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The Zinc Finger Transcription Factor SlZFP2 Negatively Regulates Abscisic Acid Biosynthesis and Fruit Ripening in Tomato

ABSTRACT: Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. The ABA biosynthesis pathway in plants has been thoroughly elucidated; however, very few transcription factors directly regulating the expression of ABA biosynthetic genes have been identified. Here we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2, which is mainly expressed in developing fruits and axillary buds, negatively regulates ABA biosynthesis. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds and faster seed germination, whereas gene silencing by RNA interference (RNAi) caused poor fruit set and inhibited seed germination. Gene expression analysis showed that SlZFP2 represses ABA biosynthesis mainly through downregulation of the ABA biosynthetic genes SITIENS (SIT), FLACCA (FLC) and aldehyde oxidase SlAO1. SlZFP2 delays the onset of ripening through suppression of the ripening regulator COLORLESS NON-RIPENING (CNR). Using bacterial one hybrid screening and a selected amplification and binding assay we identified the (A/T)(G/C)TT repeat as the core binding sequence of SlZFP2. We further identified a large number of tomato genes containing putative SlZFP2 binding sites in their promoter regions. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that SIT, FLC and SlAO1 are direct targets of SlZFP2 through binding to their promoter regions. We propose that SlZFP2 represents a novel negative regulator for fine tuning ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening.To gain further insight on transcriptome changes regulated by SlZFP2, we sequenced a representative SlZFP2 RNAi line in LA1589 background and its nontransgenic sibling (WT) on a Miseq platform.

ORGANISM(S): Solanum pimpinellifolium

PROVIDER: GSE63838 | GEO | 2015/11/23

SECONDARY ACCESSION(S): PRJNA269239

REPOSITORIES: GEO

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Project description:Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. The ABA biosynthesis pathway in plants has been thoroughly elucidated; however, very few transcription factors directly regulating the expression of ABA biosynthetic genes have been identified. Here we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2, which is mainly expressed in developing fruits and axillary buds, negatively regulates ABA biosynthesis. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds and faster seed germination, whereas gene silencing by RNA interference (RNAi) caused poor fruit set and inhibited seed germination. Gene expression analysis showed that SlZFP2 represses ABA biosynthesis mainly through downregulation of the ABA biosynthetic genes SITIENS (SIT), FLACCA (FLC) and aldehyde oxidase SlAO1. SlZFP2 delays the onset of ripening through suppression of the ripening regulator COLORLESS NON-RIPENING (CNR). Using bacterial one hybrid screening and a selected amplification and binding assay we identified the (A/T)(G/C)TT repeat as the core binding sequence of SlZFP2. We further identified a large number of tomato genes containing putative SlZFP2 binding sites in their promoter regions. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that SIT, FLC and SlAO1 are direct targets of SlZFP2 through binding to their promoter regions. We propose that SlZFP2 represents a novel negative regulator for fine tuning ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening.To gain further insight on transcriptome changes regulated by SlZFP2, we sequenced a representative SlZFP2 RNAi line in LA1589 background and its nontransgenic sibling (WT) on a Miseq platform. The RNAi line 207 showed defected fruit set and ABA biosynthesis were chosen for profiling gene expression via RNA sequencing. Its nontransgenic sibling was served as controls. Three biological replicates were conducted.

Project description:Purpose: The goals of this study was to provide genome-wide data to investigate the molecular mechanism of ABA regulation in many ripening related biological processes, including fruit color variation, antioxidant capacity, flavonoids biosynthesis and photosynthesis. Methods:By applying the next generation sequencing technology, we conducted a comparative analysis of exogenous ABA and NDGA effects on tomato fruit maturation. Results:The high throughput sequencing results showed that 25728 genes expressed across all three samples, and 10388 of them were identified as significantly differently expressed genes (DEGs). Exogenous ABA was found to enhance the transcription of genes in pigments metabolism, including carotenoids biosynthesis and chlorophyll degradation, whereas NDGA treatment inhibited these progresses. The results also revealed the crucial role of ABA in flavonoids synthesis and regulation of antioxidant system. Intriguingly, we also found that an inhibition of endogenous ABA significantly enhanced the transcriptional abundance of genes involved in fruit photosynthesis. Conclusions:next-generation sequencing enabled us to characterize the transcriptomes of tomato fruit treated with ABA and NDGA. By comparing these transcriptomes with control respectively, we observed that ABA could accelerate fruit maturation by positively regulating many genes related to ripening processes. Our study have turned spotlight on the pathways of fruit pigmentation, including carotenoid biosynthesis and chlorophyll metabolism. Exogenous ABA was able to up-regulate many genes in relation to the carotenoids accumulation and chlorophyll breakdown, thus promoting the color transition of tomato fruit. In addition, ABA has the potential to improve the genes related to antioxidant capacity, such as SODs, CATs, APXs, GSTs, GPXs, TrXs and PrxRs. Besides, the expression changes of genes involved in flavonoids biosynthesis after ABA exposure was striking, suggesting ABA could enhance the defense response by producing more secondary metabolite in tomato fruit. Moreover, the sequencing results also implied high level of ABA could negatively affect photosynthesis of tomato fruit, which needs more investigations to explore the interaction between ABA and photosynthesis in the future.

Project description:Tomato fruit ripening is under the control of ethylene as well as a group of ethylene-independent transcription factors, including NON-RIPENING (NOR) and RIPENING INHIBITOR (RIN). During ripening, the linear carotene lycopene accumulates at the expense of cyclic carotenoids. Fruit-specific overexpression of LYCOPENE β-CYCLASE (LCYb) under the control of the PHYTOENE DESATURASE (PDS) promoter resulted in increased levels of β-carotene and ABA and in decreased ethylene levels. Genes regulated by ABA, or involved in its synthesis and signaling, were overexpressed, while those associated with ethylene and cell wall remodeling were repressed. In agreement with the transcriptional data, LCYb-overexpressing fruits exhibited increased density of cell wall material containing linear, under-methylated pectins and displayed an array of additional ripening phenotypes, including delayed softening, increased turgor, enhanced shelf life and a thicker cuticle with a higher content of cutin monomers and triterpenoids. The levels of several primary metabolites and phenylpropanoids also changed in the transgenics, which could be attributed to delayed fruit ripening and to ABA respectively. Network correlation analysis suggests that ABA, acting through NOR and RIN, is responsible for many of the above phenotypes. These data reinforce suggestions that ABA plays an important role in tomato fruit ripening and provide clues that fruit b-carotene, acting as a precursor for ABA, actively participates in controlling the ripening process rather than merely being an output thereof. Overexpression of a LCYb gene from Arabidopsis under the control of the ripening-associated PDS promoter leads to ripe tomato fruits accumulating high β-carotene levels. Using several independent transgenic lines, we conducted a system-wide study of the effect of increased β-carotene levels on tomato fruit ripening and shelf life. Our data suggest that β-carotene, acting through ABA, is involved in a regulatory loop within the network controlling tomato fruit ripening.

Project description:Purpose: The goals of this study was to provide genome-wide data to investigate the molecular mechanism of the interaction between ABA and ethylene during tomato fruit ripening Methods: Regarding the fruits sampled at the 9th day as a well-characterized stage, we used RNA-seq to conducted a comparative analysis of exogenous ABA and NDGA effects on all components involved in biosynthesis and signaling of ethylene and ABA.The identified crucial genes in response to ABA were further performed a ripening time-course analysis by RT-PCR. In addition, we also detected how ethylene affected ABA action at the onset of ripening by treating the fruits with 1-MCP immediately after ABA application. Results: Our study not only illustrated how ABA regulated itself at the transcription level, but also elucidated that ABA can facilitate ethylene production and response by regulating some crucial genes such as LeACS4, LeACO1 and LeETR6 etc. In addition, investigation on the fruits treated with 1-MCP immediately after ABA exposure revealed that ethylene might be essential for the induction of ABA biosynthesis and signaling at the onset of fruit ripening. Furthermore, some specific transcription factors (TFs) known as regulators of ethylene synthesis and sensibility (e.g. MADS-RIN, TAGL1, CNR and NOR etc.) were also observed to be ABA responsive, which implied that ABA influenced ethylene action possibly through the regulation of these TFs expression. Conclusions: Our data suggested that ABA may act as an upstream regulator to modulate ethylene synthesis and signal transduction, which consequently influenced the ripening process. This comprehensive survey not only demonstrated how ABA regulated itself at the molecular level, but also indicated that ABA had a positive impact on ethylene production and action by regulating key genes such as LeACS2, LeACS4, LeACO1 and LeETR6. Besides, our results also revealed that ethylene might be of great importance to induce ABA accumulation and response at the onset of ripening. Moreover, many ripening related TFs, such as MADS-RIN, TAGL1, CNR and NOR, were observed to be affected by ABA, implying that a TF-mediated manner may be involved in the interaction between ABA and ethylene. This study extends our understanding of the mechanism that how ABA-triggered tomato fruit ripening, and illustrates the complex mechanism of reciprocity between ABA and ethylene at the transcription level.

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The Zinc Finger Transcription Factor SlZFP2 Negatively Regulates Abscisic Acid Biosynthesis and Fruit Ripening in Tomato

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