Identification and characterisation of blue light photoreceptor gene family and their expression in tomato (Solanum lycopersicum) under cold stress.

The Arabidopsis thaliana L. photoreceptor genes homologues in tomato (Solanum lycopersicum L.) genome were analysed using bioinformatic tools. The expression pattern of these genes under cold stress was also evaluated. Transcriptome analysis of the tomato sequence revealed that the photoreceptor gene family is involved in abiotic stress tolerance. They participate in various pathways and controlling multiple metabolic processes. They are structurally related to PAS, LIGHT-OXYGEN-VOLTAGE-SENSING (LOV), DNA photolyase, 5,10-methenyl tetrahydrofolate (MTHF), flavin-binding kelch F-box, GAF, PHY, Seven-bladed ß-propeller and C27 domains. They also interact with flavin adenine dinucleotide (FAD), (5S)-5-methyl-2-(methylsulfanyl)-5-phenyl-3-(phenylamino)-3,5-dihydro-4H-imidazol-4-one (FNM) and Phytochromobilin (PϕB) ligands. These interactions help to create a cascade of protein phosphorylation involving in cell defence transcription or stress-regulated genes. They localisation of these gene families on tomato chromosomes appeared to be uneven. Phylogenetic tree of tomato and Arabidopsis photoreceptor gene family were classified into eight subgroups, indicating gene expression diversity. Morphological and physiological assessment revealed no dead plant after 4h of cold treatment. All the plants were found to be alive, but there were some variations in the data across different parameters. Cold stress significantly reduced the rate of photosynthesis from 10.06 to 3.16µmolm-2 s-1 , transpiration from 4.6 to 1.3mmolm-2 s-1 , and stomatal conductance from 94.6 to 25.6mmolm-2 s-1 . The cold stressed plants also had reduced height, root/shoot length, and fresh/dry biomass weight than the control plants. Relative expression analysis under cold stress revealed that after 4h, light stimulates the transcript level of Cry2 from 1.9 to 5.7 and PhyB from 0.98 to 6.9 compared to other photoreceptor genes.

Silicon-induced alterations in the expression of aquaporins and antioxidant system activity in well-watered and drought-stressed oilseed rape.

Progressing climate change necessitates the search for solutions of plant protection against the effects of water deficit. One of these solutions could be silicon supplementation. The aim of the study was to verify the hypothesis that silicon changes aquaporin expression and antioxidant system activity in a direction which may alleviate the effects of drought stress in oilseed rape. The accumulation of BnPIP1, BnPIP2-1-7 and BnTIP1;1 aquaporins and the expression of their genes, the level of catalase, superoxide dismutase activities and hydrogen peroxide content as well as total non-enzymatic antioxidant activity were analyzed in leaf tissue from control and silicon-treated oilseed rape plants growing under well-watered and drought conditions. Silicon was applied in two forms - pure silicon and a silicon complex. It was shown that under drought conditions, both pure silicon and the silicon complex (with Fe) significantly increased the accumulation of aquaporins and improved the activity of enzymatic and non-enzymatic components of the antioxidant system, while under well-watered conditions, these effects were observed only in the case of the silicon complex. The presented study proves that silicon supplementation in oilseed rape improves the regulation of water management and contributes to the protection against oxidative stress caused by drought.

Transcriptional regulation of male-sterility in 7B-1 male-sterile tomato mutant.

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.

Identification of miRNAs with potential roles in regulation of anther development and male-sterility in 7B-1 male-sterile tomato mutant.

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.

Spatio-temporal changes in endogenous abscisic acid contents during etiolated growth and photomorphogenesis in tomato seedlings.

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.

DNA methylation and transcriptomic changes in response to different lights and stresses in 7B-1 male-sterile 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.

Endogenous abscisic acid promotes hypocotyl growth and affects endoreduplication during dark-induced growth in tomato (Solanum lycopersicum L.).

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.

MicroRNA Regulation of Abiotic Stress Response in 7B-1 Male-Sterile Tomato Mutant.

The 7B-1 tomato (Solanum lycopersicum L. 'Rutgers') is a male-sterile mutant with enhanced tolerance to abiotic stress in a blue-light (BL) specific manner compared with its wild-type (WT). This makes the 7B-1 a potential candidate for hybrid seed breeding and stress engineering. To identify small RNAs (sRNAs) linked to stress tolerance of 7B-1, two sRNA libraries from BL-grown 7B-1 and WT seedlings treated simultaneously with abscisic acid (ABA) and mannitol were sequenced, and sRNA profiles were compared. Twenty nine families of known microRNAs (miRNAs) and 27 putative novel miRNAs were identified from the two libraries. MiR5300, miR5301, miR2916, and a novel miRNA denoted miR#C were upregulated, while miR159, miR166, miR472, miR482, and two novel miRNAs, miR#A and miR#D, were downregulated in stress-treated 7B-1 seedlings. MiRNA targets with potential roles in stress regulation were validated by rapid amplification of 5' complementary DNA ends (5'-RACE) analysis. Expression of miR159, miR166, miR472, miR482, miR#A, and miR#D together with their targets were further investigated in response to ABA, mannitol, NaCl, and cold treatments and a strong negative correlation was observed between the levels of these miRNAs and expression of their targets. Only miR159 and miR166 responded to cold treatment. MiR#A and its target were regulated by ABA and mannitol as early as 0.5 h after the treatments, while other miRNAs and targets were regulated only after 2 h. This suggests a role in early response to stress for miR#A. Our data suggests that miR159, miR166, miR472, miR482, miR#A, and miR#D are likely to facilitate the BL-specific enhanced tolerance of 7B-1 to abiotic stress.

Spontaneous mutation 7B-1 in tomato impairs blue light-induced stomatal opening.

It was reported earlier that 7B-1 mutant in tomato (Solanum lycopersicum L.), an ABA overproducer, is defective in blue light (BL) signaling leading to BL-specific resistance to abiotic and biotic stresses. In this work, we examine responses of stomata to blue, red and white lights, fusicoccin, anion channel blockers (anthracene-9-carboxylic acid; 9-AC and niflumic acid; NIF) and ABA. Our results showed that the aperture of 7B-1 stomata does not increase in BL, suggesting that 7B-1 mutation impairs an element of BL signaling pathway involved in stomatal opening. Similar stomatal responses of 7B-1 and wild type (WT) to fusicoccin or 9-AC points out that activity of H(+)-ATPase and 9-AC-sensitive anion channels per se is not likely affected by the mutation. Since 9-AC restored stomatal opening of 7B-1 in BL, it seems that 9-AC and BL could block similar type of anion channels. The stomata of both genotypes did not respond to NIF neither in darkness nor in any light conditions tested. In light, 9-AC but not NIF restored stomatal opening inhibited by ABA in WT and 7B-1. We suggest that in comparison to WT, the activity of S-type anion channels in 7B-1 is more promoted by increased ABA content, and less reduced by BL, because of the mutant resistance to BL.

Effect of blue light on endogenous isopentenyladenine and endoreduplication during photomorphogenesis and de-etiolation of tomato (Solanum lycopersicum L.) seedlings.

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.

Nitric oxide is involved in light-specific responses of tomato during germination under normal and osmotic stress conditions.

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.

The 7B-1 mutation in tomato (Solanum lycopersicum L.) confers a blue light-specific lower sensitivity to coronatine, a toxin produced by Pseudomonas syringae pv. tomato.

The spontaneous mutant 7B-1 in tomato (Solanum lycopersicum=Lycopersicon esculentum) is a photoperiod-dependent male-sterile mutant previously reported as resistant to various abiotic stresses specifically under blue light. Since this finding improved the potential of 7B-1's use in breeding programmes, its susceptibility to stress induced by coronatine (COR), the phytotoxine produced by several Pseudomonas syringae strains, was assessed in this study. The 7B-1 mutant was found to be less sensitive than the corresponding wild type (WT) to COR treatment in a blue light-dependent manner. Treatment of WT and 7B-1 plants with COR induced a strong accumulation of salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) in hypocotyls. Interestingly, accumulation of ABA and SA in the 7B-1 mutant was distinctly greater than in WT, especially in blue light. Based on the cross-talk between SA- and JA-signalling pathways, expression analysis of NPR1 and COI1 genes, respectively involved in these pathways, was investigated in COR-stressed plants. The blue light-specific lower sensitivity of 7B-1 plants to COR was found to be associated with blue light-specific overexpression of the NPR1 gene. These data suggest that the SA-dependent NPR1-dependent pathway could be involved in the lower sensitivity of the 7B-1 mutant to COR. The role of anthocyanins and ABA accumulation during the response to COR is also discussed in the present study.

Development of Erect Leaves in a Modern Maize Hybrid is Associated with Reduced Responsiveness to Auxin and Light of Young Seedlings In Vitro.

Modern corn (Zea mays L.) varieties have been selected for their ability to maintain productivity in dense plantings. We have tested the possibility that the physiological consequence of the selection involves changes in responsiveness to light and auxin.Etiolated seedlings of two older corn hybrids 307 and 3306 elongated significantly more than seedlings of a modern corn hybrid 3394. The level of endogenous auxin and activity of PAT in 307 and 3394 were similar. Hybrid 3394 shows resistance to auxin- and light-induced responses at the seedling, cell and molecular levels. Intact 3394 plants exhibited less responsiveness to the inhibitory effect of R, FR and W, auxin, anti-auxin and inhibitors of PAT. In excised mesocotyl tissue 3394 seedlings also showed essentially low responsiveness to NAA. Cells of 3394 were insensitive to auxin- and light-induced hyperpolarization of the plasma membrane. Expression of ABP4 was much less in 3394 than in 307, and in contrast to 307, it was not upregulated by NAA, R and FR. Preliminary analysis of abp mutants suggests that ABPs may be involved in development of leaf angle in corn.Our results confirm the understanding that auxin interacts with light in the regulation of growth and development of young seedlings and suggest that in corn ABPs may be involved in growth of maize seedlings and development of leaf angle. We hypothesize that ABP4 plays an important role in the auxin- and/or light-induced growth responses. We also hypothesize that in the modern corn hybrid 3394, ABP4 is "mutated," which may result in the observed 3394 phenotypes, including upright leaves.

Light interacts with auxin during leaf elongation and leaf angle development in young corn seedlings.

Modern corn ( Zea mays L.) varieties have been selected for their ability to maintain productivity in dense plantings. We have tested the possibility that the physiological consequence of the selection of the modern hybrid, 3394, for increased crop yield includes changes in responsiveness to auxin and light. Etiolated seedlings in the modern line are shorter than in an older hybrid, 307, since they produce shorter coleoptile, mesocotyl, and leaves (blade as well as sheath). Etiolated 3394 seedlings, as well as isolated mesocotyl and sheath segments, were less responsive to auxin and an inhibitor of polar auxin transport, N-1-naphthylphthalamic acid (NPA). Reduced response of 3394 to auxin was associated with less reduction of elongation growth by light (white, red, far-red, blue) than in 307, whereas the activity of polar auxin transport (PAT) and its reduction by red or far-red light was similar in both genotypes. NPA reduced PAT in etiolated 3394 seedlings much less than in 307. A characteristic feature of 3394 plants is more erect leaves. In both hybrids, light (white, red, blue) increases leaf declination from the vertical, whereas NPA reduces leaf declination in 307, but not in 3394. Our results support findings that auxin and PAT are involved in elongation growth of corn seedlings, and we show that light interacts with auxin or PAT in regulation of leaf declination. We hypothesize that, relative to 307, more erect leaves in the modern hybrid may be primarily a consequence of a reduced amount of auxin receptor(s) and reduced responsiveness to light in etiolated 3394 plants. The more erect leaves in 3394 may contribute to the tolerance of the modern corn hybrid to dense planting.

The 7B-1 mutant in tomato shows blue-light-specific resistance to osmotic stress and abscisic acid.

Germination of wild-type (WT) tomato ( Lycopersicon esculentum Mill.) seed is inhibited by mannitol (100-140 mM) in light, but not in darkness, suggesting that light amplifies the responsiveness of the seed to osmotic stress (M. Fellner, V.K. Sawhney (2001) Theor Appl Genet 102:215-221). Here we report that white light (W) and especially blue light (B) strongly enhance the mannitol-induced inhibition of seed germination, and that the effect of red light (R) is weak or nil. The inhibitory effect of mannitol could be completely overcome by fluridone, an inhibitor of abscisic acid (ABA) biosynthesis, indicating that mannitol inhibits seed germination via ABA accumulation in seeds. The inhibition of WT seed germination by exogenous ABA was also amplified by W or B, but not by R. In a recessive, ABA-overproducing, 7B-1 mutant of tomato, seed germination and hypocotyl growth were resistant to inhibition by mannitol or exogenous ABA, both in W or B. Experiments with fluridone suggested that inhibition of hypocotyl growth by W or B is also partially via ABA accumulation. De-etiolation in the mutant was especially less in B compared to the WT, and there was no difference in hypocotyl growth between the two genotypes in R. Our data suggest that B amplifies the responsiveness of tomato seeds and hypocotyls to mannitol and ABA, and that W- or B-specific resistance of the 7B-1 mutant to osmotic stress or ABA is a consequence of a defect in B perception or signal transduction.