Sugar Beet Chloroplast Research Articles

Comparative proteomic analysis on chloroplast proteins provides new insights into the effects of low temperature in sugar beet

BackgroundLow temperature, which is one of the main environmental factors that limits geographical distribution and sucrose yield, is a common abiotic stress during the growth and development of sugar beet. As a regulatory hub of plant response to abiotic stress, activity in the chloroplasts is related to many molecular and physiological processes, particularly in response to low temperature stress.ResultsThe contents of chlorophyll (Chl) and malondialdehyde (MDA), relative electrical conductivity (REL), and superoxide dismutase (SOD) activity were measured. The results showed that sugar beet could manage low temperature stress by regulating the levels of Chl, REL and MDA, and the activity of SOD. The physiological responses indicated that sugar beets respond positively to low temperature treatments and are not significantly damaged. Moreover, to determine the precise time to response low temperature in sugar beet, well-known abiotic stresses-responsive transcript factor family, namely DEHYDRATION RESPONSIVE ELEMENT BINDING PROTEIN (DREB), was selected as the marker gene. The results of phylogenetic analyses showed that BvDREBA1 and BvDREBA4 were in the same branch as the cold- and drought-responsive AtDREB gene. In addition, the expression of BvDREBs reached its maximum level at 24 h after low temperature by RNA-Seq and qRT-PCR analysis. Furthermore, the changes in chloroplast proteome after low temperature at 24 h were detected using a label-free technique. A total of 416 differentially expressed proteins were identified. GO enrichment analysis showed that 16 GO terms were significantly enriched, particularly chloroplast stroma, chloroplast envelope, and chloroplast thylakoid membrane. It is notable that the transport of photosynthetic proteins (BvLTD and BvTOC100), the formation of starch granules (BvPU1, BvISA3, and BvGWD3) and the scavenging of reactive oxygen species (BvCu/Zn-SOD, BvCAT, BvPrx, and BvTrx) were the pathways used by sugar beets to respond to low temperatures at an early stage.ConclusionsThese results provide a preliminarily analysis of how chloroplasts of sugar beet respond to low temperature stress at the translational level and provide a theoretical basis for breeding low temperature resistant varieties of sugar beet.

Dynamic pattern of chlorophyll content in the leaves of sugar beet

Purpose. To determine the patterns of change in the content of the green pigment in plant chloroplasts of sugar beet and their relationship, to identify the features of pigment complex during the growing season. Methods. Field, laboratory, analytical, and statistical. Results. The peculiarities of changes in chlorophyll content in the leaves of sugar beets during the growing season are presented. It was found that the most intense green pigments synthesis occurs in the stage of closing leaves when their content reaches the maximum value: chlorophyll а of 8.620 mg/g and chlorophyll b of 4.15 mg/g. Using the ratio of chlorophyll a and b in their total weight the accumulation of a pigment depending on the stage of plant development is determined. Thus, in the first half of the growing season (the 8-true leaves stage and the stage of leaf closure between rows) the synthesis of chlorophyll b dominates, while in the second (the stage of closure leaves in rows and the technical maturity stage) chlorophyll a . Conclusions. It was established that the least intensive synthesis of chlorophyll occurs in the early stages of plant growth and development due to small leaf surface area and low assimilation of the photosynthetically active radiation. Thus, at the beginning of the growing season crops (the 8-true leaves stage and the stage of leaf closure between rows), chlorophyll b dominates in the structure of the green pigment. Reduction of chlorophyll content 1.3 times the end of the growing season (in the stage of technological maturity – 6.3 mg/g) compared with the stage of leaf closure in rows occurs due to the accumulation of sufficient sugar supply in the roots resulting in slowing physiological processes and the dying off old leaves.

Uptake and incorporation of iron in sugar beet chloroplasts

Chloroplasts contain 80-90% of iron taken up by plant cells. Though some iron transport-related envelope proteins were identified recently, the mechanism of iron uptake into chloroplasts remained unresolved. To shed more light on the process of chloroplast iron uptake, trials were performed with isolated intact chloroplasts of sugar beet (Beta vulgaris). Iron uptake was followed by measuring the iron content of chloroplasts in the form of ferrous-bathophenantroline-disulphonate complex after solubilising the chloroplasts in reducing environment. Ferric citrate was preferred to ferrous citrate as substrate for chloroplasts. Strong dependency of ferric citrate uptake on photosynthetic electron transport activity suggests that ferric chelate reductase uses NADPH, and is localised in the inner envelope membrane. The K(m) for iron uptake from ferric-citrate pool was 14.65±3.13μM Fe((III))-citrate. The relatively fast incorporation of (57)Fe isotope into Fe-S clusters/heme, detected by Mössbauer spectroscopy, showed the efficiency of the biosynthetic machinery of these cofactors in isolated chloroplasts. The negative correlation between the chloroplast iron concentration and the rate of iron uptake refers to a strong feedback regulation of the uptake.

Genetic transformation of the sugar beet plastome

It is very important for the application of chloroplast engineering to extend the range of species in which this technology can be achieved. Here, we describe the development of a chloroplast transformation system for the sugar beet (Beta vulgaris L. ssp. vulgaris, Sugar Beet Group) by biolistic bombardment of leaf petioles. Homoplasmic plastid-transformed plants of breeding line Z025 were obtained. Transformation was achieved using a vector that targets genes to the rrn16/rps12 intergenic region of the sugar beet plastome, employing the aadA gene as a selectable marker against spectinomycin and the gfp gene for visual screening of plastid transformants. gfp gene transcription and protein expression were shown in transplastomic plants. Detection of GFP in Comassie blue-stained gels suggested high GFP levels. Microscopy revealed GFP fluorescence within the chloroplasts. Our results demonstrate the feasibility of engineering the sugar beet chloroplast genome; this technology provides new opportunities for the genetic improvement of this crop and for social acceptance of genetically modified sugar beet plants.

Loss of blade photosynthetic area and of chloroplasts' photochemical capacity account for reduced CO 2 assimilation rates in zinc-deficient sugar beet leaves

The effects of zinc deficiency on sugar beet chloroplasts' ultrastructure and photochemical ability, as well as on whole-leaf CO2 assimilation rates, were evaluated. It is shown that increasing zinc shortage firstly induces progressive disorganisation of the chloroplasts' internal membrane system, followed by degradation of both lamellae and stromal components. This zinc deficiency-triggered premature senescence process leads to cell death and, ultimately, to chlorotic/necrotic blade lesions, thus reducing the photosynthetically-active leaf area; chloroplasts in the remaining leaf area show slight impairment of their capacity to reduce methyl viologen using water as electron donor. Whole-leaf CO2 assimilation rates expressed on an area basis are observed to decrease with zinc deficiency, but do not do so significantly when expressed per unit weight of chlorophyll. It is proposed that the partial loss of chloroplasts' photochemical capacity, aggravated by loss of photosynthetically-competent blade area in severely-stressed leaves, accounts for depressed CO2 assimilation rates measured in the zinc-deficient sugar beet leaves.

Time-resolved spectral studies of blue-green fluorescence of leaves, mesophyll and chloroplasts of sugar beet ( Beta vulgaris L.)

Synchrotron radiation and the time-correlated single-photon-counting technique were used to investigate the spectral and time-resolved characteristics of blue-green fluorescence of leaves, mesophyll and chloroplasts. Four kinetic components were resolved. Decay-associated spectra and comparative analysis showed that the fluorescence of leaves, on both sides, was dominated by the fast (0.3 ns) and the medium (1 ns) kinetic components, comprising fluorophores emitting principally in the blue and present in the epidermal layer. In the mesophyll, these two faster components have two maxima, in the blue and in the green part of the spectrum, with a shift of the blue maxima to longer wavelengths when compared to leaves. The slow component (3.5 ns) was green-related with a strong indication for the presence of flavins. The very slow component (9 ns) had a maximal fractional intensity in mesophyll and was blue-related. The excitation and emission characteristics and the effect of anaerobic atmosphere on the fractional intensities of the slow and very slow component showed that they contain fluorescence of flavin nucleotides and nicotinamide nucleotides, respectively. Time-resolved measurements could be a means to extract the information on nucleotide fluorescence from the overall leaf fluorescence, and therefore to evaluate changes in mesophyll redox sate.

The chloroplast genome of Beta vulgaris L.: Structural organization and transcriptional activity

The structural organization of sugarbeet chloroplast DNA (ctDNA) was studied by electron microscopic and biochemical methods. As a result, levels of ctDNA packing were demonstrated. It was observed that deoxynucleoproteid (DNP) fibrils released from the chloroplasts after a mild osmotic shock have 13–14 nm nucleosome-like globules on their fibrils. The fibrils form rosettes with loops of a contour length of 15–20 μm. Minicircular plasmid-like DNA molecules with nucleosome-like globules were found in the chloroplasts. By means of monoclonal antibodies against the β-subunit of Escherichia coli RNA polymerase and protein A-colloidal gold complexes, the sites of DNP fibrils to which chloroplast RNA polymerase are attached were localized. The highest number RNA polymerase molecules were found to be associated with the relaxed smooth DNA fibrils which are almost devoid of nucleosome-like granules. The compactly packed DNP fibrils with a high number of nucleosome-like granules were free of RNA polymerase molecules. The former regions of the chloroplast nucleoid are presumably the transcriptionally active sites of the genome, while the latter are the transcriptionally inactive. The RNA polymerase molecules were also found to be attached to the minicircular DNA.

Restriction fragment map of sugar beet (Beta vulgaris L.) chloroplast DNA

A restriction endonuclease fragment map of sugar beet chloroplast DNA (ctDNA) has been constructed with the enzymes SmaI, PstI and PvuII. The ctDNA was found to be contained in a circular molecule of 148.5 kbp. In common with many other higher plant ctDNAs, sugar beet ctDNA consists of two inverted repeat sequences of about 20.5 kbp separated by two single-copy regions of different sizes (about 23.2 and 84.3 kbp). Southern hybridization analyses indicated that the genes for rRNAs (23S+16S) and the large subunit of ribulose 1,5-bisphosphate carboxylase were located in the inverted repeats and the large single-copy regions, respectively.

Variation in Photosynthetic Pigments and Plastoquinone Contents in Sugar Beet Chloroplasts with Changes in Leaf Copper Content

The changes in the contents of chlorophyll (Chl) a, Chl b, carotenoid, and plastoquinone in sugar beet (Beta vulgaris) chloroplasts were investigated during Cu deficiency and resupply. With the onset of Cu deficiency, extrachloroplastic Cu decreased more rapidly than chloroplast Cu, which eventually accounted for all of the Cu present in the leaf. The ultrastructure of Cu-deficient chloroplasts did not differ from the control except in very young, severely deficient leaves. During Cu depletion and resupply, Chl a, Chl b, carotenoid, and plastoquinone contents changed concomitantly. Because of the concurrent changes in the contents of terpenoid-containing pigments and plastoquinone with Cu depletion and resupply, we suggest that Cu might be involved in the regulation of the early steps of terpenoid biosynthesis prior to the formation of geranyl-geranyl-pyrophosphate.

Potassium, sodium, and chloride content of isolated intact chloroplasts in relation to ionic compartmentation in leaves

Intact chloroplasts were isolated from spinach, sugarbeet, and pea leaves by a rapid procedure which included purification on a two-step Percoll gradient. The content of K +, Na +, and Cl − was determined together with the chloroplast volume from which the apparent concentration of these ions in the chloroplast was calculated. It was demonstrated by successive washing that K + leaked out of the chloroplasts during isolation whereas Na + and Cl − did not. For spinach and sugarbeet chloroplasts K + was the predominant monovalent cation (160–200 m m) with Na + present in lesser amounts (40–70 m m). Pea chloroplasts contained equal amounts of K + and Na + (40 m m). The Cl − concentration in the chloroplast was similar for the three species and was calculated to be near 100 m m. The Cl − concentration in the chloroplast was well above that of the leaf tissue implying accumulation of Cl − in the chloroplast. Qualitatively similar results were obtained for chloroplasts isolated by rapidly fractionating protoplasts of wheat and barley although the concentration of ions was lower than for the above three species. Spinach grown in 200 m m NaCl accumulated high levels of Na + and Cl − in the leaves but K + was decreased. The replacement of K + by Na + was also observed in the chloroplasts but the level of Cl − in the chloroplast was not greatly increased even at leaf Cl − in excess of 200 m m. Replacement of K + by Na + in the nutrient solution also resulted in replacement of K + by Na + both in the leaf and in the chloroplast. Spinach grown under Cl − deficiency showed a 70% decrease in growth and a similar decrease in leaf Cl − but the concentration of Cl − in the chloroplast was not significantly decreased. It is concluded that in most instances K + is the dominant monovalent cation in chloroplasts, although replacement by Na + can occur to some extent. Chloride is a major anion, being accumulated within the chloroplast at low leaf Cl − and excluded from the chloroplast at high leaf Cl −. Ionic compartmentation within the leaf cell appears to be regulated to maintain a relatively constant level of Cl − in the chloroplast.

Mineralstoffverteilung zwischen chloroplasten und übrigem blattgewebe

Summary Chloroplasts from bean, barley and sugar beet leaves were isolated in a non-aqueous media. The contents of potassium, calcium and magnesium, and in some cases of sodium and chloride, were determined both in the whole leaf tissue and in the isolated chloroplasts. Per unit dry weight the content of each of these mineral elements was lower in the chloroplasts than in the whole leaf tissue. Also the ratio of the mineral elements in the chloroplasts was different from that in the whole leaf tissue; the greatest difference was found with calcium. Sugar beet chloroplasts showed a pronounced ability for accumulation of sodium. The results demonstrate that concentration and ratio of the mineral elements in the whole leaf tissue are not representative for the conditions in the chloroplasts. More consideration should also be given to chloroplasts as compartments of ion accumulation.

Catechol oxidase: Enzymic liberation from sugar beet chloroplasts

Catechol oxidase present in isolated sugar beet chloroplasts is activated by means of proteolytic enzymes. The most effective treatment is a combination of trypsin and carboxypeptidase a. This gives a fourfold activation and releases the catechol oxidase from its bound form. The molecular weight of the enzyme liberated in this way is of the order of 10,000. Possible location of the enzyme on membranes is discussed.

Studies of plastoquinone—2. Oxidation-reduction reactions of plastoquinone in isolated chloroplasts

After illumination of isolated sugar-beet chloroplasts, there was a reduction of the endogenous plastoquinone measured as a fall in the oxidation level of the quinone. The value of the photo-reduction varied between 10 and 30 per cent in different preparations of chloroplasts. The reduction was inhibited by either o-phenanthroline or DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). Addition of ascorbic acid plus 2,6-dichlorophenol indophenol stimulated the photo-reduction and the stimulation was not appreciably affected by either o-phenanthroline or DCMU. These results are interpreted on the assumption that plastoquinone photo-reduction occurs via the light reaction involved in water photolysis. The possible site of action of plastoquinone in the photosynthetic electron transport system is discussed in relation to these and other experimental results reported.

Occurrence of inclusions of beet yellows viruses in chloroplasts

Abstract Some chloroplasts of sugar beet ( Beta vulgaris L.) plants infected with either beet yellows virus or Western yellows virus were found to contain inclusions with a regular pattern. Two kinds of pattern were recognized, parallel-linear and cross hatched, and both occurred in the same section of a given inclusion.

Fatty Acids of Spinach Chloroplasts

The photosynthetic apparatus of higher plants consists of discrete cytoplasmic organelles, the chloroplasts, which contain proteins, lipids, pigments, water, and other substances. Relatively little is known, apparently, concerning the composition of the lipid fraction of the chloroplast. Menke (8), studying chloroplasts of spinach, Spinacia oleracea, found that essentially all the lipids of the leaf were located in the chloroplasts. The chloroplasts had an average lipid content of 30.9 % of the dry weight, while the cytoplasm had a lipid content of only 0.5 %. For this reason, a number of earlier studies of the lipid content and composition of green leaves as a whole are pertinent to the present problem. Chibnall and Channon (3), studying the fatty acids of leaves of cabbage, Brassica oleracea, found that approximately 10.7 % of the fatty acid fraction consisted of saturated acids, and 89.3 % of unsaturated acids. The saturated fraction was composed of 70 % palmitic acid and 30 % stearic acid. The unsaturated fraction included large quantities of linoleic and linolenic acids. Speer et al. (13) examined the lipids of spinach leaves, finding that 53 % of the fatty acidls occurred in the free form, with 47 % as glycerides. The saturated acids consisted chiefly of palmitic and stearic acids. Oleic acid made up 26.3 %, linoleic acid 34.7 %, and linolenic acid 12.7 % of the unsaturated lipid fraction. The fatty acids of the forage grasses Dactylis glomierata and Lolium perenne were examined by Smith and Chibnall (12). Saturated acids composed 11 % of the total in Dactylis and 12 % in Lolium; the corresponding figures for oleic acid were 16 and 22 %, for linoleic acid 31 and 26 %, and for linolenic acid 42 and 40 %, respectively. According to Chibnall (2), cabbage leaves contain 17.5 % glycerides, 12.3 % waxes, 4.5 % sterols, and 18.4 % phospholipids. Comparable figures for cocksfoot grass, Dactylis glomnerata, were 38.0 % glycerides, 23.5 % waxes, 3.0 % sterols, and 1.5 % phospholipids. Menke (9) isolated chloroplasts from spinach leaves by centrifugation; he found that their lipid content was approximately 37 %. The ether-soluble fraction of the chloroplasts is a complex mixture of pigments, fatty acids, glycerides, phosphatides, and sterols. Bot (1) isolated grana from chloroplasts of Lathyrus odorattis and Spinacia oleracea. These were shown to have an ether-soluble fraction, excluding chlorophyll, of 18 to 30 % of the dry weight in sweet pea and 26 to 32 % in spinach. Comar (4) determined the lipid content of spinach chloroplasts as 34%. Menke and Jacob (10) extracted isolated chloroplasts of spinach with ether or ether-alcohol mixtures. The extracts were fractionated into a larger acetonesoluble fraction, making up about 80 % of the total, and containing the pigments, triglycerides and sterols, and an acetone-insoluble fraction including phosphatides and waxes. The pigment-free portion of the acetone-soluble fraction consisted of 5.1 % glycerol, 42.2 % fatty acids, 49.0 % triglycerides, and 2.1 % sterols. Sisakyan and Smirnov (11) found that fatty acids composed 57.8 % of the dry weight of the chloroplasts of sugar beet, 48.8 % in sunflower, and 60.0 % in red clover. Isolated chloroplasts of sunflower incorporated acetate-C'4 into higher fatty acids in either light or darkness, but bean chloroplasts did not. In manometric experiments, chloroplasts of bean oxidized palmitic, oleic, linoleic, and linolenic acids. Therefore, the chloroplasts are metabolically active in both fatty acid synthesis and oxidation. Zill and Harmon (14) investigated the chloroplast lipids of spinach by the technique of chromatography on silicic acid columns. Of the crude whole spinach extract 24 % was represented by two monoglycerides, which were not specifically identified. Further studies of chloroplast lipids by these workers are in progress (personal communication). Debuch (5) has recently analyzed the fatty acid composition of chloroplasts of Antirrhinum majus, a plastid mutant of this species, and Alliumn porrum by means of gas chromatography. C1l6 fatty acids composed 10 to 20 % and C18 fatty acids composed 80 to 90 % of the total. Linolenic acid was the component present in greatest amount (44.1-71.3 % of the total), linoleic acid (15.0-29.2 %) and palmitic acid (9.519.7 %) were the other principal constituents. The studies reported in this paper were designed to determine the fatty acid composition of chloroplasts obtained from spinach leaves.

Reduction of low-potential compounds by illuminated leaf homogenate and chloroplasts

1. 1. Illuminated sugar beet leaf homogenate reduced both indigo carmine and FMN in the presence of DPIP and ascorbate. DPIP was absolutely required for the reaction. Reconstitution experiments indicated a requirement for some heat-labile factor present in the supernatant fluid following centrifugation. 2. 2. Balance studies revealed that one mole of ascorbate was photooxidized for each mole of indigo carmine photoreduced. 3. 3. The data are consistent with the view that the DPIP-ascorbate couple serves to prevent reoxidation of reduced indigo carmine by either reacting with the photochemical [OH] system or by depleting the oxygen content in the solution. 4. 4. Sugar beet chloroplasts are capable of effecting DPN photoreduction when they are present in high concentration and are illuminated for long periods. This photoreduction does not require added ascorbate and DPIP.