Thermostability of photosynthesis in two new chlorophyllb-less rice mutants

Leaves of the two new chlorophyllb-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48°C in the dark for 30 min or gradually elevated temperature from 30°C to 80°C at 0.5°C/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H₂O₂ accumulation. There were different patterns of F_o-temperature curves between the Chlb-less mutants and the wild type plant, and the temperature of F_o rising threshold was shifted 3°C lower in the Chlb-less mutants (48°C) than in the wild type (51°C). At temperature up to about 45°C, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chlb-less mutants, but chloroplast ultrastructure in the wild type showed no obvious alteration. After 55°C exposure, the disordered thylakoid and significant H₂O₂ accumulation in leaves were found in the two Chlb-less mutants, whereas in the wild type plant, less H₂O₂ was accumulated and the swollen thylakoid still maintained a certain extent of stacking. A large extent of the changes in qP, NPQ and F_v/F_m was consistent with the Pn decreasing rate in the Chlb-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chlb-less mutants showed a tendency for higher thermosensitivity, and loss of Chlb in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the internal oxidative stress produced at severely high temperature.     

Thermostability of photosynthesis in two new chlorophyllb-less rice mutants

Leaves of the two new chlorophyll b-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48℃ in the dark for 30 min or gradually elevated temperature from 30℃ to 80℃ at 0.5℃/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H2O2 accumulation. There were different patterns of Fo-temperature curves between the Chl b-less mutants and the wild type plant, and the temperature of F_o rising threshold was shifted 3℃ lower in the Chl b-less mutants (48℃) than in the wild type (51℃). At temperature up to about 45℃, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chl b-less mutants, but chloroplast ultrastruc-ture in the wild type showed no obvious alteration. After 55℃ exposure, the disordered thylakoid and significant H₂O₂ accumulation in leaves were found in the two Chl _b-less mutants, whereas in the wild type plant, less H₂O₂ was accumulated and the swollen thylakoid still maintained a cer-tain extent of stacking. A large extent of the changes in qP, NPQ and F_v/F_m was consistent with the Pn decreasing rate in the Chl b-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chl b-less mutants showed a tendency for higher thermosensitivity, and loss of Chl b in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the in-ternal oxidative stress produced at severely high temperature.     

Storage protein characteristics of proline-requiring mutants of Zea mays (L.)

Summary Protein and amino acid composition of mature karnels from three allelic proline-requiring mutants in maize, pro _1-1, pro _1-2, and pro _1-3 were analyzed and compared to kernels of the stock A 188 containing the wild type allele. The amount of free proline was specifically reduced in the embryos of all three mutants, while in the endosperm such a reduction was only found for pro _1-2 and pro _1-3 Accumulation of the proline-rich zeins was strongly reduced in the mutants, but in contrast to opaque-2 the reduction affected all major zein polypeptides to the same extent, possibly as a consequence of the defective proline metabolism. Albumins and globulins as well as free amino acids were more abundant in the endosperms of the mutants than in the wild type. Analysis of the albumins and globulins by SDS-PAGE revealed specific increases as well as reductions of certain polypeptides in the endosperms and embryos of the mutants.     

Some properties of carbohydrate and C4-dicarboxylic acid utilization negative mutants ofRhizobium leguminosarum biovarphaseoli strain P121

After NTG treatment of the very effective wild type strain P121 ofRhizobium leguminosarum biovarphaseoli, mutants defective in the utilization of sugars or organic acids were obtained. All the mutants nodulated the cultivar Goldie ofPhaseolus vulgaris. The arabinose, fructose, glucose and pyruvate utilization mutants formed nodules similar in shape and size to the nodules formed by the wild type strain. These mutants exhibited an acetylene reduction activity significantly lower than the activity observed with the wild type strain. All the C₄-dicarboxylic acid utilization mutatns, formed ineffective nodules that did not show a significant acetylene reduction activity. The C₄-dicarboxylic acids uptake system is apparently inducible in the free-living bacteria of strain P121. When P121 cells were grown on glucose in the presence of 2.5 mM malate, the rate of glucose-dependent O₂ consumption significantly decreased suggesting the presence of a catabolite repression-like phenomenon. Isolated bacteroids of strain P121, under the experimental conditions used, were able to oxidize succinate, fumarate or malate but did not oxidize pyruvate, glucose, fructose or sucrose.     

Catalases CAT1 and CAT3 Are not Key Enzymes in Alleviating Gamma Irradiation-Induced DNA Damage,H2O2 Accumulation,or Lipid Peroxidation in Arabidopsis thaliana

Gamma irradiation increased catalase activities at 0.1 kGy and decreased them at 10 kGy in Arabidopsis wild type and catalase-deficient mutants, cat3-1 and cat1 cat3. Irradiation induced DNA damage, H₂O₂ accumulation, and lipid peroxidation in both mutants as well as the wild type. Thus catalases might not be key enzymes protecting gamma irradiation-induced damage.     

The effect of the locus pstB on phosphate binding in the phosphate specific transport (PST) system of Escherichia coli

Summary The periplasmic phosphate binding protein is a product of the phoS gene and is an essential component of the phosphate specific transport (PST) system, which mediates Pi uptake in Escherichia coli. The binding of Pi to periplasmic protein(s) and the kinetic parameters of Pi uptake were studied in phoT and pstB mutants of E. coli. These mutants are impaired in Pi uptake but have a periplasmic Pi-binding protein whose Pi-binding acpacity was estimated by the retention kinetics. The Pi-binding activity in two pstB mutants was found to be weaker as compared to phoT9 and the wild type. The K _D values for Pi binding to periplasmic protein were determined by equilibrium dialysis. In the pstB mutants the K _D value was found to be 9–31 times higher than the values obtained for the wild type and the phoT mutant. The apparent K _m values for Pi uptake in one pstB mutant is 14.3 times higher than in the wild type. V _max of the mutant is 8.3 times lower that of the wild type. The data indicate that pstB, an essential gene of the PST transport system, is promoting the binding capacity of the Pi-binding protein.Abbreviations AP alkaline phosphatase - Pi inorganic orthophosphate - Km kanamycin     

Activity of delta-endotoxin protein crystals in Bacillus thuringiensis mutants when influenced by UV treatments

The ability of some isolates of Bacillus thuringiensis to produce dark brown pigment was measured as an indicator to UV resistance. M₅ as an indigenous Egyptian isolate was used as wild type to improve its resistance to UV. It was exposed to UV irradiation for different periods ranging between 1 and 10?h. The induced mutants were examined morphologically by phase contrast microscope. One hundred and forty four mutants were obtained; 10 of them were selected and tested for their toxicity against Spodoptera littoralis. The results showed that mutants 62, 65 and 85 were the most toxic ones. These three mutants and the wild type were examined by transmission electron microscope. Crystal proteins with bipyramidal shape and active against Lepidopteran insects were detected in all the selected mutants.     

Low expression of lipid-linked oligosaccharide due to a functionally altered Dol-P-Man synthase reduces protein glycosylation in cAMP-dependent protein kinase deficient Chinese hamster ovary cells

Chinese hamster ovary cells express a wide variety of glycoproteins with M_r ranging from 15,000 to 200,000 dalton and higher. Glycosylation of these proteins was much less in cAMP-dependent protein kinase (PKA)-deficient mutants which expressed either (i) a defective C-subunit with altered substrate specificity and having no detectable type II kinase (mutant 10215); or (ii) an altered RI subunit and having no detectable type II kinase (mutant 10248); or (iii) exhibited the lowest level of total kinase with no detectable type I kinase but having a small amount of type II kinase (mutant 10260). Addition of 8Br-cAMP enhanced protein glycosylation index in wild type cells 10001 by 120% but only 7 to 23% in the mutant cells. The rate of lipid-linked oligosaccharide (LLO) biosynthesis was linear for 1 h in all cell types, but the total amount of LLO expressed was much less in PKA-deficient mutants. Pulse-chase experiments indicated that the t_1/2 for LLO turnover was also twice as high in PKA-deficient cells as in the wild type. Size exclusion chromatography of the mild-acid released oligosaccharide confirmed that both wild type and the mutant cells synthesized Glc₃Man₉GlcNAc₂-PP-Dol as the most predominating species with no accumulation of Man₅GlcNAc₂-PP-Dol in the mutants. Kinetic studies exhibited a reduced mannosylphosphodolichol synthase (DPMS) activity in mutant cells with a K_m for GDP-mannose 160 to 400% higher than that of the wild type. In addition, the k_cat for DPMS was also reduced 2 to 4-fold in these mutant cells. Exogenously added Dol-P failed to rescue the k_cat for DPMS in CHO cell mutants; however, in vitro protein phosphorylation with a cAMP-dependent protein kinase restored their kinetic activity to the level of the wild type. Published in 2004.     

Mutational and kinetic analysis of basic amino acid transport in the cyanobacterium Synechocystis sp. PCC 6803

Two nitrogen-deregulated mutants of Phanerochaete chrysosporium, der8-2 and der8-5, were isolated by subjecting wild type conidia to gamma irradiation, plating on Poly-R medium containing high levels of nitrogen, and identifying colonies that are able to decolorize Poly-R. The mutants showed high levels of ligninolytic activity (¹⁴C-synthetic lignin ¹⁴CO₂), and lignin peroxidase, manganese peroxidase and glucose oxidase activities in both low nitrogen (2.4 mM) and high nitrogen (24 mM) media. The wild type on the otherhand displayed these activities in low nitrogen medium but showed little or no activities in high nitrogen medium. Fast protein liquid chromatographic analyses showed that the wild type as well as the der mutants produce three major lignin peroxidase peaks (designated L1, L2 and L3) with lignin peroxidase activity in low nitrogen medium. Furthermore, in low nitrogen medium, mutant der8-5 produced up to fourfold greater lignin peroxidase activity than that produced by the wild type. In high nitrogen medium, the wild type produced no detectable lignin peroxidase peaks whereas the mutants produced peaks L1 and L2, but not L3, and a new lignin peroxidase protein peak designated LN. Mutants der8-2 and der8-5 also produced high levels of glucose oxidase, an enzyme known to be associated with secondary metabolism and an important source of H₂O₂ in ligninolytic cultures, both in low and high nitrogen media. In contrast, the wild type produced high levels of glucose oxidase in low nitrogen medium and only trace amounts of this enzyme in high nitrogen medium. The results of this study indicate that the der mutants are nitrogen-deregulated for the production of a set of secondary metabolic activities associated with lignin degradation such as lignin peroxidases, manganese peroxidases and glucose oxidase.     

Physiological and genetic analysis of the glucose-fructose permeation system in two Synechocystis species

Fructose is toxic for Synechocystis PCC 6714 and 6803, strains which grow chemoheterotrophically on glucose. This toxicity, as well as fructose uptake, were inhibited by glucose or by its non-metabolized analogue 3-O-methyl-glucose. The results suggested that both sugars were transported by the same permeation system, the affinity for fructose, estimated from the corresponding K _m and K _i, being very low. The unicity of the permeation system was further established by the isolation of spontaneous mutants showing the expected pleiotropic phenotype, Glu^–, Fru^r, transport^–, and by the simultaneous re-acquisition of the relevant wild type characteristics in mutant cells transformed by wild type DNA. The genetic nature of this mutation is discussed in view of the impossibility to isolate spontaneously reversed wild type clones from the transport deficient mutants.Abbreviations OMG 3-O-methyl-glucose - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethylurea     

The kinetics and feedback inhibition of cytidine 5′-triphosphate synthetase in wild-type and mutant Chinese hamster cells

The kinetics and cytidine 5-triphosphate (CTP) feedback inhibition of CTP synthetase in wild-type and four mutants of Chinese hamster V79 cells have been studied. The enzymes of the wild type and three of the four mutants exhibited positive cooperativity with the substrate uridine 5-triphosphate (UTP). Three of the mutants had K _{m app} and S ₅₀ valuves distinctly greater than those of the wild type, while the fourth mutant had values similar to those of the wild type. all four mutants exhibited resistance to CTP feedback inhibition, while the wild type was sensitive to such inhibition. It is postulated that a single mutational event in each mutant had caused a concomitant change of the enzyme in its binding both to the substrate UTP and to the end-product CTP.This work was supported by Grant GM 20608 from the U.S. Public Health Service.     

The effect of radiation sensitivity and cell stage on liquid holding response in Schizosaccharomyces pombe

Summary The response of the wild type strain and 20 different radiation sensitive mutants of S. pombe to liquid holding after ultraviolet irradiation was ivestigated. Three of the sensitive mutants tested showed appreciable liquid holding recovery, as opposed to the negative liquid holding effect observed in the wild type cells. One of these mutants is reported to be recombination-deficient while the other two have a normal recombination capability. Further experiments were carried out by using G₁ cells and ascospores to test the possible role of a recombinational type of repair pathway in the failure of wild type S. pombe to show liquid holding recovery. Data from such studies indicated that the negative liquid holding effect observed in the wild type cannot be ascribed to this particular pathway. This conclusion is further supported by the observation that caffeine which is believed to inhibit mainly the recombinational repair in this yeast, did not alter the negative liquid holding effect in the wild type. This observation implies that the caffeine-sensitive repair process occurs only in a rich medium and not in the non-nutrient solution. Data have been discussed as these relate to possible cause(s) of negative liquid holding effect in this organism.     

Mechanism of gene conversion in Ascobolus immersus

Summary Sixty two ascospore colour mutants have been induced in Ascobolus immersus: 25 by an acridine (ICR₁₇₀), 18 by N-methyl-N-nitro-N-nitrosoguanidine (NG) and 19 by ethyl methanesulfonate (EMS). All these mutants have been crossed to the wild type strain and their conversion spectrum has been determined. It appears that the conversion spectrum is closely related to the origin of the mutants studied with respect to the mutagen by which they were induced. All NG mutants gave numerous asci with postmeiotic segregation and an excess of conversion to wild type over conversion to the mutant type. ICR mutants gave no postmeiotic segregation and an excess of conversion to the mutant type. The majority of EMS mutants behave like NG mutants, but some showed only meiotic segregation with either an excess of conversion to the mutant type or an excess of conversion to wild type. These data are in good agreement with the hypothesis that the nature of the mutation has a strong influence on the conversion spectrum.     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

Three <Emphasis Type="Italic">nth</Emphasis> homologs are all required for efficient repair of spontaneous DNA damage in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans is a bacterium that can survive extreme DNA damage. To understand the role of endonuclease III (Nth) in oxidative repair and mutagenesis, we constructed nth single, double and triple mutants. The nth mutants showed no significant difference with wild type in both IR resistance and H₂O₂ resistance. We characterized these strains with regard to mutation rates and mutation spectrum using the rpoB/Rif^r system. The Rif^r frequency of mutant MK1 (△dr0289) was twofold higher than that of wild type. The triple mutant of nth (ME3)generated a mutation frequency 34.4-fold, and a mutation rate 13.8-fold higher than the wild type. All strains demonstrated specific mutational hotspots. Each single mutant had higher spontaneous mutation frequency than wild type at base substitution (G:C → A:T). The mutational response was further increased in the double and triple mutants. The higher mutation rate and mutational response in ME3 suggested that the three nth homologs had non-overlapped and overlapped substrate spectrum in endogenous oxidative DNA repair.     

Genetic control of repair of radiation damage produced under euoxic and anoxic conditions in diploid yeastSaccharomyces cerevisiae

Summary Diploid wild type yeast strains 211, X2180 and the radiation sensitive mutantsrad2, 6, 9, 18, 50–55, and57 were exposed to cobalt-60 gamma radiation, in the presence and absence of oxygen, in order to identify the RAD loci involved in the repair of sublethal damage (SLD), recovery from potentially lethal damage (PLD) and oxygen enhancement ratio (OER). Response of wild type and mutants were compared in terms of survival curve parameters D_q, D₁₀, D₁, and D₀. As compared to wild type the mutants showed increased sensitivity to radiation lethality, both under euoxic and hypoxic conditions, as judged by the reduction in D_q and D₀ values. OER was reduced in therad2, 9, 18, 50, 51, and57 mutants indicating that these genes could be associated with the repair of gamma radiation damage produced under hypoxic condition.Shoulder (D_q) a measure of the ability of the cells to repair SLD, was reduced in therad6, 9, 18, 50, 53, and57 strains and was almost absent in therad51, 52, 54, and55 mutants. The ability to recover from PLD was equal to that of wild type strain in therad2, 6, 9, and18 strains, reduced in therad53, 55, and57 strains and was absent in therad50–52 and54 strains. In the mutants with liquid holding recovery ability, the extent of recovery from PLD produced under euoxic and hypoxic conditions was the same. These observations suggest that different groups of loci are involved in the control of different repair processes and that the expression of therad50–57 loci play a very important role in the repair of ionising radiation damage.On the basis of the liquid holding recovery data presented here and the observations made by others it is suggested that the unrepaired DSB constitute the PLD and that the repair of DSB involves recombination between homologous chromosomes.     

The mitochondrial genome of Chlamydomonas. II. Genetic analysis of non-mendelian obligate photautotrophic mutants

Summary Among a collection of obligate photoautotrophic (dark-dier,dk) mutants isolated inChlamydomonas reinhardtii, two have been found which are inherited in crosses to wild type in a non-Mendelian, biparental and apparently random fashion. F₁ progeny include not only cells which show thedk and wildtype parental phenotypes but also many which possess intermediate phenotypes between wild type anddk. When F₁ progeny withdk, intermediate or wild-type phenotype were backcrossed to wild type, thedk phenotype continued to be inherited in a biparental and random fashion. Upon selection, neither mutant formed stable clones producing onlydk progeny, suggesting that the two mutants segregatedk and wild-type progeny somatically and that the homozygousdk condition may be lethal. The biparental transmission of these two non-Mendeliandk mutations resembles the transmission of acriflavin-inducedminute mutations ofChlamydomonas and is distinct from the uniparentally inherited chloroplast mutations of this alga. Both thedk andminute mutations may alter mitochondrial DNA and thereby alter mitochondrial functions.     

MSX-resistant mutants of Anabaena 7120 with derepressed heterocyst development and nitrogen fixation

To investigate the role of ammonium-assimilating enzyme in heterocyst differentiation, pattern formation and nitrogen fixation, MSX-resistant and GS-impaired mutants of Anabaena 7120 were isolated using transposon (Tn5-1063) mutagenesis. Mutant Gs1 and Gs2 (impaired in GS activity) exhibited a similar rate of nitrogenase activity compared to that of the wild type under dinitrogen aerobic conditions in the presence and absence of MSX. Filaments of Gs1 and Gs2 produced heterocysts with an evenly spaced pattern in N₂-grown conditions, while addition of MSX altered the interheterocyst spacing pattern in wild type as well as in mutant strains. The wild type showed complete repression of heterocyst development and nitrogen fixation in the presence of NO₃ ^– or NH₄ ⁺, whereas the mutants Gs1 and Gs2 formed heterocysts and fixed nitrogen in the presence of NO₃ ^– and NH₄ ⁺. Addition of MSX caused complete inhibition of glutamine synthetase activity in wild type but Gs1 and Gs2 remained unaffected. These results suggest that glutamine but not ammonium is directly involved in regulation of heterocyst differentiation, interheterocyst spacing pattern and nitrogen fixation in Anabaena.     

Comparative study on energy partitioning in photosystem II of two <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> mutants with reduced non-photochemical quenching capacity

Lhcb1-2 and PsbS proteins of photosystem II (PSII) have important roles in photoprotective thermal energy dissipation of the absorbed excess light energy. The light responses of chlorophyll fluorescence parameters were analyzed to examine how the absence of Lhcb1-2 or PsbS proteins can modify the energy allocation patterns of absorbed light energy in PSII using an antisense construct of lhcb2 and a psbS deletion (npq4-1) mutant of Arabidopsis thaliana. Both mutants exhibit reduced Stern–Volmer non-photochemical chlorophyll fluorescence quenching (NPQ). Here, we have adopted an approach, presented by Hendrickson et al. (Photosynth Res 82:73–81, 2004), to gain a better insight into the mechanism of the NPQ in these mutants. We have found no significant differences in the quantum yields of photochemical energy conversion (Φ_PSII) between the mutants and the wild type. Nevertheless, as it was expected, the fraction of the energy, which is dissipated as heat via regulated pathways in PSII (Φ_NPQ) for both mutants, were reduced as compared to the wild type. In a complementary way, the extent of non-regulated non-photochemical energy loss in PSII (Φ_NO) for both mutants was significantly higher than that in the wild type. This reflects, together with the lower Φ_NPQ (or NPQ) values, suboptimal capacity of photoprotective reactions at higher light intensities.