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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

Involvement of V-H+-ATPase during de-etiolation of tomato seedlings. a Analysis by qPCR of V- ATPase subunit c2 (B-D5) during de-etiolation. 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. b Analysis by qPCR of VHA-A1 subunit during de-etiolation. 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. c Effect of bafilomycin A1 on hypocotyl growth of tomato seedlings grown in BL . Germinated seeds were grown either in darkness or under constant BL on Murashige and Skoog medium containing varying concentrations of BafA1. After 5 days of growth, the length of hypocotyl was measured with a ruler to the nearest millimeter. The data are presented as boxes and whiskers. The whiskers represent the range of the data; the white dot within the box indicates the median value, while the boxes’ lower and upper boundaries indicate the first and third quartiles, respectively. An average of 45 plantlets coming from independent replicates was measured. The non-parametric Kruskal-Wallis Anova with multiple comparison of mean rank was used for statistical significance of the data (Software: Statistica 12); a: statistically different from the control condition with p-value ≤ 0.01
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Fig6: Involvement of V-H+-ATPase during de-etiolation of tomato seedlings. a Analysis by qPCR of V- ATPase subunit c2 (B-D5) during de-etiolation. 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. b Analysis by qPCR of VHA-A1 subunit during de-etiolation. 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. c Effect of bafilomycin A1 on hypocotyl growth of tomato seedlings grown in BL . Germinated seeds were grown either in darkness or under constant BL on Murashige and Skoog medium containing varying concentrations of BafA1. After 5 days of growth, the length of hypocotyl was measured with a ruler to the nearest millimeter. The data are presented as boxes and whiskers. The whiskers represent the range of the data; the white dot within the box indicates the median value, while the boxes’ lower and upper boundaries indicate the first and third quartiles, respectively. An average of 45 plantlets coming from independent replicates was measured. The non-parametric Kruskal-Wallis Anova with multiple comparison of mean rank was used for statistical significance of the data (Software: Statistica 12); a: statistically different from the control condition with p-value ≤ 0.01

Mentions: In tomato, three ESTs encoding vacuolar H + -ATPase (V-ATPase: 9, B-D5, E169) subunits were found to be up-regulated during PHOT1-mediated inhibition of hypocotyl growth. This was confirmed by qPCR for the V-type H+-ATPase subunit c2 (B-D5; Fig. 6a). Considering the role of V-ATPase during de-etiolationis important if one considers that hypocotyl growth in darkness does not require the division of cortical or epidermal cells and cells elongate along an acropetal spatial and temporal gradient [39]. Cell expansion is achieved by: i) increase in cell ploidy via endoreduplication, and ii) osmotic water uptake into the vacuole, creating the turgor pressure necessary for the irreversible extension of the cell wall caused by the synthesis, incorporation, and cross-linking of new cell wall components. The cell expansion is restricted by cell wall extensibility [40], [41]. V-ATPases are potentially involved in creating or regulating turgor pressure. They represent a major fraction of the total tonoplast proteins. V-ATPases also are present in the trans-Golgi network (TGN), where they are essential for its proper function [42]. Whereas inhibition of the tonoplast-localized V-ATPase does not affect cell expansion, inhibition of that which is TGN-localized is sufficient to restrict cell expansion [43]. Moreover, the det3 mutant, a possible negative regulator of photomorphogenesis affected in V-ATPase function, was originally proposed to be impaired in vacuolar solute uptake resulting in adequate turgor pressure for cell expansion [3]. Recent evidence has shown that a cell wall defect in the mutant is responsible for its reduced hypocotyl cell expansion [43]. Together, these data indicate that V-ATPase plays a role in cell wall integrity/synthesis through its function in the TGN-mediated secretory pathway, thereby participating in the restriction of cell expansion.Fig. 6


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)

Involvement of V-H+-ATPase during de-etiolation of tomato seedlings. a Analysis by qPCR of V- ATPase subunit c2 (B-D5) during de-etiolation. 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. b Analysis by qPCR of VHA-A1 subunit during de-etiolation. 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. c Effect of bafilomycin A1 on hypocotyl growth of tomato seedlings grown in BL . Germinated seeds were grown either in darkness or under constant BL on Murashige and Skoog medium containing varying concentrations of BafA1. After 5 days of growth, the length of hypocotyl was measured with a ruler to the nearest millimeter. The data are presented as boxes and whiskers. The whiskers represent the range of the data; the white dot within the box indicates the median value, while the boxes’ lower and upper boundaries indicate the first and third quartiles, respectively. An average of 45 plantlets coming from independent replicates was measured. The non-parametric Kruskal-Wallis Anova with multiple comparison of mean rank was used for statistical significance of the data (Software: Statistica 12); a: statistically different from the control condition with p-value ≤ 0.01
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Related In: Results  -  Collection

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Fig6: Involvement of V-H+-ATPase during de-etiolation of tomato seedlings. a Analysis by qPCR of V- ATPase subunit c2 (B-D5) during de-etiolation. 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. b Analysis by qPCR of VHA-A1 subunit during de-etiolation. 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. c Effect of bafilomycin A1 on hypocotyl growth of tomato seedlings grown in BL . Germinated seeds were grown either in darkness or under constant BL on Murashige and Skoog medium containing varying concentrations of BafA1. After 5 days of growth, the length of hypocotyl was measured with a ruler to the nearest millimeter. The data are presented as boxes and whiskers. The whiskers represent the range of the data; the white dot within the box indicates the median value, while the boxes’ lower and upper boundaries indicate the first and third quartiles, respectively. An average of 45 plantlets coming from independent replicates was measured. The non-parametric Kruskal-Wallis Anova with multiple comparison of mean rank was used for statistical significance of the data (Software: Statistica 12); a: statistically different from the control condition with p-value ≤ 0.01
Mentions: In tomato, three ESTs encoding vacuolar H + -ATPase (V-ATPase: 9, B-D5, E169) subunits were found to be up-regulated during PHOT1-mediated inhibition of hypocotyl growth. This was confirmed by qPCR for the V-type H+-ATPase subunit c2 (B-D5; Fig. 6a). Considering the role of V-ATPase during de-etiolationis important if one considers that hypocotyl growth in darkness does not require the division of cortical or epidermal cells and cells elongate along an acropetal spatial and temporal gradient [39]. Cell expansion is achieved by: i) increase in cell ploidy via endoreduplication, and ii) osmotic water uptake into the vacuole, creating the turgor pressure necessary for the irreversible extension of the cell wall caused by the synthesis, incorporation, and cross-linking of new cell wall components. The cell expansion is restricted by cell wall extensibility [40], [41]. V-ATPases are potentially involved in creating or regulating turgor pressure. They represent a major fraction of the total tonoplast proteins. V-ATPases also are present in the trans-Golgi network (TGN), where they are essential for its proper function [42]. Whereas inhibition of the tonoplast-localized V-ATPase does not affect cell expansion, inhibition of that which is TGN-localized is sufficient to restrict cell expansion [43]. Moreover, the det3 mutant, a possible negative regulator of photomorphogenesis affected in V-ATPase function, was originally proposed to be impaired in vacuolar solute uptake resulting in adequate turgor pressure for cell expansion [3]. Recent evidence has shown that a cell wall defect in the mutant is responsible for its reduced hypocotyl cell expansion [43]. Together, these data indicate that V-ATPase plays a role in cell wall integrity/synthesis through its function in the TGN-mediated secretory pathway, thereby participating in the restriction of cell expansion.Fig. 6

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