The 7B-1 tomato (Solanum lycopersicum L. cv Rutgers) is a male-sterile mutant with enhanced tolerance to abiotic stress, which makes it a potential candidate for hybrid seed breeding and stress engineering. To underline the molecular mechanism regulating the male-sterility in 7B-1, transcriptomic profiles of the 7B-1 male-sterile and wild type (WT) anthers were studied using mRNA sequencing (RNA-Seq). In total, 768 differentially expressed genes (DEGs) were identified, including 132 up-regulated and 636 down-regulated transcripts. Gene ontology (GO) enrichment analysis of DEGs suggested a general impact of the 7B-1 mutation on metabolic processes, such as proteolysis and carbohydrate catabolic process. Sixteen candidates with key roles in regulation of anther development were subjected to further analysis using qRT-PCR and in situ hybridization. Cytological studies showed several defects associated with anther development in the 7B-1 mutant, including unsynchronized anther maturation, dysfunctional meiosis, arrested microspores, defect in callose degradation and abnormal tapetum development. TUNEL assay showed a defect in programmed cell death (PCD) of tapetal cells in 7B-1 anthers. The present study provides insights into the transcriptome of the 7B-1 mutant. We identified several genes with altered expression level in 7B-1 (including beta-1,3 glucanase, GA2oxs, cystatin, cysteine protease, pectinesterase, TA29, and actin) that could potentially regulate anther developmental processes, such as meiosis, tapetum development, and cell-wall formation/degradation.
- MeSH
- genetická transkripce * MeSH
- genová ontologie MeSH
- glukany metabolismus MeSH
- mutace * MeSH
- neplodnost rostlin genetika MeSH
- regulace genové exprese u rostlin * MeSH
- Solanum lycopersicum genetika metabolismus MeSH
- stanovení celkové genové exprese MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The 7B-1 tomato line (Solanum lycopersicum cv. Rutgers) is a photoperiod-sensitive male-sterile mutant, with potential application in hybrid seed production. Small RNAs (sRNAs) in tomato have been mainly characterized in fruit development and ripening, but none have been studied with respect to flower development and regulation of male-sterility. Using sRNA sequencing, we identified miRNAs that are potentially involved in anther development and regulation of male-sterility in 7B-1 mutant. RESULTS: Two sRNA libraries from 7B-1 and wild type (WT) anthers were sequenced and thirty two families of known miRNAs and 23 new miRNAs were identified in both libraries. MiR390, miR166, miR159 were up-regulated and miR530, miR167, miR164, miR396, miR168, miR393, miR8006 and two new miRNAs, miR#W and miR#M were down-regulated in 7B-1 anthers. Ta-siRNAs were not differentially expressed and likely not associated with 7B-1 male-sterility. miRNA targets with potential roles in anther development were validated using 5'-RACE. QPCR analysis showed differential expression of miRNA/target pairs of interest in anthers and stem of 7B-1, suggesting that they may regulate different biological processes in these tissues. Expression level of most miRNA/target pairs showed negative correlation, except for few. In situ hybridization showed predominant expression of miR159, GAMYBL1, PMEI and cystatin in tapetum, tetrads and microspores. CONCLUSION: Overall, we identified miRNAs with potential roles in anther development and regulation of male-sterility in 7B-1. A number of new miRNAs were also identified from tomato for the first time. Our data could be used as a benchmark for future studies of the molecular mechanisms of male-sterility in other crops.
The role of abscisic acid (ABA) during early development was investigated in tomato seedlings. The endogenous content of ABA in particular organs was analyzed in seedlings grown in the dark and under blue light. Our results showed that in dark-grown seedlings, the ABA accumulation was maximal in the cotyledons and elongation zone of hypocotyl, whereas under blue-light, the ABA content was distinctly reduced. Our data are consistent with the conclusion that ABA promotes the growth of etiolated seedlings and the results suggest that ABA plays an inhibitory role in de-etiolation and photomorphogenesis in tomato.
We reported earlier that 7B-1 mutant in tomato (Solanum lycopersicum L., cv. Rutgers), an ABA overproducer, is defective in blue light (B) signaling leading to B-specific resistance to abiotic and biotic stresses. Using a methylation-sensitive amplified polymorphism (MSAP) assay, a number of genes were identified, which were differentially methylated between 7B-1 and its wild type (WT) seedlings in white (W), blue (B), red (R) lights and dark (D) or in response to exogenous ABA and mannitol-induced stresses. The genomic methylation level was almost similar in different lights between 7B-1 and WT seedlings, while significant differences were observed in response to stresses in D, but not B. Using a cDNA-AFLP assay, several transcripts were identified, which were differentially regulated between 7B-1 and WT by B or D or in response to stresses. Blue light receptors cryptochrome 1 and 2 (CRY1 and CRY2) and phototropin 1 and 2 (PHOT1 and PHOT2) were not affected by the 7B-1 mutation at the transcriptional level, instead the mutation had likely affected downstream components of the light signaling pathway. 5-azacytidine (5-azaC) induced DNA hypomethylation, inhibited stem elongation and differentially regulated the expression of a number of genes in 7B-1. In addition, it was shown that mir167 and mir390 were tightly linked to auxin signaling pathway in 5-azaC-treated 7B-1 seedlings via the regulation of auxin-response factor (ARF) transcripts. Our data showed that DNA methylation remodeling is an active epigenetic response to different lights and stresses in 7B-1 and WT, and highlighted the differences in epigenetic and transcriptional regulation of light and stress responses between 7B-1 and WT. Furthermore, it shed lights on the crosstalk between DNA hypomethylation and miRNA regulation of ARFs expression. This information could also be used as a benchmark for future studies of male-sterility in other crops.
- MeSH
- genetická transkripce * genetika účinky záření MeSH
- metylace DNA * genetika účinky záření MeSH
- mutace MeSH
- neplodnost rostlin * MeSH
- rostlinné proteiny * biosyntéza genetika MeSH
- Solanum lycopersicum * genetika metabolismus MeSH
- světlo * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Dark-induced growth (skotomorphogenesis) is primarily characterized by rapid elongation of the hypocotyl. We have studied the role of abscisic acid (ABA) during the development of young tomato (Solanum lycopersicum L.) seedlings. We observed that ABA deficiency caused a reduction in hypocotyl growth at the level of cell elongation and that the growth in ABA-deficient plants could be improved by treatment with exogenous ABA, through which the plants show a concentration dependent response. In addition, ABA accumulated in dark-grown tomato seedlings that grew rapidly, whereas seedlings grown under blue light exhibited low growth rates and accumulated less ABA. We demonstrated that ABA promotes DNA endoreduplication by enhancing the expression of the genes encoding inhibitors of cyclin-dependent kinases SlKRP1 and SlKRP3 and by reducing cytokinin levels. These data were supported by the expression analysis of the genes which encode enzymes involved in ABA and CK metabolism. Our results show that ABA is essential for the process of hypocotyl elongation and that appropriate control of the endogenous level of ABA is required in order to drive the growth of etiolated seedlings.
- MeSH
- cyklin-dependentní kinasy antagonisté a inhibitory MeSH
- cytokininy biosyntéza metabolismus MeSH
- endoreduplikace účinky záření MeSH
- homeostáza účinky záření MeSH
- hypokotyl cytologie růst a vývoj metabolismus účinky záření MeSH
- inhibitory proteinkinas farmakologie MeSH
- klíčení účinky léků účinky záření MeSH
- kyselina abscisová metabolismus MeSH
- Solanum lycopersicum cytologie růst a vývoj metabolismus účinky záření MeSH
- tma * MeSH
- vývoj rostlin účinky léků účinky záření MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Light is one of the most important factor influencing plant growth and development all through their life cycle. One of the well-known light-regulated processes is de-etiolation, i.e. the switch from skotomorphogenesis to photomorphogenesis. The hormones cytokinins (CKs) play an important role during the establishment of photomorphogenesis as exogenous CKs induced photomorphogenesis of dark-grown seedlings. Most of the studies are conducted on the plant model Arabidopsis, but no or few information are available for important crop species, such as tomato (Solanum lycopersicum L.). In our study, we analyzed for the first time the endogenous CKs content in tomato hypocotyls during skotomorphogenesis, photomorphogenesis and de-etiolation. For this purpose, two tomato genotypes were used: cv. Rutgers (wild-type; WT) and its corresponding mutant (7B-1) affected in its responses to blue light (BL). Using physiological and molecular approaches, we identified that the skotomorphogenesis is characterized by an endoreduplication-mediated cell expansion, which is inhibited upon BL exposure as seen by the accumulation of trancripts encoding CycD3, key regulators of the cell cycle. Our study showed for the first time that iP (isopentenyladenine) is the CK accumulated in the tomato hypocotyl upon BL exposure, suggesting its specific role in photomorphogenesis. This result was supported by physiological experiments and gene expression data. We propose a common model to explain the role and the relationship between CKs, namely iP, and endoreduplication during de-etiolation and photomorphogenesis.
- MeSH
- buněčný cyklus účinky léků genetika MeSH
- cyklin D3 genetika metabolismus MeSH
- cytokininy metabolismus MeSH
- endoreduplikace fyziologie účinky záření MeSH
- fylogeneze MeSH
- hypokotyl fyziologie účinky záření MeSH
- isopentenyladenosin metabolismus MeSH
- morfogeneze fyziologie účinky záření MeSH
- ploidie MeSH
- rostlinné proteiny genetika metabolismus MeSH
- semenáček fyziologie účinky záření MeSH
- Solanum lycopersicum fyziologie účinky záření MeSH
- světlo MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND AND AIMS: Nitric oxide (NO) is involved in the signalling and regulation of plant growth and development and responses to biotic and abiotic stresses. The photoperiod-sensitive mutant 7B-1 in tomato (Solanum lycopersicum) showing abscisic acid (ABA) overproduction and blue light (BL)-specific tolerance to osmotic stress represents a valuable model to study the interaction between light, hormones and stress signalling. The role of NO as a regulator of seed germination and ABA-dependent responses to osmotic stress was explored in wild-type and 7B-1 tomato under white light (WL) and BL. METHODS: Germination data were obtained from the incubation of seeds on germinating media of different composition. Histochemical analysis of NO production in germinating seeds was performed by fluorescence microscopy using a cell-permeable NO probe, and endogenous ABA was analysed by mass spectrometry. KEY RESULTS: The NO donor S-nitrosoglutathione stimulated seed germination, whereas the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) had an inhibitory effect. Under WL in both genotypes, PTIO strongly suppressed germination stimulated by fluridone, an ABA inhibitor. The stimulatory effect of the NO donor was also observed under osmotic stress for 7B-1 seeds under WL and BL. Seed germination inhibited by osmotic stress was restored by fluridone under WL, but less so under BL, in both genotypes. This effect of fluridone was further modulated by the NO donor and NO scavenger, but only to a minor extent. Fluorescence microscopy using the cell-permeable NO probe DAF-FM DA (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) revealed a higher level of NO in stressed 7B-1 compared with wild-type seeds. CONCLUSIONS: As well as defective BL signalling, the differential NO-dependent responses of the 7B-1 mutant are probably associated with its high endogenous ABA concentration and related impact on hormonal cross-talk in germinating seeds. These data confirm that light-controlled seed germination and stress responses include NO-dependent signalling.
- MeSH
- biologické modely MeSH
- cyklické N-oxidy farmakologie MeSH
- donory oxidu dusnatého farmakologie MeSH
- fluoresceiny analýza MeSH
- fyziologický stres * účinky léků účinky záření MeSH
- imidazoly farmakologie MeSH
- kinetika MeSH
- klíčení * účinky léků účinky záření MeSH
- kyselina abscisová metabolismus MeSH
- mutace MeSH
- osmóza účinky léků účinky záření MeSH
- oxid dusnatý farmakologie MeSH
- pyridony farmakologie MeSH
- regulace genové exprese u rostlin účinky léků účinky záření MeSH
- regulátory růstu rostlin metabolismus MeSH
- S-nitrosoglutathion farmakologie MeSH
- scavengery volných radikálů farmakologie MeSH
- semena rostlinná účinky léků genetika fyziologie účinky záření MeSH
- signální transdukce účinky léků účinky záření MeSH
- Solanum lycopersicum účinky léků genetika fyziologie účinky záření MeSH
- světlo * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
1. čes. vyd. xviii, 389 s. : il., tab. ; 25 cm
