| Anthocyanin is ubiquitously produced in plant root, stem, leaf and fruit, which is a important class of secondary metabolites synthesized during photomorphogenesis or as a response to different stress signals. This study focused on its likely protective effect on tomato(L402) photosynthetic apparatus to justify further research on its attenuation of damage caused by stress environments.Spectra of sulfur lamp and xenon lamp differ in red/far-red ratio (R/FR), which is 1.5 for sulfur lamp and 1.0 for xenon lamp. Spectrophotometric analysis suggested that tomato seedling leaves grown under sulfur lamp could normally synthesize anthocyanin as that grown in sunlight, while the anthocyanin synthesis in tomato seedling leaves grown under xenon lamp was reduced to 1/9 of that under sulfur lamp. Further anaylsis of emission spectra of sulfur and xenon lamp suggested that anthocyanin synthesis process was influenced by red/far-red ratio. However, there was no significant difference in total flavonol content in tomato seedling leaves grown under sulfur lamp and xenon lamp, indicating either or both of DFR and LDOX-mediated steps in anthocyanin synthesis pathway were inhibited in tomato seedling leaves grown under xenon lamp with low red/far red ratio.Wildtype(WT) and phytochrome mutant phyA, phyB1, phyB2, double mutant phyBlphyB2 and triple mutant phyAphyBlphyB2 tomato were used to study the effects of phytochrome on hypocotyl length, anthocyanin content and inflorescence initiation time. The results indicated that hypocotyl length and anthocyanin biosynthesis varied with phytochrome mutants. phyBl significantly inhibited hypocotyl elongation but promoted anthocyanidin biosynthesis. phyA and phyB2 influenced the two traits to a relatively much less extent. However, phyA and phyB2 obviously interacted with phyBl to control hypocotyl elongation and anthocyanin biosynthesis. HPLC coupled with DAD was employed to find that the double mutant phyB1phyB2 and triple mutant phyAphyB1phyB2 could accumulate flavonone, namely eridictyol, indicating that in the two mutants the metabolic step(s) mediated by flavonone-3-hydroxylase or other enzymes after flavonone-3-hydroxylase was(were)inhibited . Double mutant phyBlphyB2 significantly promoted inflorescence initiation. 1-4* inflorescences of triple mutant phyAphyBlphyB2 aborted at its early developmental stage, and resulted in extremely tiny ones.The difference of leaf anthocyanin content between tomato seedlings grown under sulfur lamp and xenon lamp, and the difference between WT and photomorphogenetic mutant phyBlphyB2 were taken advantage to study if anthocyanin could act as a protectant for photosynthetic apparatus under stress environment. Chlorophyll fluorescence, known as one of the most powerful and widely-used techniques available to plant physiologist and ecophysiologist, was chosen to be the methodology for photosynthesis activity assessment. The results indicated that, exposure to low temperature and low light (5°C, 100 umol photons m"2 s'1) and UV-C irradiation markedly inhibited PS II activity, but hardly affected PSI activity. No difference was observed in light saturation point, light compensation point, CO2 saturation point and CO2 compensation point of tomato seedlings grown under sulfur lamp and xenon lamp. After treartment with low temperature and low light for 3hr, PS II Fv/Fm in anthocyanin-rich tomato leaves under sulfur lamps decreased to 76.3% of pretreatment, while under xenon lamp 63.3%; after exposure to UV-C for 3 hr, Fv/Fm of leaves under sulfur and xenon leaves reduced to 72.5 and 62.2%, respectively. Similarly, after treatment with low temperature and low light for 3hr, Fv/Fm decreased to 64.3% of pretreatment for WT seedlings and 53.7% for phyBlphyB2 seedlings, 3 hr after exposure to UV-C, Fv/Fm was reduced to 69.5% and 46.8% of pretreatment for WT and double mutant seedlings, respectively. After tomato plants grown under sulfur lamp or that of widetype was not able to synthesize anthocyanin, plants exposed to low light at low temperature or UV-C irradiation, no significant difference was observed between plants grown under different light environment, and between the two genetypes. It indicated that anthocyanin protected photosynthesis apparatus in the two stress environment. For all seedlings treated with the two stresses, decrease in Fv/Fm was caused by reduction of Fm, not by increase of Fo. So it could be concluded that the impairment to PS II efficiency resulted from photodamage in photosynthetic apparatus, anthocyanin could attenuate the photodamage by mechanism without xanthophylls involved.HPLC-MS technique was employed to characterize structural properties of anthocyanins in tomato seedling leaves. Reverse phase column of Zorbax-C18 and solvent system composed of water-methanol-formic acid were proved to be suitable for tomato anthocyanin analysis. The results showed that there were 3 different anthocyanin molecular species in tamato seedling leaves, their retention time were 52. 365, 53. 418 and 58. 388min, respectively. DAD revealed that the three anthocyanins shared similar absorbance spectrumwith maximum absorbance wavelength at 535nm and a signature peak at 302nm for p-coumaric acyl group. In addition, lack of 440nm shoulder peaks implied that all anthocyanins in the tomato seedling leaves were 3,5-diglucosidated anthocyanidin derivatives, the first fraction in the HPLC profile was mono-acylated, the second and third were diacylated. HPLC-ESI-MS analysis with quadrapole mass spectrometer indicated that the first fraction is cyanindin 3-glucosyl rutinosido-5-(p-coumaroyl) glucoside or cyanindin 3-(p-coumaroyl) glucosido-5-glucosylrutinoside,the second is pelargonidin 3,5-(p-coumaroyl)glucosido-rutinoside,and the third is malvidin 3-( ;?-coumaroyl) rutinosido-5-(p-coumaroyl)sambubioside or malvidin 3-(p-coumaroyl) sambubiosido (p-coumaroyl) rutinoside. According to the absorbance spectra and structure characteristics, the three anthocyanins in tomato seedling leaves not only had high stability and ability to remove reactive oxygen species, but also filtered unrequired and excessive irradiation for photosynthesis, which helped to protect photosynthetic apparatus from serious photodamage under stress environment.Anthocyanins played important role in plant resistance to stress environment, and it has been well-documented that the resistance contributed by anthocyanin varies with structure. Thereby it was suggested that molecular biological approach should be used to elucidate the factors controlling anthocyanin metabolism in plant tissues or organs, and the factors affecting modification including acylation in order to enhance accumulation of protective anthocyanin in special tissue or organ for increased resistance to stress environment. |
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