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A subtracted cDNA library identifies genes up-regulated during PHOT1-mediated early step of de-etiolation in tomato (Solanum lycopersicum L.).

Hloušková P, Bergougnoux V - BMC Genomics (2016)

Bottom Line: Our conclusions based on bioinformatics data were supported by qRT-PCR analyses the specific investigation of V-H(+)-ATPase during de-etiolation in tomato.The profound induction of transcription/translation, as well as modification of chromatin structure, is relevant in regard to the fact that the entry into photomorphogenesis is based on a deep reprograming of the cell.Also, we postulated that BL restrains the cell expansion by the rapid modification of the cell wall.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research and Faculty of Science, Palacký University in Olomouc, Šlechtitelů 11, CZ-783 71, Olomouc, Czech Republic.

ABSTRACT

Background: De-etiolation is the switch from skoto- to photomorphogenesis, enabling the heterotrophic etiolated seedling to develop into an autotrophic plant. Upon exposure to blue light (BL), reduction of hypocotyl growth rate occurs in two phases: a rapid inhibition mediated by phototropin 1 (PHOT1) within the first 30-40 min of illumination, followed by the cryptochrome 1 (CRY1)-controlled establishment of the steady-state growth rate. Although some information is available for CRY1-mediated de-etiolation, less attention has been given to the PHOT1 phase of de-etiolation.

Results: We generated a subtracted cDNA library using the suppression subtractive hybridization method to investigate the molecular mechanisms of BL-induced de-etiolation in tomato (Solanum lycopersicum L.), an economically important crop. We focused our interest on the first 30 min following the exposure to BL when PHOT1 is required to induce the process. Our library generated 152 expressed sequence tags that were found to be rapidly accumulated upon exposure to BL and consequently potentially regulated by PHOT1. Annotation revealed that biological functions such as modification of chromatin structure, cell wall modification, and transcription/translation comprise an important part of events contributing to the establishment of photomorphogenesis in young tomato seedlings. Our conclusions based on bioinformatics data were supported by qRT-PCR analyses the specific investigation of V-H(+)-ATPase during de-etiolation in tomato.

Conclusions: Our study provides the first report dealing with understanding the PHOT1-mediated phase of de-etiolation. Using subtractive cDNA library, we were able to identify important regulatory mechanisms. The profound induction of transcription/translation, as well as modification of chromatin structure, is relevant in regard to the fact that the entry into photomorphogenesis is based on a deep reprograming of the cell. Also, we postulated that BL restrains the cell expansion by the rapid modification of the cell wall.

No MeSH data available.


Related in: MedlinePlus

Analysis by quantitative real-time PCR of expression of selected genes belonging to different functional categories. The data represent the average fold change of 3 independent biological replicates ± SEM. Normalization was done using the pp2ase gene as housekeeping gene. Fold change was calculated compared to the value obtained for the dark control sample. The non-parametric Mann-Whitney U test (Statistica 12) was used to determine the significance of the results
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Fig3: Analysis by quantitative real-time PCR of expression of selected genes belonging to different functional categories. The data represent the average fold change of 3 independent biological replicates ± SEM. Normalization was done using the pp2ase gene as housekeeping gene. Fold change was calculated compared to the value obtained for the dark control sample. The non-parametric Mann-Whitney U test (Statistica 12) was used to determine the significance of the results

Mentions: In order to study the molecular events of the rapid inhibition of tomato hypocotyl growth observed within the first 30 min following exposure to BL, a SSH library was constructed and screened for genes whose expression is stimulated by BL. Two contrasting mRNA samples were extracted. One sample, extracted from the elongating zone of the hypocotyl of seedlings grown in darkness and exposed for 30 min to BL (10 μmol.m−2.s−1), potentially containing differentially expressed genes, was used as the tester. The second sample, isolated from the elongating zone of the hypocotyl of seedlings grown only in darkness, constituted the driver that should express transcripts common to both samples and eliminated during the process of subtraction. Due to technical limitation, 500 putative subtracted clones were randomly picked and used in cDNA dot-plot array for differential screening. Clones were considered for sequencing when they hybridized only to the BL-specific probes or showed higher intensity with the BL-specific probe than with dark-specific probe. In these conditions, we determined that 168 ESTs were potentially differentially expressed. After BLAST analysis, 17 sequences from 168 were found to be redundant, bringing to 151 the number of expressed sequence tag (ESTs) encoding proteins. The ontology annotation was performed using Blast2GO according to plant-specific Gene Ontology terms [21]. Computational analysis using the software Blast2GO enabled annotation of the expressed sequences according to the terms of the three main Gene Ontology vocabularies (i.e., cellular compartment, molecular function and biological process; Fig. 2). Concerning molecular function, the most represented categories were those of binding and catalytic activities (Fig. 2b). Regarding cellular compartments, the most represented were ribosome, plastid, and nucleus, together accounting for more than 50 % of total annotations (Fig. 2c). When taking into consideration the most relevant level of distribution for the biological process (i.e., level 8, as shown in Fig. 2a), more than 40 categories were found for the biological process vocabulary (data not shown). The number of categories was therefore simplified to level 2 of the distribution (Fig. 2d). The functional annotation was performed with Mercator, using the last updated version of the tomato annotation (ITAG2.4) [22]. Identical description of the ESTs was obtained when the annotation was performed by Blast2Go, KOG attribution or Blast against the specific annotated tomato genome ITAG2.4 (Additional file: 1 Table S1). The Table 2 shows the number of sequences which enter the different categories. Twenty seven sequences could not be annotated. The functional annotations “Protein: synthesis, targeting, postranslation modification, degradation” and “RNA: processing, transcription, regulation of transcription” were the most represented, including 33 and 12 sequences, respectively. More detailed information can be found in Additional file 1: Table S1. For all genes tested, qPCR confirmed the differential expression detected by the screening of the cDNA library, meaning the up-regulation of the expression of ESTs as soon as 30 min after exposure to BL (Fig. 3). Below, we discuss the potential involvement of various genes in the rapid inhibition of hypocotyl growth induced by 30 min of exposure to BL and mediated by PHOT1.Fig. 2


A subtracted cDNA library identifies genes up-regulated during PHOT1-mediated early step of de-etiolation in tomato (Solanum lycopersicum L.).

Hloušková P, Bergougnoux V - BMC Genomics (2016)

Analysis by quantitative real-time PCR of expression of selected genes belonging to different functional categories. The data represent the average fold change of 3 independent biological replicates ± SEM. Normalization was done using the pp2ase gene as housekeeping gene. Fold change was calculated compared to the value obtained for the dark control sample. The non-parametric Mann-Whitney U test (Statistica 12) was used to determine the significance of the results
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4835860&req=5

Fig3: Analysis by quantitative real-time PCR of expression of selected genes belonging to different functional categories. The data represent the average fold change of 3 independent biological replicates ± SEM. Normalization was done using the pp2ase gene as housekeeping gene. Fold change was calculated compared to the value obtained for the dark control sample. The non-parametric Mann-Whitney U test (Statistica 12) was used to determine the significance of the results
Mentions: In order to study the molecular events of the rapid inhibition of tomato hypocotyl growth observed within the first 30 min following exposure to BL, a SSH library was constructed and screened for genes whose expression is stimulated by BL. Two contrasting mRNA samples were extracted. One sample, extracted from the elongating zone of the hypocotyl of seedlings grown in darkness and exposed for 30 min to BL (10 μmol.m−2.s−1), potentially containing differentially expressed genes, was used as the tester. The second sample, isolated from the elongating zone of the hypocotyl of seedlings grown only in darkness, constituted the driver that should express transcripts common to both samples and eliminated during the process of subtraction. Due to technical limitation, 500 putative subtracted clones were randomly picked and used in cDNA dot-plot array for differential screening. Clones were considered for sequencing when they hybridized only to the BL-specific probes or showed higher intensity with the BL-specific probe than with dark-specific probe. In these conditions, we determined that 168 ESTs were potentially differentially expressed. After BLAST analysis, 17 sequences from 168 were found to be redundant, bringing to 151 the number of expressed sequence tag (ESTs) encoding proteins. The ontology annotation was performed using Blast2GO according to plant-specific Gene Ontology terms [21]. Computational analysis using the software Blast2GO enabled annotation of the expressed sequences according to the terms of the three main Gene Ontology vocabularies (i.e., cellular compartment, molecular function and biological process; Fig. 2). Concerning molecular function, the most represented categories were those of binding and catalytic activities (Fig. 2b). Regarding cellular compartments, the most represented were ribosome, plastid, and nucleus, together accounting for more than 50 % of total annotations (Fig. 2c). When taking into consideration the most relevant level of distribution for the biological process (i.e., level 8, as shown in Fig. 2a), more than 40 categories were found for the biological process vocabulary (data not shown). The number of categories was therefore simplified to level 2 of the distribution (Fig. 2d). The functional annotation was performed with Mercator, using the last updated version of the tomato annotation (ITAG2.4) [22]. Identical description of the ESTs was obtained when the annotation was performed by Blast2Go, KOG attribution or Blast against the specific annotated tomato genome ITAG2.4 (Additional file: 1 Table S1). The Table 2 shows the number of sequences which enter the different categories. Twenty seven sequences could not be annotated. The functional annotations “Protein: synthesis, targeting, postranslation modification, degradation” and “RNA: processing, transcription, regulation of transcription” were the most represented, including 33 and 12 sequences, respectively. More detailed information can be found in Additional file 1: Table S1. For all genes tested, qPCR confirmed the differential expression detected by the screening of the cDNA library, meaning the up-regulation of the expression of ESTs as soon as 30 min after exposure to BL (Fig. 3). Below, we discuss the potential involvement of various genes in the rapid inhibition of hypocotyl growth induced by 30 min of exposure to BL and mediated by PHOT1.Fig. 2

Bottom Line: Our conclusions based on bioinformatics data were supported by qRT-PCR analyses the specific investigation of V-H(+)-ATPase during de-etiolation in tomato.The profound induction of transcription/translation, as well as modification of chromatin structure, is relevant in regard to the fact that the entry into photomorphogenesis is based on a deep reprograming of the cell.Also, we postulated that BL restrains the cell expansion by the rapid modification of the cell wall.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research and Faculty of Science, Palacký University in Olomouc, Šlechtitelů 11, CZ-783 71, Olomouc, Czech Republic.

ABSTRACT

Background: De-etiolation is the switch from skoto- to photomorphogenesis, enabling the heterotrophic etiolated seedling to develop into an autotrophic plant. Upon exposure to blue light (BL), reduction of hypocotyl growth rate occurs in two phases: a rapid inhibition mediated by phototropin 1 (PHOT1) within the first 30-40 min of illumination, followed by the cryptochrome 1 (CRY1)-controlled establishment of the steady-state growth rate. Although some information is available for CRY1-mediated de-etiolation, less attention has been given to the PHOT1 phase of de-etiolation.

Results: We generated a subtracted cDNA library using the suppression subtractive hybridization method to investigate the molecular mechanisms of BL-induced de-etiolation in tomato (Solanum lycopersicum L.), an economically important crop. We focused our interest on the first 30 min following the exposure to BL when PHOT1 is required to induce the process. Our library generated 152 expressed sequence tags that were found to be rapidly accumulated upon exposure to BL and consequently potentially regulated by PHOT1. Annotation revealed that biological functions such as modification of chromatin structure, cell wall modification, and transcription/translation comprise an important part of events contributing to the establishment of photomorphogenesis in young tomato seedlings. Our conclusions based on bioinformatics data were supported by qRT-PCR analyses the specific investigation of V-H(+)-ATPase during de-etiolation in tomato.

Conclusions: Our study provides the first report dealing with understanding the PHOT1-mediated phase of de-etiolation. Using subtractive cDNA library, we were able to identify important regulatory mechanisms. The profound induction of transcription/translation, as well as modification of chromatin structure, is relevant in regard to the fact that the entry into photomorphogenesis is based on a deep reprograming of the cell. Also, we postulated that BL restrains the cell expansion by the rapid modification of the cell wall.

No MeSH data available.


Related in: MedlinePlus