulating the anthocyanin-related bHLH TFs in response to a glucose signal in plants.
Asunto(s)
Antocianinas/biosíntesis , Frutas/genética , Hexoquinasa/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos/genética , Antocianinas/genética , Frutas/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Glucosa/metabolismo , Hexoquinasa/biosíntesis , Malus/genética , Malus/crecimiento & desarrollo , Fosforilación , Plantas Modificadas Genéticamente , Homología de Secuencia de Aminoácido , Factores de Transcripción/biosíntesisRESUMEN
Malate, the predominant organic acid in many fruits, is a crucial component of the organoleptic quality of fruit, including taste and flavor. The genetic and environmental mechanisms affecting malate metabolism in fruit cells have been studied extensively. However, the transcriptional regulation of malate-metabolizing enzymes and vacuolar transporters remains poorly understood. Our previous studies demonstrated that MdMYB1 modulates anthocyanin accumulation and vacuolar acidification by directly activating vacuolar transporters, including MdVHA-B1, MdVHA-E, MdVHP1 and MdtDT. Interestingly, we isolated and identified a MYB transcription factor, MdMYB73, a distant relative of MdMYB1 in this study. It was subsequently found that MdMYB73 protein bound directly to the promoters of MdALMT9 (aluminum-activated malate transporter 9), MdVHA-A (vacuolar ATPase subunit A) and MdVHP1 (vacuolar pyrophosphatase 1), transcriptionally activating their expression and thereby enhancing their activities. Analyses of transgenic apple calli demonstrated that MdMYB73 influenced malate accumulation and vacuolar pH. Furthermore, MdCIbHLH1 interacted with MdMYB73 and enhanced its activity upon downstream target genes. These findings help to elucidate how MdMYB73 directly modulates the vacuolar transport system to affect malate accumulation and vacuolar pH in apple.
Asunto(s)
Malatos/metabolismo , Malus/metabolismo , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Vacuolas/metabolismo , Antocianinas/metabolismo , Regulación de la Expresión Génica de las Plantas , Malus/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: The roles in photosystem I (PSI) assembly of the nucleus-encoded thylakoid protein Y3IP1 who interacts with the plastid-encoded Ycf3 protein that has been well-characterized in plants. However, its function and potential mechanisms in other aspects remain poorly understood. RESULTS: We identified the apple MdY3IP1 gene, which encodes a protein highly homologous to the Arabidopsis Y3IP1 (AtY3IP1). Ectopic expression of MdY3IP1 triggered early-flowering and enhanced salt tolerance in Arabidopsis plants. MdY3IP1 controlled floral transition by accelerating sugar metabolism process in plant cells, thereby influencing the expression of flowering-associated genes. The increase in salt stress tolerance in MdY3IP1-expressing plants correlated with reduced reactive oxygen species (ROS) accumulation, and an increase in lateral root development by regulating both auxin biosynthesis and transport, as followed by enhancement of salt tolerance in Arabidopsis. Overall, these findings provide new evidences for additional functions of Y3IP1-like proteins and their underlying mechanisms of which Y3IP1 confers early-flowering and salt tolerance phenotypes in plants. CONCLUSIONS: These observations suggest that plant growth and stress resistance can be affected by the regulation of the MdY3IP1 gene. Further molecular and genetic approaches will accelerate our knowledge of MdY3IP1 functions in PSI complex formation and plants stress resistance, and inform strategies for creating transgenic crop varieties with early maturity and high-resistant to adverse environmental conditions.
Asunto(s)
Arabidopsis/genética , Expresión Génica Ectópica , Flores/fisiología , Malus/genética , Proteínas de Plantas/genética , Tolerancia a la Sal/genética , Arabidopsis/metabolismo , Flores/genética , Flores/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Tilacoides/metabolismoRESUMEN
MYB transcription factors (TFs) participate in many biological processes. However, the molecular mechanisms by which MYB TFs affect plant resistance to apple ring rot remain poorly understood. Here, the R2R3-MYB gene MdMYB73 was cloned from "Royal Gala" apples and functionally characterized as a positive regulator of the defense response to Botryosphaeria dothidea. qRT-PCR and GUS staining demonstrated that MdMYB73 was strongly induced in apple fruits and transgenic calli after inoculation with B. dothidea. MdMYB73 overexpression improved resistance to B. dothidea in apple calli and fruits, while MdMYB73 suppression weakened. Increased resistance to B. dothidea was also observed in MdMYB73-expressing Arabidopsis thaliana. Interestingly, salicylic acid (SA) contents and the expression levels of genes related with SA synthesis and signaling were greater in MdMYB73-overexpressing plant materials compared to wild-type controls after inoculation, suggesting that MdMYB73 might enhance resistance to B. dothidea via the SA pathway. Finally, we discovered that MdMYB73 interacts with MdWRKY31, a positive regulator of B. dothidea. Together, MdWRKY31 and MdMYB73 enhanced B. dothidea resistance in apples. Our results clarify the mechanisms by which MdMYB73 improves resistance to B. dothidea and suggest that resistance may be affected by regulating the SA pathway.
Asunto(s)
Ascomicetos/fisiología , Malus/inmunología , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/inmunología , Ácido Salicílico/inmunología , Factores de Transcripción/inmunología , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Malus/genética , Malus/microbiología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Factores de Transcripción/genéticaRESUMEN
Basic helix-loop-helix (bHLH) domain-containing transcription factors are known for their roles in regulating various plant growth and developmental processes. Previously, we showed that MdbHLH3 from apple (Malus domestica) has multiple functions, modulating both anthocyanin biosynthesis and cell acidification. Here, we show that MdbHLH3 also regulates ethylene biosynthesis and leaf senescence by promoting the expression of dehydratase-enolase-phosphatase complex 1 (MdDEP1). Therefore, we propose a model whereby MdbHLH3 acts as a crucial factor that modulates anthocyanin biosynthesis and cell acidification in addition to fruit ripening and leaf senescence by regulating distinct target genes.
RESUMEN
Ethylene response factor (ERF) is a plant-specific transcription factor involved in many biological processes including root formation, hypocotyl elongation, fruit ripening, organ senescence and stress responses, as well as fruit quality formation. However, its underlying mechanism in plant pathogen defense against Botryosphaeria dothidea (B. dothidea) remains poorly understood. Here, we isolate MdERF11, an apple nucleus-localized ERF transcription factor, from apple cultivar 'Royal Gala'. qRT-PCR assays show that the expression of MdERF11 is significantly induced in apple fruits after B. dothidea infection. Overexpression of MdERF11 gene in apple calli significantly increases the resistance to B.dothidea infection, while silencing MdERF11 in apple calli results in reduced resistance. Ectopic expression of MdERF11 in Arabidopsis also exhibits enhanced resistance to B. dothidea infection compared to that of wild type. Infections in apple calli and Arabidopsis leaves by B. dothidea respectively cause an increase in endogenous levels of salicylic acid (SA) followed by induction of SA synthesis-related and signaling-related gene expression. Taken together, these findings illustrate a potential mechanism by which MdERF11 elevates plant pathogen defense against B. dothidea by regulating SA synthesis pathway.
Asunto(s)
Ascomicetos/fisiología , Malus/genética , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Factores de Transcripción/genética , Resistencia a la Enfermedad/genética , Malus/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
As an important primary metabolite, malate plays a key role in regulating osmotic pressure, pH homeostasis, stress tolerance, and fruit quality of apple. The R2R3-MYB transcription factor (TF) MdMYB73 was identified as a protein that plays a critical role in determining malate accumulation and vacuolar acidification by directly regulating the transcription of aluminum-activated malate transporter 9 (MdALMT9), vacuolar ATPase subunit A (MdVHA-A), and vacuolar pyrophosphatase 1 (MdVHP1) in apple. In addition, the bHLH TF MdCIbHLH1 interacts with MdMYB73 and enhances the transcriptional activity of MdMYB73. Our previous studies demonstrated that the BTB-BACK-TAZ domain protein MdBT2 can degrade MdCIbHLH1 to influence malate accumulation and vacuolar acidification. However, the potential upstream regulators of MdMYB73 are currently unknown. In this study, we found that MdBT2 directly interacts with and degrades MdMYB73 through the ubiquitin/26S proteasome pathway to regulate malate accumulation and vacuolar acidification. A series of functional assays with apple calli and fruit showed that MdBT2 controls malate accumulation and vacuolar acidification in an MdMYB73-dependent manner. Overall, our findings shed light on the mechanism by which the BTB-BACK-TAZ domain protein MdBT2 regulates malate accumulation and vacuolar acidification by targeting MdMYB73 and MdCIbHLH1 for ubiquitination in apple. This information may help guide traditional breeding programs and fruit tree molecular breeding, and lead to improvements in fruit quality and stress tolerance.
RESUMEN
Plant root systems are essential for many physiological processes, including water and nutrient absorption. MADS-box transcription factor (TF) genes have been characterized as the important regulators of root development in plants; however, the underlying mechanism is largely unknown, including chrysanthemum. Here, it was found that the overexpression of CmANR1, a chrysanthemum MADS-box TF gene, promoted both adventitious root (AR) and lateral root (LR) development in chrysanthemum. Whole transcriptome sequencing analysis revealed a series of differentially expressed unigenes (DEGs) in the roots of CmANR1-transgenic chrysanthemum plants compared to wild-type plants. Functional annotation of these DEGs by alignment with Gene Ontology (GO) terms and biochemical pathway Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that CmANR1 TF exhibited "DNA binding" and "catalytic" activity, as well as participated in "phytohormone signal transduction". Both chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) and gel electrophoresis mobility shift assays (EMSA) indicated the direct binding of CmPIN2 to the recognition site CArG-box motif by CmANR1. Finally, a firefly luciferase imaging assay demonstrated the transcriptional activation of CmPIN2 by CmANR1 in vivo. Overall, our results provide novel insights into the mechanisms of MADS-box TF CmANR1 modulation of both AR and LR development, which occurs by directly regulating auxin transport gene CmPIN2 in chrysanthemum.
RESUMEN
Vacuolar pH is important and involves in many different physiological processes in plants. A recent paper published in Plant Physiology reveals that MdMYB1 regulates vacuolar pH by directly transcriptionally regulating proton pump genes and malate transporters genes, such as V-ATPase subunit gene MdVHA-B1. Here, we found that MdSOS2L1 in vitro did not directly interact with MdMYB1, however, in vivo formed a complex with MdMYB1 in the nucleus to regulate MdVHA-B1-mediated vacuolar acidification. This finding shed light on the role of MdSOS2L1 in transcriptionally regulating MdVHA-B1 in addition to its post-modified function in apples.