Limits...
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

Schematic representation of the suppression subtractive hybridization adapted to the present study from [15]
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Schematic representation of the suppression subtractive hybridization adapted to the present study from [15]

Mentions: For all experiments, the elongating zone of the hypocotyl of 3-day-old etiolated seedlings was excised from the rest of the seedling. either under green safety light (dark control) or under BL after 30 min of exposure to BL. The elongating zone corresponding to the portion of hypocotyl situated beneath the hook and cotyledons was limited to the upper third of the hypocotyl as described in [14]. Samples were immediately frozen in liquid nitrogen and stored at −80 °C before RNA isolation. Frozen tissues were ground in liquid nitrogen using a mortar and pestle. Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen). Remaining traces of DNA were removed with a recombinant DNAseI (Takara) and RNA were subsequently purified by a phenol:chloroform:isoamyl alcohol (25:24:1) step. PolyA+ mRNA were purified using the Straight A’s mRNA isolation system (Novagen). Quantity and quality of mRNA were checked by spectrophotometer and electrophoresis. The suppression subtractive hybridization (SSH) library was constructed according to the instructions of the PCR-Select cDNA Subtraction Kit (Clontech). The principle of SSH library is illustrated by the Fig. 1. In order to identify genes up-regulated by the exposure to BL, subtractive hybridization was performed using cDNA from hypocotyls exposed for 30 min to BL as tester against cDNA from control hypocotyls (not exposed to BL) as driver. The subtraction efficiency was evaluated by a PCR reaction amplifying a region of the tomato EF1α gene and the PCR product was analyzed after 15, 20, 25, 30, and 35 cycles.Fig. 1


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)

Schematic representation of the suppression subtractive hybridization adapted to the present study from [15]
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Schematic representation of the suppression subtractive hybridization adapted to the present study from [15]
Mentions: For all experiments, the elongating zone of the hypocotyl of 3-day-old etiolated seedlings was excised from the rest of the seedling. either under green safety light (dark control) or under BL after 30 min of exposure to BL. The elongating zone corresponding to the portion of hypocotyl situated beneath the hook and cotyledons was limited to the upper third of the hypocotyl as described in [14]. Samples were immediately frozen in liquid nitrogen and stored at −80 °C before RNA isolation. Frozen tissues were ground in liquid nitrogen using a mortar and pestle. Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen). Remaining traces of DNA were removed with a recombinant DNAseI (Takara) and RNA were subsequently purified by a phenol:chloroform:isoamyl alcohol (25:24:1) step. PolyA+ mRNA were purified using the Straight A’s mRNA isolation system (Novagen). Quantity and quality of mRNA were checked by spectrophotometer and electrophoresis. The suppression subtractive hybridization (SSH) library was constructed according to the instructions of the PCR-Select cDNA Subtraction Kit (Clontech). The principle of SSH library is illustrated by the Fig. 1. In order to identify genes up-regulated by the exposure to BL, subtractive hybridization was performed using cDNA from hypocotyls exposed for 30 min to BL as tester against cDNA from control hypocotyls (not exposed to BL) as driver. The subtraction efficiency was evaluated by a PCR reaction amplifying a region of the tomato EF1α gene and the PCR product was analyzed after 15, 20, 25, 30, and 35 cycles.Fig. 1

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