Application of fast chlorophyll a fluorescence transient (OJIP) analysis to monitor functional integrity of pea (Pisum sativum) mesophyll protoplasts during isolation (original) (raw)
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Electrophoretic separation and characterization of pea protoplasts
Physiologia Plantarum, 1992
The thylakoid membranesof higher plant chloroplasts contain pigment-complexes that function to harvest light and perform the primary photochemical event.1} These include: the reaction center P700 chlorophyll a-protein complex of photosystem I (PS I)2) and its peripheral antenna of chlorophyll a/b-protein complex (light harvesting complex LHC-I);3) the reaction center P680 chlorophyll a-protein complex of photosystem II (PS II)4) and its peripheral antenna of chlorophyll a/b-protQms LHC-II).5) The chlorophyll-protein complexes are only
Physiologia Plantarum, 2012
To study if etiolation symptoms exist in plants grown under natural illumination conditions, under-soil epicotyl segments of light-grown pea (Pisum sativum) plants were examined and compared to those of hydroponically dark-grown plants. Light-, fluorescence-and electron microscopy, 77 K fluorescence spectroscopy, pigment extraction and pigment content determination methods were used. Etioplasts with prolamellar bodies and/or prothylakoids, protochlorophyll (Pchl) and protochlorophyllide (Pchlide) forms (including the flash-photoactive 655 nm emitting form) were found in the (pro)chlorenchyma of epicotyl segments under 3 cm soil depth; their spectral properties were similar to those of hydroponically grown seedlings. However, differences were found in etioplast sizes and Pchlide:Pchl molar ratios, which indicate differences in the developmental rates of the under-soil and of hydroponically developed cells. Tissue regions closer to the soil surface showed gradual accumulation of chlorophyll, and in parallel, decrease of Pchl and Pchlide. These results proved that etioplasts and Pchlide exist in soil-covered parts of seedlings even if they have a 3-4-cm long photosynthetically active shoot above the soil surface. This underlines that etiolation symptoms do develop under natural growing conditions, so they are not merely artificial, laboratory phenomena. Consequently, dark-grown laboratory plants are good models to study the early stages of etioplast differentiation and the Pchlide-chlorophyllide phototransformation.
Functional Plant Biology, 2020
Developing seeds of some higher plants are photosynthetically active and contain chlorophylls (Chl), which are typically destroyed at the late stages of seed maturation. However, in some crop plant cultivars, degradation of embryonic Chl remains incomplete, and mature seeds preserve green colour, as it is known for green-seeded cultivars of pea (Pisum sativum L.). The residual Chl compromise seed quality and represent a severe challenge for farmers. Hence, comprehensive understanding of the molecular mechanisms, underlying incomplete Chl degradation is required for maintaining sustainable agriculture. Therefore, here we address dynamics of plastid conversion and photochemical activity alterations, accompanying degradation of Chl in embryos of yellow- and green-seeded cultivars Frisson and Rondo respectively. The yellow-seeded cultivar demonstrated higher rate of Chl degradation at later maturation stage, accompanied with termination of photochemical activity, seed dehydration and co...
Preparation of Arabidopsis mesophyll protoplasts with high rates of photosynthesis
Physiologia Plantarum, 2007
A simple and quick method is described for rapid isolation of metabolically active mesophyll protoplasts from leaves of Arabidopsis thaliana. The optimal composition of the digestion medium, period of digestion and stability of protoplast preparation were examined. A large number of protoplasts could be prepared within an hour. The isolated protoplasts were intact, stable and metabolically very active, as indicated by their high rates of photosynthetic oxygen evolution. The important factors during the preparation of protoplasts are short time of digestion, composition of medium, use of nylon nets for filtration, centrifugation at low speed and use of pH 7.0 for storage. The highest rate of photosynthesis obtained in these experiments was 130 AE 4 mmol O 2 evolved mg 21 Chl h 21 , at 1 mM sodium bicarbonate and at a light intensity of 600 mE m 22 s 21 . The present technique of isolation can be very useful for making Arabidopsis protoplasts for studies on not only metabolic processes, such as photosynthesis, but also metabolomics, proteomics and genomics.
Planta, 1992
To measure transcript levels for individual members of the Cab (chlorophyll a/b protein) multigene family in pea under a range of developmental situations, we developed a system using cDNA synthesis, the polymerase chain reaction (PCR), and chemiluminescence detection. In order to design gene-specific PCR primers for all genes, a partial genomic clone for a fifth, Type I LHCII (light-harvesting complex of photosystem II) gene, Cab-9 The Cab-9 sequence appears in the Genbank/EMBL databases under the accession number M86906 , was isolated and sequenced. All seven known Cab genes in pea are expressed in light-grown buds and leaves, including several genes previously known only from genomic clones. There appear to be at least two groups of Cab genes in pea which differ in their response to light and development. The first group (consisting of Cab-8, AB96, Cab-215 and Cab-315) includes Type I, Type II and Type III genes, shows a relatively strong response to red light, and has bud transc...
Expression of the chlorophyll-a/b-protein multigene family in pea (Pisum sativum L.)
Planta, 1992
To measure transcript levels for individual members of the Cab (chlorophyll a/b protein) multigene family in pea under a range of developmental situations, we developed a system using cDNA synthesis, the polymerase chain reaction (PCR), and chemiluminescence detection. In order to design gene-specific PCR primers for all genes, a partial genomic clone for a fifth, Type I LHCII (light-harvesting complex of photosystem II) gene, Cab-91, was isolated and sequenced. All seven known Cab genes in pea are expressed in light-grown buds and leaves, including several genes previously known only from genomic clones. There appear to be at least two groups of Cab genes in pea which differ in their response to light and development. The first group (consisting of Cab-8, AB96, Cab-215 and Cab-315) includes Type I, Type II and Type III genes, shows a relatively strong response to red light, and has bud transcript levels similar to or slightly higher than leaves. The second group, consisting of the Type I genes Cab-9, ABSO and AB66, shows little or no transcript accumulation 24 h after a red light pulse, and has higher transcript levels in leaves than in buds. Transcript levels for genes in this second group appear to be lower than those of the first group in all developmental situations examined. These data indicate that there has been an evolutionary divergence of the responses to light and development among the Type I LHCII genes.
1999
We analysed the changes of the chlorophyll (Chl) a fluorescence rise kinetic (from 50 µs to 1 s) that occur when leaves or chloroplasts of pea (Pisum sativum L.) are incubated under anaerobic conditions in the dark. In control leaves, Chl a fluorescence followed a typical O-J-I-P polyphasic rise [Strasser et al. (1995) Photochem Photobiol 61: 32-42]. Anaerobiosis modified the shape of the transient with the main effect being a time-dependent increase in the fluorescence yield at the J-step (2 ms). Upon prolongation of the anaerobic treatment (> 60 min), the O-J-I-P fluorescence rise was eventually transformed to an O-J (J = P) rise. A similar transformation was observed when pea leaves were treated with DCMU or sodium dithionite. Anaerobiosis resulted in a 10-20% reduction in the maximum quantum yield of the primary photochemistry of Photosystem II, as measured by the ratio of the maximal values of variable and total fluorescence (F V /F M). When the leaves were returned to the air in the dark, the shape of the fluorescence transient showed a time-dependent recovery from the anaerobiosis-induced change. The original O-J-I-P shape could also be restored by illuminating the anaerobically treated samples with far-red light but not with blue or white light. Osmotically broken chloroplasts displayed under anaerobic conditions fluorescence transients similar to those observed in anaerobically treated leaves, but only when they were incubated in a medium comprising reduced pyridine nucleotides (NADPH or NADH). As in intact leaves, illumination of the anaerobically treated chloroplasts by far-red light restored the original O-J-I-P transient, although only in the presence of methyl viologen. The results provide additional evidence for the existence of a chlororespiratory pathway in higher plant cells. Furthermore, they suggest that the J-level of the fluorescence transient is strongly determined by the redox state of the electron carriers at the PS II acceptor side. Abbreviations: Chl a-chlorophyll a; DCMU-3-(3,4-dichlorophenyl)-1,1-dimethylurea; EDTAethylendediaminetetraacetic acid; F M-maximal fluorescence yield; F 0-minimal fluorescence yield; F V-maximal variable fluorescence yield, equal to F M-F 0 ; FR-far-red light; HEPES-4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid; I and J-intermediate steps in the Chl a fluorescence transient appearing between F 0 and F M ; methyl viologen-1,1-dimethyl-4,4-bipyridylium dichloride; MOPS-3-morpholinopropanesulfonic acid;
Temperature dependence of chlorophyll fluorescence parameters of pea seedlings
Journal of plant physiology, 1993
The thermosensitivity of the photosynthetic apparatus of 12 day-old seedlings of Pisum sativum cv. Ran was investigated within the temperature range 2-50°C at intervals of 2 0c. The parameters of prompt chlorophyll fluorescence were used as a criteria of photosynthetic capacity, measured after 5 min temperature treatment (simultaneously with dark adaptation) at the respective temperature. It was established that in the temperature interval 2-42 °C the ground fluorescence (Fo) did not change significantly, but above 42°C it increased dramatically. This gave us reason to suppose that temperatures higher than 44 °C induced irreversible injuring of pea thylakoid membranes. The variable fluorescence (Fv) remained unchanged from 2°C to 20°C, after 22 °C started to decrease first monotonously, then sharply and at 50°C it comprised only 6 % of its initial level. It was also shown that in the temperature range 2-30 °C the ratio FjF m , considered as a measure of PS2 activity, did not change significantly, but began to decrease at higher temperatures. After 42°C its level strongly decreased and at 50 °C it was only about 10 % of the initial value of FjF m • Similar changes were found for the FjF o ratio. It is concluded that the physiological state and capacity of PS2 for electron transport do not changes significantly in a wide temperature range.
Journal of Photochemistry and Photobiology B: Biology, 2008
Low-temperature (77 K) fluorescence emission spectra of 100 dark-grown pea (Pisum sativum L.) seedlings of various ages were measured. The spectra of the 100 leaf samples were collected into a separate data group and those of epicotyls formed another one. This group was divided into three subgroups as spectra of uppermost, middle and lowermost 3 cm sections. Further subgroups were formed on the basis of the ages of the plants. The spectra were normalized to their total integral values (within the 580-780 nm region) then the AVERAGE (arithmetic mean function) and AVEDEV (average of the absolute deviations of data points of their mean function) spectra were calculated. Very sharp bands were found in the AVEDEV spectra. Even the strongly overlapped 629 and 636 nm emission bands appeared as separate peaks, due to the decrease of their half-bandwidth values in the AVEDEV function. Both types of spectra were resolved into Gaussian components. The results showed that the variabilities of the 633 and 655 nm protochlorophyllide forms were similar in the leaves. In epicotyls, the protochlorophyllide forms had different variabilities, especially in the middle sections. The most variable was the amplitude of the 636 nm band and the variabilities of the 629 and 655 nm bands were smaller but still remarkable. The calculation of AVEDEV spectra is an effective method to study the biological variability and spectral resolution of biological samples containing chromophores with multiple spectral properties.