Dataset Information

Negative feedback regulation of ABA biosynthesis in peanut (Arachis hypogaea): a transcription factor complex inhibits AhNCED1 expression during water stress.

ABSTRACT: Abscisic acid (ABA), a key plant stress-signaling hormone, is produced in response to drought and counteracts the effects of this stress. The accumulation of ABA is controlled by the enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). In Arabidopsis, NCED3 is regulated by a positive feedback mechanism by ABA. In this study in peanut (Arachis hypogaea), we demonstrate that ABA biosynthesis is also controlled by negative feedback regulation, mediated by the inhibitory effect on AhNCED1 transcription of a protein complex between transcription factors AhNAC2 and AhAREB1. AhNCED1 was significantly down-regulated after PEG treatment for 10?h, at which time ABA content reached a peak. A ChIP-qPCR assay confirmed AhAREB1 and AhNAC2 binding to the AhNCED1 promoter in response to ABA. Moreover, the interaction between AhAREB1 and AhNAC2, and a transient expression assay showed that the protein complex could negatively regulate the expression of AhNCED1. The results also demonstrated that AhAREB1 was the key factor in AhNCED1 feedback regulation, while AhNAC2 played a subsidiary role. ABA reduced the rate of AhAREB1 degradation and enhanced both the synthesis and degradation rate of the AhNAC2 protein. In summary, the AhAREB1/AhNAC2 protein complex functions as a negative feedback regulator of drought-induced ABA biosynthesis in peanut.

SUBMITTER: Liu S

PROVIDER: S-EPMC5124963 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Negative feedback regulation of ABA biosynthesis in peanut (Arachis hypogaea): a transcription factor complex inhibits AhNCED1 expression during water stress.

Liu Shuai S Li Meijuan M Su Liangchen L Ge Kui K Li Limei L Li Xiaoyun X Liu Xu X Li Ling L

Scientific reports 20161128

Abscisic acid (ABA), a key plant stress-signaling hormone, is produced in response to drought and counteracts the effects of this stress. The accumulation of ABA is controlled by the enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). In Arabidopsis, NCED3 is regulated by a positive feedback mechanism by ABA. In this study in peanut (Arachis hypogaea), we demonstrate that ABA biosynthesis is also controlled by negative feedback regulation, mediated by the inhibitory effect on AhNCED1 transcription ...[more]

PMID: 27892506

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Project description:Purpose: to screen the candidate genes involved in the peanut drought stress response, we conducted global transcriptome analysis of peanut plants challenged with water deficit and ABA, using the Illumina HiSeq2000 sequencing platform. Methods: a sequences library of Yueyou7 were constructed at first. Then the profile of diffentialy expressed genes (DEGs) under three different treatments (control, water deficit without ABA, and water deficit with ABA) were conducted based on above sequence library. For sequencing library construction, plants were grown under normal conditions, as described previously , Seeds were planted in soil and kep in the greenhouse at temperature of 25-30℃ and water well. Three tissues (leaves, roots, and stems) were collected at three development stages (four-leaf, flowering and podding stages), respectively. Then all of these tissues were mixed to extract the total RNA for sequence library construction. For DEGs study, two-week-old plants were divided into three groups with three independent replication: (1). Water deficit without ABA groups. Plants directly steeped in water containing 30% PEG600 for 30 min in this groups. (2) Water deficit with ABA groups. Plant was subjected to 100 µmol/L ABA for 30 min and then steeped in water containing 30% PEG6000 for 30 min in this groups, (3) Control. Plants steeped in H2O. All treatments were conducted in parallel. After treatments, Total RNA was extracted from 100 mg of plant material, and RNA integrity was checked by gel electrophoresis. Also RNA quality was checked and RNA was quantified using the Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA) and Nanodrop ND-1000 (Thermo Scientific, Waltham, USA). Results: we generated 4.96×107 raw sequence reads and assembled the high quality reads into 92,390 unique genes. Compared with the control, we found that 621 genes (≥1.5 fold change) responded rapidly to water deficit and 2665 genes (≥1.5 fold change) responded rapidly to ABA. We found 279 genes that overlapped between water deficit and ABA responses, 264 genes that showed the same trend in expression while 15 genes expression that showed opposite trend. Among the identified genes, 257 showed high induction by ABA (>5 fold), and 19 showed high induction by drought (>5 fold). In addition, we identified 100 transcription factor genes among the ABA-inducible genes but only 22 transcription factor genes among the water deficit-inducible genes. Conclusions: we identified genes differentially expressed in the early response to water deficit or ABA. These genes were annotated with GO functional categories for water deficit (33 categories) or ABA (31 categories). We found that only 19 genes were highly induced by water deficit, but 257 genes were highly induced by ABA. Our previous work has examined many of these genes and our future work will reveal their functions and relationships. These data will facilitate functional genomic studies and have established a biotechnological platform for examination of the early drought- and ABA-responsive transcriptome regulatory network in peanut.

Project description:Purpose: to screen the candidate genes involved in the peanut drought stress response, we conducted global transcriptome analysis of peanut plants challenged with water deficit and ABA, using the Illumina HiSeq2000 sequencing platform. Methods: a sequences library of Yueyou7 were constructed at first. Then the profile of diffentialy expressed genes (DEGs) under three different treatments (control, water deficit without ABA, and water deficit with ABA) were conducted based on above sequence library. For sequencing library construction, plants were grown under normal conditions, as described previously , Seeds were planted in soil and kep in the greenhouse at temperature of 25-30M-bM-^DM-^C and water well. Three tissues (leaves, roots, and stems) were collected at three development stages (four-leaf, flowering and podding stages), respectively. Then all of these tissues were mixed to extract the total RNA for sequence library construction. For DEGs study, two-week-old plants were divided into three groups with three independent replication: (1). Water deficit without ABA groups. Plants directly steeped in water containing 30% PEG600 for 30 min in this groups. (2) Water deficit with ABA groups. Plant was subjected to 100 M-BM-5mol/L ABA for 30 min and then steeped in water containing 30% PEG6000 for 30 min in this groups, (3) Control. Plants steeped in H2O. All treatments were conducted in parallel. After treatments, Total RNA was extracted from 100 mg of plant material, and RNA integrity was checked by gel electrophoresis. Also RNA quality was checked and RNA was quantified using the Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA) and Nanodrop ND-1000 (Thermo Scientific, Waltham, USA). Results: we generated 4.96M-CM-^W107 raw sequence reads and assembled the high quality reads into 92,390 unique genes. Compared with the control, we found that 621 genes (M-bM-^IM-%1.5 fold change) responded rapidly to water deficit and 2665 genes (M-bM-^IM-%1.5 fold change) responded rapidly to ABA. We found 279 genes that overlapped between water deficit and ABA responses, 264 genes that showed the same trend in expression while 15 genes expression that showed opposite trend. Among the identified genes, 257 showed high induction by ABA (>5 fold), and 19 showed high induction by drought (>5 fold). In addition, we identified 100 transcription factor genes among the ABA-inducible genes but only 22 transcription factor genes among the water deficit-inducible genes. Conclusions: we identified genes differentially expressed in the early response to water deficit or ABA. These genes were annotated with GO functional categories for water deficit (33 categories) or ABA (31 categories). We found that only 19 genes were highly induced by water deficit, but 257 genes were highly induced by ABA. Our previous work has examined many of these genes and our future work will reveal their functions and relationships. These data will facilitate functional genomic studies and have established a biotechnological platform for examination of the early drought- and ABA-responsive transcriptome regulatory network in peanut. Two-week-old plants were divided into three groups with three independent replication: (1). Water deficit without ABA groups. Plants directly steeped in water containing 30% PEG600 for 30 min in this groups. (2) Water deficit with ABA groups. Plant was subjected to 100 M-BM-5mol/L ABA for 30 min and then steeped in water containing 30% PEG6000 for 30 min in this groups, (3) Control. Plants steeped in H2O. All treatments were conducted in parallel.

Project description:BACKGROUND:Microsatellites, or simple sequence repeats (SSRs), represent important DNA variations that are widely distributed across the entire plant genome and can be used to develop SSR markers, which can then be used to conduct genetic analyses and molecular breeding. Cultivated peanut (A. hypogaea L.), an important oil crop worldwide, is an allotetraploid (AABB, 2n = 4× = 40) plant species. Because of its complex genome, genomic marker development has been very challenging. However, sequencing of cultivated peanut genome allowed us to develop genomic markers and construct a high-density physical map. RESULTS:A total of 8,329,496 SSRs were identified, including 3,772,653, 4,414,961, and 141,882 SSRs that were distributed in subgenome A, B, and nine scaffolds, respectively. Based on the flanking sequences of the identified SSRs, a total of 973,984 newly developed SSR markers were developed in subgenome A (462,267), B (489,394), and nine scaffolds (22,323), with an average density of 392.45 markers per Mb. In silico PCR evaluation showed that an average of 88.32% of the SSR markers generated only one in silico-specific product in two tetraploid A. hypogaea varieties, Tifrunner and Shitouqi. A total of 39,599 common SSR markers were identified among the two A. hypogaea varieties and two progenitors, A. duranensis and A. ipaensis. Additionally, an amplification effectiveness of 44.15% was observed by real PCR validation. Moreover, a total of 1276 public SSR loci were integrated with the newly developed SSR markers. Finally, a previously known leaf spot quantitative trait locus (QTL), qLLS_T13_A05_7, was determined to be in a 1.448-Mb region on chromosome A05. In this region, a total of 819 newly developed SSR markers were located and 108 candidate genes were detected. CONCLUSIONS:The availability of these newly developed and public SSR markers both provide a large number of molecular markers that could potentially be used to enhance the process of trait genetic analyses and improve molecular breeding strategies for cultivated peanut.

Project description:BACKGROUND: Lack of sufficient molecular markers hinders current genetic research in peanuts (Arachis hypogaea L.). It is necessary to develop more molecular markers for potential use in peanut genetic research. With the development of peanut EST projects, a vast amount of available EST sequence data has been generated. These data offered an opportunity to identify SSR in ESTs by data mining. RESULTS: In this study, we investigated 24,238 ESTs for the identification and development of SSR markers. In total, 881 SSRs were identified from 780 SSR-containing unique ESTs. On an average, one SSR was found per 7.3 kb of EST sequence with tri-nucleotide motifs (63.9%) being the most abundant followed by di- (32.7%), tetra- (1.7%), hexa- (1.0%) and penta-nucleotide (0.7%) repeat types. The top six motifs included AG/TC (27.7%), AAG/TTC (17.4%), AAT/TTA (11.9%), ACC/TGG (7.72%), ACT/TGA (7.26%) and AT/TA (6.3%). Based on the 780 SSR-containing ESTs, a total of 290 primer pairs were successfully designed and used for validation of the amplification and assessment of the polymorphism among 22 genotypes of cultivated peanuts and 16 accessions of wild species. The results showed that 251 primer pairs yielded amplification products, of which 26 and 221 primer pairs exhibited polymorphism among the cultivated and wild species examined, respectively. Two to four alleles were found in cultivated peanuts, while 3-8 alleles presented in wild species. The apparent broad polymorphism was further confirmed by cloning and sequencing of amplified alleles. Sequence analysis of selected amplified alleles revealed that allelic diversity could be attributed mainly to differences in repeat type and length in the microsatellite regions. In addition, a few single base mutations were observed in the microsatellite flanking regions. CONCLUSION: This study gives an insight into the frequency, type and distribution of peanut EST-SSRs and demonstrates successful development of EST-SSR markers in cultivated peanut. These EST-SSR markers could enrich the current resource of molecular markers for the peanut community and would be useful for qualitative and quantitative trait mapping, marker-assisted selection, and genetic diversity studies in cultivated peanut as well as related Arachis species. All of the 251 working primer pairs with names, motifs, repeat types, primer sequences, and alleles tested in cultivated and wild species are listed in Additional File 1.

Dataset Information

Negative feedback regulation of ABA biosynthesis in peanut (Arachis hypogaea): a transcription factor complex inhibits AhNCED1 expression during water stress.

Publications

Negative feedback regulation of ABA biosynthesis in peanut (Arachis hypogaea): a transcription factor complex inhibits AhNCED1 expression during water stress.

Similar Datasets

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