RESUMEN
Salt stress is a critical limiting factor for fruit yield and quality of apples. Brassinosteroids (BRs) play an important role in response to abiotic stresses. In the present study, application of 2,4- Epicastasterone on seedlings of Malus 'M9T337' and Malus domestica 'Gala3' alleviated the physiological effects, such as growth inhibition and leaf yellowing, induced by salt stress. Further analysis revealed that treatment with NaCl induced expression of genes involved in BR biosynthesis in 'M9T337' and 'Gala3'. Among which, the expression of BR biosynthetic gene MdBR6OX2 showed a three-fold upregulation upon salt treatment, suggesting its potential role in response to salt stress in apple. MdBR6OX2, belonging to the CYP450 family, contains a signal peptide region and a P450 domain. Expression patterns analysis showed that the expression of MdBR6OX2 can be significantly induced by different abiotic stresses. Overexpressing MdBR6OX2 enhanced the tolerance of apple callis to salt stress, and the contents of endogenous BR-related compounds, such as Typhastero (TY), Castasterone (CS) and Brassinolide (BL) were significantly increased in transgenic calli compared with that of wild-type. Extopic expression of MdBR6OX2 enhanced tolerance to salt stress in Arabidopsis. Genes associated with salt stress were significantly up-regulated, and the contents of BR-related compounds were significantly elevated under salt stress. Our data revealed that BR-biosynthetic gene MdBR6OX2 positively regulates salt stress tolerance in both apple calli and Arabidopsis.
Asunto(s)
Arabidopsis , Brasinoesteroides , Regulación de la Expresión Génica de las Plantas , Malus , Proteínas de Plantas , Tolerancia a la Sal , Malus/genética , Malus/metabolismo , Malus/efectos de los fármacos , Brasinoesteroides/metabolismo , Brasinoesteroides/biosíntesis , Brasinoesteroides/farmacología , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efectos de los fármacos , Tolerancia a la Sal/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Estrés Salino/genética , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismoRESUMEN
The Tetratricopeptide repeat (TPR)-like superfamily with TPR conserved domains is widely involved in the growth and abiotic stress in many plants. In this report, the gene MdTPR16 belongs to the TPR family in apple (Malus domestica). Promoter analysis reveal that MdTPR16 incorporated various stress response elements, including the drought stress response elements. And different abiotic stress treatments, drought especially, significantly induce the response of MdTPR16. Overexpression of MdTPR16 result in better drought tolerance in apple and Arabidopsis by up-regulating the expression levels of drought stress-related genes, achieving a higher chlorophyll content level, more material accumulation, and overall better growth compared to WT in the drought. Under drought stress, the overexpressed MdTPR16 also mitigate the oxidative damage in cells by reducing the electrolyte leakage, malondialdehyde content, and the H2O2 and O2- accumulation in apples and Arabidopsis. In conclusion, MdTPR16 act as a beneficial regulator of drought stress response by regulating the expression of related genes and the cumulation of reactive oxygen species (ROS).
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Malus , Proteínas de Plantas , Malus/genética , Malus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sequías , Arabidopsis/genética , Arabidopsis/metabolismo , Estrés Fisiológico/genética , Plantas Modificadas Genéticamente/genética , Repeticiones de Tetratricopéptidos/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Although brassinolide (BR) and jasmonic acid (JA) play essential roles in the regulation of cold stress responses, the molecular basis of their crosstalk remains elusive. Here, we show a key component of BR signaling in apple (Malus × domestica), BR INSENSITIVE1 (BRI1)-EMS-SUPPRESSOR1 (BES1)-INTERACTING MYC-LIKE PROTEIN1 (MdBIM1), increases cold tolerance by directly activating expression of C-REPEAT BINDING FACTOR1 (MdCBF1) and forming a complex with C-REPEAT BINDING FACTOR2 (MdCBF2) to enhance MdCBF2-activated transcription of cold-responsive genes. Two repressors of JA signaling, JAZMONATE ZIM-DOMAIN1 (MdJAZ1) and JAZMONATE ZIM-DOMAIN2 (MdJAZ2), interact with MdBIM1 to integrate BR and JA signaling under cold stress. MdJAZ1 and MdJAZ2 reduce MdBIM1-promoted cold stress tolerance by attenuating transcriptional activation of MdCBF1 expression by MdBIM1 and interfering with the formation of the MdBIM1-MdCBF2 complex. Furthermore, the E3 ubiquitin ligase ARABIDOPSIS TÓXICOS en LEVADURA73 (MdATL73) decreases MdBIM1-promoted cold tolerance by targeting MdBIM1 for ubiquitination and degradation. Our results not only reveal crosstalk between BR and JA signaling mediated by a JAZ-BIM1-CBF module but also provide insights into the posttranslational regulatory mechanism of BR signaling.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Malus , Brasinoesteroides/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Malus/genética , Malus/metabolismo , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
PHR1 (PHOSPHATE STARVATION RESPONSE1) plays key roles in the inorganic phosphate (Pi) starvation response and in Pi deficiency-induced anthocyanin biosynthesis in plants. However, the post-translational regulation of PHR1 is unclear, and the molecular basis of PHR1-mediated anthocyanin biosynthesis remains elusive. In this study, we determined that MdPHR1 was essential for Pi deficiency-induced anthocyanin accumulation in apple (Malus × domestica). MdPHR1 interacted with MdWRKY75, a positive regulator of anthocyanin biosynthesis, to enhance the MdWRKY75-activated transcription of MdMYB1, leading to anthocyanin accumulation. In addition, the E3 ubiquitin ligase SEVEN IN ABSENTIA1 (MdSINA1) negatively regulated MdPHR1-promoted anthocyanin biosynthesis via the ubiquitination-mediated degradation of MdPHR1. Moreover, the protein kinase apple BRASSINOSTEROID INSENSITIVE2 (MdBIN2) phosphorylated MdPHR1 and positively regulated MdPHR1-mediated anthocyanin accumulation by attenuating the MdSINA1-mediated ubiquitination degradation of MdPHR1. Taken together, these findings not only demonstrate the regulatory role of MdPHR1 in Pi starvation induced anthocyanin accumulation, but also provide an insight into the post-translational regulation of PHR1.
Asunto(s)
Malus , Malus/genética , Malus/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Antocianinas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ubiquitinación , Regulación de la Expresión Génica de las PlantasRESUMEN
Although DELLA protein destabilization mediated by post-translational modifications is essential for gibberellin (GA) signal transduction and GA-regulated anthocyanin biosynthesis, the related mechanisms remain largely unknown. In this study, we report the ubiquitination and phosphorylation of an apple DELLA protein MdRGL2a in response to GA signaling and its regulatory role in anthocyanin biosynthesis. MdRGL2a could interact with MdWRKY75 to enhance the MdWRKY75-activated transcription of anthocyanin activator MdMYB1 and interfere with the interaction between anthocyanin repressor MdMYB308 and MdbHLH3 or MdbHLH33, thereby promoting anthocyanin accumulation. A protein kinase MdCIPK20 was found to phosphorylate and protect MdRGL2a from degradation, and it was essential for MdRGL2a-promoting anthocyanin accumulation. However, MdRGL2a and MdCIPK20 were ubiquitinated and degraded by E3 ubiquitin ligases MdSINA1 and MdSINA2, respectively, both of which were activated in the presence of GA. Our results display the integration of SINA1/2 with CIPK20 to dynamically regulate GA signaling and will be helpful toward understanding the mechanism of GA signal transduction and GA-inhibited anthocyanin biosynthesis. The discovery of extensive interactions between DELLA and SINA and CIPK proteins in apple will provide reference for the study of ubiquitination and phosphorylation of DELLA proteins in other species.
Asunto(s)
Arabidopsis , Malus , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Antocianinas/metabolismo , Arabidopsis/metabolismo , Proteínas Quinasas/metabolismo , Ubiquitinación , Malus/genética , Malus/metabolismo , Giberelinas/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Apomixis is the asexual reproduction through seeds that leads to the production of genetically uniform progeny. It has become an important tool in plant breeding because it facilitates the retention of genotypes with desirable traits and allows seeds to be obtained directly from mother plants. Apomixis is rare in most economically important crops, but it occurs in some Malus species. Here, the apomictic characteristics of Malus were examined using four apomictic and two sexually reproducing Malus plants. Results from transcriptome analysis showed that plant hormone signal transduction was the main factor affecting apomictic reproductive development. Four of the apomictic Malus plants examined were triploid, and pollen was either absent or present in very low densities in the stamen. Variation in the presence of pollen was associated with variation in the apomictic percentage; specifically, pollen was absent in the stamens of tea crabapple plants with the highest apomictic percentage. Furthermore, pollen mother cells failed to progress normally into meiosis and pollen mitosis, a trait mostly observed in apomictic Malus plants. The expression levels of meiosis-related genes were upregulated in apomictic plants. Our findings indicate that our simple method of detecting pollen abortion could be used to identify apple plants that are capable of apomictic reproduction.
RESUMEN
The AP2/ERF family is an important class of transcription factors involved in plant growth and various biological processes. One of the AP2/ERF transcription factors, RAP2.6L, participates in various stresses responses. However, the function of RAP2.6L is largely unknown in apples (Malus domestica). In this study, an apple gene homologous to Arabidopsis AtRAP2.6L, MdERF113, was analyzed by bioinformatic characterization, gene expression analysis and subcellular localization assessment. MdERF113 was highly expressed in the sarcocarp and was responsive to hormonal signals and abiotic stresses. MdERF113-overexpression apple calli were less sensitive to low temperature, drought, salinity, and abscisic acid than wild-type. Subcellular localization revealed that MdERF113 was a nuclear-localized transcription factor, and yeast experiments confirmed that MdERF113 has no autonomous activation activity. Overall, this study indicated that MdERF113 plays a role in regulating plant growth under abiotic conditions.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Malus , Malus/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/metabolismo , Ácido Abscísico/metabolismo , Estrés Fisiológico/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Filogenia , Sequías , Proteínas de Arabidopsis/metabolismoRESUMEN
Trihelix transcription factors consist of five subfamilies, including GT-1, GT-2, SH4, GTγ, and SIP1, which play important roles in the responses to biotic and abiotic stresses, however, seldom is known about the role of the SIP1 genes in apples. In this study, 12 MdSIP1 genes were first identified in apples by genome-wide analysis, and contained conserved MYB/SANT-like domains. Expression patterns analyses showed that the MdSIP1 genes had different tissue expression patterns, and different transcription levels in response to abiotic stresses, indicating that MdSIP1s may play multiple roles under various abiotic stresses. Among them, the MdSIP1-2 gene was cloned and ectopic transformed into Arabidopsis, and its biology function was identified. The subcellular localization showed that MdSIP1-2 protein was specifically localized in the nucleus, and that overexpression of MdSIP1-2 promoted the development of lateral roots, increased abscisic acid (ABA) sensitivity, and improved salt and drought tolerance. These findings suggested that MdSIP1-2 plays an important role in root development, ABA synthesis, and salt and drought stress tolerance. In conclusion, these results lay a solid foundation for determining the role of MdSIP1 in the growth and development and abiotic stress tolerance of apples.
Asunto(s)
Arabidopsis , Malus , Malus/genética , Malus/metabolismo , Arabidopsis/metabolismo , Ácido Abscísico/farmacología , Ácido Abscísico/metabolismo , Genoma de Planta/genética , Regulación de la Expresión Génica de las Plantas , Estrés Fisiológico/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sequías , Plantas Modificadas Genéticamente/genéticaRESUMEN
BACKGROUND: Cyclophilin (CYP) belongs to the immunophilin family and has peptidyl-prolyl cis-trans isomerase (PPIase) activity, which catalyzes the cis-trans isomerization process of proline residues. CYPs widely exist in eukaryotes and prokaryotes, and contain a conserved cyclophilin-like domain (CLD). Plant cyclophilins are widely involved in a range of biological processes including stress response, metabolic regulation, and growth and development. RESULT: In this study, 30 cyclophilin genes on 15 chromosomes were identified from the 'Golden Delicious' apple (M. domestica) genome. Phylogenetic analysis showed that the cyclophilin family genes can be divided into three clades in Malus. Collinear analysis showed that ten gene pairs were the result of segmental duplication. Analysis of gene and protein structure further supported the phylogenetic tree and collinearity analysis. The expression of MdCYPs in different organs was higher in leaves, flowers, and fruits. Ten and eight CYPs responded to drought and salt stress, respectively. MdCYP16, a nuclear-localized MD CYP, was screened from the intersection of the two expression profiling datasets and was highly sensitive to drought and salt stress. GUS staining of transgenic Arabidopsis indicated that MdCYP16 may be involved in the regulation of abiotic stress. CONCLUSION: This study systematically analyzed members of the apple cyclophilin family and confirmed the involvement of MdCYP16 as a nuclear-localized MD cyclophilin that acts in response to salt and drought stress in apple. Our work identifies members of the apple cyclophilin gene family, and provides an important theoretical basis for in-depth study of cyclophilin function. Additionally, the analysis provides candidate genes that may be involved in stress response in apple.
Asunto(s)
Ciclofilinas , Malus , Ciclofilinas/genética , Malus/genética , FilogeniaRESUMEN
Cold stress limits plant growth, geographical distribution, and crop yield. The MYC-type bHLH transcription factor ICE1 is recognized as the core positive regulator of the cold-stress response. However, how ICE1 protein levels are regulated remains to be further studied. In this study, we observed that a U-box-type E3 ubiquitin ligase, MdPUB23, positively regulated the cold-stress response in apple. The expression of MdPUB23 increased at both the transcriptional and post-translational levels in response to cold stress. Overexpression of MdPUB23 in transgenic apple enhanced sensitivity to cold stress. Further study showed that MdPUB23 directly interacted with MdICE1, promoting the ubiquitination-mediated degradation of the MdICE1 protein through the 26S-proteasome pathway and reducing the MdICE1-improved cold-stress tolerance in apple. Our results reveal that MdPUB23 regulates the cold-stress response by directly mediating the stability of the positive regulator MdICE1. The PUB23-ICE1 ubiquitination module may play a role in maintaining ICE1 protein homeostasis and preventing overreactions from causing damage to plants. The discovery of the ubiquitination regulatory pathway of ICE1 provides insights for the further exploration of plant cold-stress-response mechanisms.
RESUMEN
Salt stress restricts plant growth and productivity worldwide. Zinc finger proteins play important roles in response to various abiotic plant stresses. In this research, we identified and characterized the ZAT17 gene in Malus domestica, which encodes a C2H2-type zinc finger protein. MdZAT17 has two typical conserved zinc finger domains and an ERF-associated amphiphilic repression (EAR) motif. Promoter analysis showed that MdZAT17 contains several stress-related response elements (ABRE, CGTCA-motif, and TC-rich repeats), and qRT-PCR analysis showed that the expression level of MdZAT17 was induced by various abiotic stress treatments. The overexpression of MdZAT17 improved tolerance to salt stress in apple calli. The ectopic expression of MdZAT17 in Arabidopsis enhanced salt stress tolerance and led to lower malondialdehyde (MDA) content, lower reactive oxygen species (ROS) accumulation, and greater anthocyanin accumulation under salt stress. Moreover, the overexpression of MdZAT17 transgenic apple calli and Arabidopsis reduced the sensitivity to abscisic acid (ABA). In conclusion, our results indicate that MdZAT17 plays a positive regulatory role in salt tolerance, providing a theoretical basis for further research on its molecular mechanisms.
Asunto(s)
Arabidopsis , Malus , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Malus/genética , Malus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Estrés Salino , Estrés Fisiológico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de ZincRESUMEN
Zinc finger proteins are widely involved and play an important role in plant growth and abiotic stress. In this research, MdZAT5, a gene encoding C2H2-type zinc finger protein, was cloned and investigated. The MdZAT5 was highly expressed in flower tissues by qRT-PCR analyses and GUS staining. Promoter analysis showed that MdZAT5 contained multiple response elements, and the expression levels of MdZAT5 were induced by various abiotic stress treatments. Overexpression of MdZAT5 in apple calli positively regulated anthocyanin accumulation by activating the expressions of anthocyanin biosynthesis-related genes. Overexpression of MdZAT5 in Arabidopsis also enhanced the accumulation of anthocyanin. In addition, MdZAT5 increased the sensitivity to salt stress in apple calli. Ectopic expression of MdZAT5 in Arabidopsis reduced the expression of salt-stress-related genes (AtNHX1 and AtABI1) and improved the sensitivity to salt stress. In conclusion, these results suggest that MdZAT5 plays a positive regulatory role in anthocyanin accumulation and negatively regulates salt resistance.
Asunto(s)
Antocianinas/metabolismo , Arabidopsis/crecimiento & desarrollo , Malus/crecimiento & desarrollo , Proteínas Represoras/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Clonación Molecular , Flores/genética , Flores/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Malus/genética , Malus/metabolismo , Modelos Moleculares , Filogenia , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/química , Proteínas Represoras/genética , Estrés Salino , Regulación hacia ArribaRESUMEN
Various abiotic stressors, particularly drought stress, affect plant growth and yield. Zinc finger proteins play an important role in plant abiotic stress tolerance. Here, we isolated the apple MdZAT10 gene, a C2H2-type zinc finger protein, which is a homolog of Arabidopsis STZ/ZAT10. MdZAT10 was localized to the nucleus and highly expressed in leaves and fruit. Promoter analysis showed that MdZAT10 contained several response elements and the transcription level of MdZAT10 was induced by abiotic stress and hormone treatments. MdZAT10 was responsive to drought treatment both at the transcriptional and post-translational levels. MdZAT10-overexpressing apple calli decreased the expression level of MdAPX2 and increased sensitivity to PEG 6000 treatment. Moreover, ectopically expressed MdZAT10 in Arabidopsis reduced the tolerance to drought stress, and exhibited higher water loss, higher malondialdehyde (MDA) content and higher reactive oxygen species (ROS) accumulation under drought stress. In addition, MdZAT10 reduced the sensitivity to abscisic acid in apple. Ectopically expressed MdZAT10 in Arabidopsis promoted seed germination and seedling growth. These results indicate that MdZAT10 plays a negative regulator in the drought resistance, which can provide theoretical basis for further molecular mechanism research.
Asunto(s)
Dedos de Zinc CYS2-HIS2 , Malus , Ácido Abscísico/farmacología , Sequías , Regulación de la Expresión Génica de las Plantas , Malus/genética , Malus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Estrés Fisiológico/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Jasmonic acid (JA) plays an important role in regulating leaf senescence. However, the molecular mechanisms of leaf senescence in apple (Malus domestica) remain elusive. In this study, we found that MdZAT10, a C2H2-type zinc finger transcription factor (TF) in apple, markedly accelerates leaf senescence and increases the expression of senescence-related genes. To explore how MdZAT10 promotes leaf senescence, we carried out liquid chromatography/mass spectrometry screening. We found that MdABI5 physically interacts with MdZAT10. MdABI5, an important positive regulator of leaf senescence, significantly accelerated leaf senescence in apple. MdZAT10 was found to enhance the transcriptional activity of MdABI5 for MdNYC1 and MdNYE1, thus accelerating leaf senescence. In addition, we found that MdZAT10 expression was induced by methyl jasmonate (MeJA), which accelerated JA-induced leaf senescence. We also found that the JA-responsive protein MdBT2 directly interacts with MdZAT10 and reduces its protein stability through ubiquitination and degradation, thereby delaying MdZAT10-mediated leaf senescence. Taken together, our results provide new insight into the mechanisms by which MdZAT10 positively regulates JA-induced leaf senescence in apple.