A subtracted cDNA library identifies genes up-regulated during PHOT1-mediated early step of de-etiolation in tomato (Solanum lycopersicum L.)
© Hloušková and Bergougnoux. 2016
Received: 1 October 2015
Accepted: 6 April 2016
Published: 18 April 2016
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.
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.
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.
Light is one of the most important environmental factors influencing plants throughout their life spans. Blue and red/far-red portions of light can be considered as the most active rays within the light spectrum for regulating plant growth and development. As sessile organisms, plants have evolved highly sophisticated unique photoreceptors to sense light. They possess three main classes of photoreceptors: phytochromes (PHY), cryptochromes (CRY), and phototropins (PHOT), capable of absorbing red/far-red, blue, and blue light, respectively . Not only is light the primary source of energy for photosynthesis, but it also regulates numerous physiological responses, such as shade avoidance, flowering, germination, tropisms, and de-etiolation . De-etiolation occurs during early seedling development. In dicotyledonous plants, the hypocotyl (embryonic stem) connects the two cotyledons (embryonic leaves) to the root. When germinated in darkness in the soil, the hypocotyl expands toward the surface in order to place the shoot apical meristem in an environment suitable to ensure photoautotrophic growth. When the seedling emerges from the soil, it perceives light; the hypocotyl stops growing, the cotyledons unfold and green, the chloroplasts differentiate, and finally photosynthetic growth is initiated . As almost all of the hypocotyl’s cells are formed during embryogenesis; only a few cell divisions occur in the hypocotyl during etiolation, being limited to the development of stomata . For example, in Arabidopsis, the hypocotyl consisting of only 20 epidermal cells elongates more than 100-fold its embryonic length .
Hypocotyl de-etiolation is regulated by the three mentioned photoreceptor families. Nevertheless, at equal irradiances, blue light (BL) is more effective than red light as it inhibits growth more quickly and to a greater extent . Using cry1 Arabidopsis mutant defective in BL-induced de-etiolation, studies have demonstrated that CRY1 is the BL receptor involved in the control of hypocotyl elongation [5, 6]. Using computer-assisted electronic image capture, however, Parks and co-authors  demonstrated that in cry1 seedlings hypocotyl growth inhibition begins to develop within approximately 30 sec of BL irradiation and reaches the same maximum level displayed by wild-type seedlings after approximately 30 min of BL treatment. At this point, cry1 seedling growth accelerates, soon attaining the growth rate observed for darkness-grown seedlings. This experiment demonstrated that BL-mediated hypocotyl inhibition in Arabidopsis occurs in two genetically independent phases . A few years later, while applying the same method to different Arabidopsis photoreceptor mutants, Folta and Spalding  identified PHOT1 as being involved in the rapid phase of BL-mediated hypocotyl growth inhibition.
The PHOT1 signaling pathway has been studied extensively in the phase of stomata opening. In response to BL, plasma membrane H+-ATPases in the guard cells are activated. This induces a negative electrical potential across the plasma membrane and drives K+ uptake. Ions and metabolites enter the cell concomitantly with water uptake, thereby increasing turgor pressure and resulting in the opening of the stomata. The plasma membrane H+-ATPase is activated by phosphorylation of its C-terminus with a concomitant binding of the 14-3-3 proteins . By comparison, the mechanisms involved in PHOT1-mediated de-etiolation are still poorly understood. Nevertheless, genetic, biochemical, and physiological studies have begun to delineate the signaling pathway initiated after the onset of BL excitation. Evidence has accumulated to prove that excitation of PHOT1 induces a rapid activation of Ca2+ channels at the plasma membrane, leading to an increased concentration of cytosolic Ca2+ , . To our knowledge, few events acting downstream of PHOT1 have been identified during de-etiolation , . Therefore, it remains challenging to identify the PHOT1-signaling pathway during de-etiolation. All analyses to date have been performed on plant models, most notably in Arabidopsis. Little or no information is available from important crop species. For several years, we have focused on understanding the role of BL in the growth and development of tomato (Solanum lycopersicum L.), an economically important crop . We previously demonstrated that in etiolated tomato seedlings exposed to BL the reduction of the hypocotyl growth rate is a two-step process . Based on the knowledge coming from studies on Arabidopsis, we hypothesized that the first rapid inhibition might be triggered by PHOT1, and that the steady-state rate of growth might be established by CRY1.
Suppression subtractive hybridization (SSH) is a powerful approach which allows the comparison of two samples (tester and driver) and the identification of differentially regulated genes . Indeed, SSH is a combination of normalization which equalizes the abundance of cDNA within the target population and subtraction which excludes sequences common to both the tester and the driver . Therefore, SSH identifies not only abundant differentially expressed genes but also rare transcripts which were enriched during the process. This latest category is of high interest as it can represent a pool of unknown genes. This method does not require an in-depth knowledge of the genome under study and can thus be applied easily to non-model species , , , . In the present study, we used the SSH approach to identify the molecular mechanisms of the PHOT1-mediated rapid inhibition of hypocotyl elongation in tomato. Our current results provide evidence that a complex network is quickly activated by exposure to BL in order to induce de-etiolation.
Plant materials and light treatment
The tomato cultivar Rutgers was used in this study. Sterile cultures were obtained as described in . After germination in darkness at 23 °C, germinated seeds were transferred in the dark for 3 additional days to a culture chamber maintained at 23 °C. For BL-induced de-etiolation, dishes containing 3-day-old etiolated seedlings were transferred for 30 min under BL provided by fluorescent lamps (BL; TL-D 36 W/Blue, Phillips; total photon fluence rate 10 μmol.m−2.s−1).
RNA extraction and subtractive library construction
Cloning, screening for differential expression, sequencing and analysis
Secondary SSH-PCR products were inserted into pGEM-T Easy Vector (Promega) and cloned into Escherichia coli DH5α strain. A blue–white screening was performed in order to obtain a bank of subtracted ESTs. White colonies were picked and grown in 96-well microtiter plates in a lysogeny broth medium containing ampicillin (100 mg.L−1). Screen for differentially expressed ESTs was performed by dot blot hybridization as described in the PCR-select cDNA subtraction kit (Clontech). For this purpose, plasmids were isolated, quantified and transferred to Hybond-N+ nylon membranes. Membranes were prepared in duplicates with equal amounts of plasmids and were hybridized either with the BL-specific tester probe or the dark-specific driver probe, both DIG-labelled. Detection was performed with an anti-digoxigenin antibody coupled with a horse radish peroxidase. Cold detection was performed by enhanced chemiluminescent detection and exposure to X-ray films. Autoradiographies were scanned and the intensity of the dots was determined using ImageJ software. The dot intensity of a specific clone obtained with the BL-specific probe was compared to that obtained with the dark-specific probe. All clones showing higher intensity with BL-specific probe compared to dark-specific probe were selected and sequenced by Macrogen (Korea). A total of 168 ESTs were found to be differentially expressed. Their sizes ranged from 128 to 1387 bp, with an average size of 447 bp. Because during the process of library preparation the cDNA were restricted by RsaI, the first step of the analysis was to retrieve the full-length of the gene and the corresponding protein for downstream analyses by BLAST against the tomato database at SolGenomics Network. The gene ontology annotation was performed with Blast2GO according to plant-specific Gene Ontology terms . Concurrently, the functional annotation was performed by Mercator/MapMan which allows attributing DEGs a functional pathway , .
Quantitative real time-PCR
Primers used in quantitative real-time PCR
Description of the gene
Mitogen-activated protein kinase
F: 5′- GAAGATGAGAAACCACAAGCG
R: 5′- CATTCTGAGGAACTTGGAGAGG
Importin subunit alpha1a
F: 5′- GAACTCATTTTGTGCCCCATC
R: 5′- GCTGAGGGATTGGAAAAGATTG
Intracellular Ras-group-related LRR protein 9
F: 5′- GAGAGGCAGGATTGGAGATTG
R: 5′- TCCGCATCCTTCAACATCTTC
Polyadenylate-binding protein RBP47
F: 5′- TCCTAATGAGCCTAACAAACCTG
R: 5′- TCCGTCTTATTGCCTTCCAC
V-ATPase catalytic subunit A1
F: 5’- CGAGAAGGAAAGCGAGTATGG
R: 5’- TCATTCACCATCAGACCAGC
Vacuolar H + -ATPase V0 sector
F: 5′- GCAGTCATTATCAGTACCGGG
F: 5′- TTGGTAACAGCCTTAGTTCCTC
R: 5′- AAAGCCTACCATCACTTCTCG
PROTEIN PHOSPHATASE 2A catalytic subunit
F: 5’- CGATGTGTGATCTCCTATGGTC
R: 5’- AAGCTGATGGGCTCTAGAAATC
The non-parametric Mann-Whitney U test (Statistica 12) was used to determine the significance of the results.
Bafilomycin A1 treatment
Sterile cultures were obtained as described in . After germination in darkness, germinated seeds were transferred on a Murashige and Skoog medium containing varying concentrations of bafilomycin A1. For condition of darkness, dishes were wrapped in aluminum foil and placed in the culture chamber; for light conditions, dishes were cultivated in a culture chamber illuminated with BL (total photon fluence rate 10 μmol.m−2.s−1). After 5 days, the length of the hypocotyl was measured to the nearest millimeter with a ruler. The graph represents the mean ± SEM; an average of 45 seedlings were measured for each condition. The non-parametric Kruskal-Wallis ANOVA (Statistica 12) was performed in order to support the statistical significance of the data.
Results and discussion
Construction of the subtracted cDNA library and analysis
Functional categories of up-regulated genes
Number of sequences
Mitochondrial electron transport/ATP synthesis
Amino acid metabolism
Miscellaneous enzyme families
RNA: processing, transcription, regulation of transcription
DNA: synthesis/chromatin structure, repair
Protein: synthesis, targeting, postranslation modification, degradation
Cell, vesicle transport
Translation and transcription
Sixteen differentially expressed sequences were predicted to encode proteins involved in translation, RNA processing and modification (ribosomal proteins), transcription (eukaryotic initiation factors), and chromatin structure and dynamics (Histone 2B – H2B, Histone H2A).
Cell wall modification
Based on our data and the analysis of the literature, we can hypothesize that exposure to BL rapidly induces changes in cell wall properties, namely extensibility. It would be thus interesting to validate this hypothesis through physico-chemical measurement of the cell wall of tomato seedlings’ hypocotyl during BL-induced de-etiolation.
Role of vacuolar H+-ATPase during de-etiolation
In eukaryotes, V-ATPase consists of at least 12 distinct subunits organized in two large subcomplexes: the cytosolic V1 and membrane Vo subcomplexes. The cytosolic V1 complex is constituted of subunits A through H and catalyzes the hydrolysis of ATP which is associated with the pumping of protons into a compartment via the membrane-bound Vo complex. The Vo complex includes three integral proteins, named subunits a, c, c”, and one hydrophilic subunit d . In tomato, two isoforms of the subunits A (A1 and A2) were isolated. Whereas VHA-A2 isoform was found to be specifically expressed in roots, VHA-A1 isoform was ubiquitously expressed in all tissues and up-regulated by salinity stress . The analysis of expression of the VHA-A1 isoform in the elongating zone of the tomato hypocotyl during BL-induced de-etioation revealed the accumulation of VHA-A1 transcripts during the time-course of the experiment (Fig. 6b). When tomato seedlings were grown in BL on a medium containing varying concentrations of bafilomycin A1, a specific inhibitor of V-ATPases, the length of hypocotyl increased with increased concentration of bafilomycin A1 (Fig. 6c). These results indicated that in tomato, like in barley, BL induces accumulation of V-ATPase as well as its activation . Both events appear to be required to trigger the restriction of cell expansion occurring during de-etiolation. To conclude, we found a strong evidence that V-ATPases play a role during BL-mediated inhibition of hypocotyl growth. It nevertheless would be interesting to verify if V-ATPase participates in elaborating the turgor pressure required for cell expansion or if it contributes to cell wall integrity.
BL-induced de-etiolation is a sequential process depending first on PHOT1 during the first 30–40 min of exposure to BL, with CRY1 being later responsible for the establishment of the steady-state growth rate. Whereas CRY1-mediated de-etiolation has been characterized at the molecular level , no information had been available concerning the PHOT1-mediated phase of de-etiolation.
Our analysis contributes to the understanding of PHOT1-mediated de-etiolation in plants and more particularly in important crop species. Using a subtracted cDNA library, we were able to identify 152 genes quickly up-regulated by BL. Their annotation revealed deep changes in chromatin modelling, transcription, and translation, but also in cellular processes and signaling such as cell wall integrity/synthesis, cytoskeleton, and trafficking/secretion. By using a high-throughput RNAseq method we could obtain more precise information concerning genes differentially expressed during PHOT1-mediated de-etiolation. We are currently developing such analysis including also a tomato mutant depleted of PHOT1 generated in our laboratory by artificial microRNA. Nevertheless, the current study already opens the doors toward processes upon which to focus our attention, notably chromatin modelling and the potential role of histone 2B, as well as the involvement of V-ATPase in either generating appropriate turgor pressure or participating in cell wall integrity/synthesis. It is also noteworthy that an array of sequences encodes for protein of unknown function and represents a pool of proteins with novel functions in PHOT1-mediated de-etiolation.
Availability of data and material
The authors would like to thank M. Čudejková for help in bioinformatic analysis and J.F. Humplík for critical reading of the manuscript. Editing of the manuscript was provided by English Editorial Services, s.r.o.
Funding was provided by the grant L01204 of the National Program of Sustainability I from the Ministry of Education, Youth and Sports, Czech Republic.
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