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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 qPCR of the expression of two genes encoding proteins involved in cell wall modification: xyloglucan endotransglucosylase-hydrolase/XTH (a) and pectin acetylesterase (b). 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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Fig5: Analysis by qPCR of the expression of two genes encoding proteins involved in cell wall modification: xyloglucan endotransglucosylase-hydrolase/XTH (a) and pectin acetylesterase (b). 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 our study, seven tomato EST encoding proteins involved in cell wall modification were found to be up-regulated in the etiolated hypocotyl of tomato seedlings exposed for 30 min to BL: pectin acetylesterase, pectinesterase, xyloglucan endotransglucosylase-hydrolase 1 (XTH), or endoglucanase. This suggested that de-etiolation induced a strong modification of the cell wall structure and/or composition. Plant cell walls consist of a complex network of cellulose microfibrils embedded in a matrix of hemicelluloses (mainly xyloglucans), pectins, and glycoproteins [29]. During cell maturation, cell walls lose the ability to expand [30]. Growth cessation is accompanied by cell wall tightening [31]. Various modifications of cell wall structure during maturation have been proposed, including changes in hemicellulose. For example, the maturation of pea tissues is characterized by an increase in the total amount of xyloglucan [32]. Xyloglucan endotransglucosylase/hydrolases (XTH) are enzymes capable of modifying xyloglucan during cell expansion. They comprise a subgroup of the glycoside hydrolase family 16. XTH proteins characterized to date have endotransglycosylase (XET) or hydrolase (XEH) activities towards xyloglucans, or both. Their phylogenic study indicates that they are organized into three groups: I/II, III-a, and III-b. Only members of the III-a group are strict XEH [33]. Transgenic tomatoes with altered levels of XTH gene showed higher XET activity, lower hemicellulose depolymerization and reduced fruit softening during ripening. This suggests that XET could have a role in maintaining the structural integrity of the cell wall [34], [35]. Thus, whereas some XTH members are critical in promoting cell wall expansion, others are required for wall strengthening in cells that have completed the expansion process [36]. The analysis by qPCR of the XTH identified by SSH screening confirmed that it is up-regulated by BL (Clone 12; Fig. 5a). Based on the aforementioned literature, we can assume its role in cell wall strengthening during de-etiolation. Pectins, comprising another important cell wall component, are synthesized in the cis-Golgi, methyl-esterified in the medial-Golgi, substituted in the trans-Golgi, and then secreted into the cell wall. Zhao and co-authors [37] reported that de-esterification of methyl-esterified pectin may also be associated with growth cessation in both grasses and dicotyledons and may contribute to wall tightening by strengthening pectin–calcium networks. Pectin acetylation is another modification of pectins which probably occurs between the Golgi and the cell wall during pectin exocytosis. Its occurrence and function are poorly understood. The degree of O-acetylation of pectin changes during growth and differentiation of plant tissues, but also in response to environmental conditions. Pectin acetylesterases trigger the deacetylation of pectin. The overexpression of the black cottonwood (Populus trichocarpa) PAE1 gene in tobacco has been shown to impair the cellular elongation of floral organs. Thus, it appears that pectin acetylesterases function as an important regulator of pectin acetylation status to affect the physiochemical properties of the cell wall’s polysaccharides and consequently to affect cell extensibility [38]. The confirmation by qPCR that pectin acetylesterase (B-E2; Fig. 5b) is up-regulated by BL supports the hypothesis that they are actors of the inhibition of cell expansion which occurs during de-etiolation.Fig. 5


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 qPCR of the expression of two genes encoding proteins involved in cell wall modification: xyloglucan endotransglucosylase-hydrolase/XTH (a) and pectin acetylesterase (b). 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

Fig5: Analysis by qPCR of the expression of two genes encoding proteins involved in cell wall modification: xyloglucan endotransglucosylase-hydrolase/XTH (a) and pectin acetylesterase (b). 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 our study, seven tomato EST encoding proteins involved in cell wall modification were found to be up-regulated in the etiolated hypocotyl of tomato seedlings exposed for 30 min to BL: pectin acetylesterase, pectinesterase, xyloglucan endotransglucosylase-hydrolase 1 (XTH), or endoglucanase. This suggested that de-etiolation induced a strong modification of the cell wall structure and/or composition. Plant cell walls consist of a complex network of cellulose microfibrils embedded in a matrix of hemicelluloses (mainly xyloglucans), pectins, and glycoproteins [29]. During cell maturation, cell walls lose the ability to expand [30]. Growth cessation is accompanied by cell wall tightening [31]. Various modifications of cell wall structure during maturation have been proposed, including changes in hemicellulose. For example, the maturation of pea tissues is characterized by an increase in the total amount of xyloglucan [32]. Xyloglucan endotransglucosylase/hydrolases (XTH) are enzymes capable of modifying xyloglucan during cell expansion. They comprise a subgroup of the glycoside hydrolase family 16. XTH proteins characterized to date have endotransglycosylase (XET) or hydrolase (XEH) activities towards xyloglucans, or both. Their phylogenic study indicates that they are organized into three groups: I/II, III-a, and III-b. Only members of the III-a group are strict XEH [33]. Transgenic tomatoes with altered levels of XTH gene showed higher XET activity, lower hemicellulose depolymerization and reduced fruit softening during ripening. This suggests that XET could have a role in maintaining the structural integrity of the cell wall [34], [35]. Thus, whereas some XTH members are critical in promoting cell wall expansion, others are required for wall strengthening in cells that have completed the expansion process [36]. The analysis by qPCR of the XTH identified by SSH screening confirmed that it is up-regulated by BL (Clone 12; Fig. 5a). Based on the aforementioned literature, we can assume its role in cell wall strengthening during de-etiolation. Pectins, comprising another important cell wall component, are synthesized in the cis-Golgi, methyl-esterified in the medial-Golgi, substituted in the trans-Golgi, and then secreted into the cell wall. Zhao and co-authors [37] reported that de-esterification of methyl-esterified pectin may also be associated with growth cessation in both grasses and dicotyledons and may contribute to wall tightening by strengthening pectin–calcium networks. Pectin acetylation is another modification of pectins which probably occurs between the Golgi and the cell wall during pectin exocytosis. Its occurrence and function are poorly understood. The degree of O-acetylation of pectin changes during growth and differentiation of plant tissues, but also in response to environmental conditions. Pectin acetylesterases trigger the deacetylation of pectin. The overexpression of the black cottonwood (Populus trichocarpa) PAE1 gene in tobacco has been shown to impair the cellular elongation of floral organs. Thus, it appears that pectin acetylesterases function as an important regulator of pectin acetylation status to affect the physiochemical properties of the cell wall’s polysaccharides and consequently to affect cell extensibility [38]. The confirmation by qPCR that pectin acetylesterase (B-E2; Fig. 5b) is up-regulated by BL supports the hypothesis that they are actors of the inhibition of cell expansion which occurs during de-etiolation.Fig. 5

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