Gas exchange in intact isolated chloroplasts from Chlamydomonas reinhardtii during starch degradation in the dark

During starch degradation in intact isolated chloroplasts from Chlamydomonas reinhardtii gas exchange was studied with a mass spectrometer. Oxygen uptake by intact chloroplasts in the dark never exceeded 1.5% of the starch degradation rate [maximum 15 nmol O₂ (mg Chl)⁻¹ h⁻¹ consumed. 1 000 nmol glucose (mg Chl)⁻¹h⁻¹ degraded]. Evolution of CO₂ under aerobic conditions [9.8–28 nmol (mg Chl)⁻¹ h⁻¹] was stimulated by addition of 0.1–0.5 m M oxaloacetate [393–425 nmol CO₂ (mg Chl)⁻¹ h⁻¹]. Pyridoxal phosphate (5 m M ) inhibited starch degradation by more than 80%, but had no effect on O₂ uptake. Starch degradation rates and CO₂ evolution did not differ under acrobic and anaerobic conditions. Increasing P_i in the reaction medium from 0.5 m M to 5.0 m M stimulated starch degradation by 230 and 260% under aerobic and anaerobic conditions, respectively. A rapid autooxidation of reduced ferredoxin was observed in a reconstituted system consisting of purified Chlamydomonas ferredoxin, purified Chlamydomonas NADP-ferredoxin oxidoreductase (EC 1.6.7.1) and NADPH. Addition of isolated thylakoids from C. reinhardtii did not affect the rate of O₂ uptake. Our results clearly indicate the absence of any oxygen requirement during starch degradation in isolated chloroplasts.     

Inhibition of starch synthesis results in overproduction of lipids in Chlamydomonas reinhardtii

Starch and neutral lipids are two major carbon storage compounds in many microalgae and plants. Lipids are more energy rich and have often been used as food and fuel feedstocks. Genetic engineering of the lipid biosynthesis pathway to overproduce lipid has achieved only limited success. We hypothesize that through blocking the competing pathway to produce starch, overproduction of neutral lipid may be achieved. This hypothesis was tested using the green microalga Chlamydomonas reinhardtii and its low starch and starchless mutants. We discovered that a dramatic increase in neutral lipid content and the neutral lipid/total lipid ratio occurred among the mutants under high light and nitrogen starvation. BAFJ5, one of the mutants defective in the small subunit of ADP‐glucose pyrophosphorylase, accumulated neutral and total lipid of up to 32.6% and 46.4% of dry weight (DW) or 8‐ and 3.5‐fold higher, respectively, than the wild‐type. These results confirmed the feasibility of increasing lipid production through redirecting photosynthetically assimilated carbon away from starch synthesis to neutral lipid synthesis. However, some growth impairment was observed in the low starch and starchless mutants, possibly due to altered energy partitioning in PSII, with more excitation energy dissipated as heat and less to photochemical conversion. This study demonstrated that biomass and lipid production by the selected mutants can be improved by physiological manipulation. Biotechnol. Bioeng. 2010;107: 258–268. © 2010 Wiley Periodicals, Inc.     

淀粉合成受限的莱茵衣藻的甘油酯酰基的变化

【目的】基于突变藻株本身属性和意义出发,考察在两种常用培养方式下莱茵衣藻淀粉突变株(CC-4326)与野生型藻株(CC-137)在甘油酯中酰基随生长的变化差异,为进一步认识莱茵衣藻突变株提供参考信息。【方法】分别在柱状鼓泡式反应器和摇瓶中培养CC-4326和CC-137,比较两株藻在正常培养和氮胁迫培养状态下甘油酯中酰基相对含量和其在甘油三酯(TAG)含量的差异。【结果】正常培养状态下,CC-4326和CC-137中多不饱和酰基C16:4和C18:3相对含量占总酰基45%左右,CC-4326在两种培养方式下这两个酰基含量及变化无差别,而CC-137在摇瓶中培养二者相对含量增加幅度和含量均高于反应器。缺氮条件下两种藻株积累TAG,但程度不同,CC-4326在反应器中培养TAG含量达到CC-137的1.5倍,在摇瓶中培养含量与CC-137无显著差异,两株藻的甘油酯和TAG中C18:1含量显著增加,CC-4326在反应器中培养C18:1增加幅度大于摇瓶,比摇瓶培养更快速积累TAG。而CC-137在摇瓶中培养TAG含量与反应器接近,单不饱和酰基增加幅度却高于反应器,表明CC-137在摇瓶中培养比反应器更利于积累TAG。最终,CC-4326在光生物反应器中缺氮培养实现了TAG 12倍的增加。【结论】通过对淀粉合成抑制,与CC-137相比,缺氮光生物反应器培养条件下,CC-4326能够实现TAG的高效积累。     

Multiple forms of starch phosphorylase from banana leaves

Two forms (A and B) of starch phosphorylase were found in the mature banana leaf by polyacrylamide gel electrophoresis and DEAE-cellulose chromatography. The young leaf contained only form A and the appearance of form B with leaf development was accompanied by a decrease in the content of form A. At a later stage of leaf maturity only form B could be found. The MWs of forms A and B were 450 000, and 220 000 respectively.     

New arginine-requiring mutants in Chlamydomonas reinhardtii

Twelve arginine-requiring mutants of the unicellular green alga Chlamydomonas reinhardtii previously isolated in our laboratory were investigated to find new blocks in the biosynthetic pathway of arginine. In addition to the already described mutants lacking acetylglutamyl phosphate reductase (arg 1), ornithine carbamoyltransferase (arg4) and argininosuccinate lyase (arg7), three new types of mutants were found lacking acetylornithine aminotransferase (arg9-1, arg9-2), acetylornithine glutamate transacetylase (arg10) and argininosuccinate synthetase (arg8-1, arg8-2, arg8-3) respectively. The genetic analysis of these new mutants showed that arg9 and arg8 are unlinked to the other arginine markers and that arg10 probably carries a chromosomal mutation inducing a very high lethality of meiotic products.Abbreviations WT wild-type - mt mating-type - SP spore plating - ZP zygote plating     

Starch phosphorylase: Role in starch metabolism and biotechnological applications

The α-glucan phosphorylases of the glycosyltransferase family are important enzymes of carbohydrate metabolism in prokaryotes and eukaryotes. The plant α-glucan phosphorylase, commonly called starch phosphorylase (EC 2.4.1.1), is largely known for the phosphorolytic degradation of starch. Starch phosphorylase catalyzes the reversible transfer of glucosyl units from glucose-1-phosphate to the nonreducing end of α-1,4-d-glucan chains with the release of phosphate. Two distinct forms of starch phosphorylase, plastidic phosphorylase and cytosolic phosphorylase, have been consistently observed in higher plants. Starch phosphorylase is industrially useful and a preferred enzyme among all glucan phosphorylases for phosphorolytic reactions for the production of glucose-1-phosphate and for the development of engineered varieties of glucans and starch. Despite several investigations, the precise functional mechanisms of its characteristic multiple forms and the structural details are still eluding us. Recent discoveries have shed some light on their physiological substrates, precise biological functions, and regulatory aspects. In this review, we have highlighted important developments in understanding the role of starch phosphorylases and their emerging applications in industry.     

A bioluminescent assay for glycogen phosphorylase in cultured cells

A new method for the determination of glycogen phosphorylase (1,4 alpha-D-glucose:orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) in cultured cells is described. The assay utilizes bacterial luciferase (EC 2.7) in a liquid scintillation spectrometer to measure NAD(P)H formed in a coupled enzyme reaction comprising glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and phosphoglucomutase (EC 2.7.5.1). This assay is highly sensitive, easily detecting as little as 10 microU phosphorylase, fast and simple to perform. With modifications this procedure can be extended to measure other glycogenolytic enzymes and intermediates.     

Glycogen phosphorylase is involved in stress endurance and biofilm formation in Azospirillum brasilense Sp7

Here we report the identification of a glycogen phosphorylase ( glgP ) gene in the plant growth-promoting rhizobacterium Azospirillum brasilense , Sp7, and the characterization of a glgP marker exchange mutant of this strain. The glgP mutant showed a twofold reduction of glycogen phosphorylase activity and an increased glycogen accumulation as compared with wild-type Sp7, indicating that the identified gene indeed encodes a protein with glycogen phosphorylase activity. Interestingly, the glgP mutant had higher survival rates than the wild type after exposure to starvation, desiccation and osmotic pressure. The mutant was shown to be compromised in its biofilm formation ability. Analysis of the exopolysaccharide sugar composition of the glgP mutant revealed a decrease in the amount of glucose, accompanied by increases in rhamnose, fucose and ribose, as compared with the Sp7 exopolysaccharide. To the best of our knowledge, this is the first study that demonstrates GlgP activity in A. brasilense , and shows that glycogen accumulation may play an important role in the stress endurance of this bacterium.     

Ferritin is required for rapid remodeling of the photosynthetic apparatus and minimizes photo-oxidative stress in response to iron availability in Chlamydomonas reinhardtii

Ferritin is a key player in the iron homeostasis due to its ability to store large quantities of iron. Chlamydomonas reinhardtii contains two nuclear genes for ferritin ( ferr1 and ferr2 ) that are induced when Chlamydomonas cells are shifted to iron-deficient conditions. In response to the reduced iron availability, degradation of photosystem I (PSI) and remodeling of its light-harvesting complex occur. This active PSI degradation slows down under photo-autotrophic conditions where photosynthesis is indispensable. We observed a strong induction of ferritin correlated with the degree of PSI degradation during iron deficiency. The PSI level can be restored to normal within 24 h after iron repletion at the expense of the accumulated ferritin, indicating that the ferritin-stored iron allows fast adjustment of the photosynthetic apparatus with respect to iron availability. RNAi strains that are significantly reduced in the amount of ferritin show a striking delay in the degradation of PSI under iron deficiency. Furthermore, these strains are more susceptible to photo-oxidative stress under high-light conditions. We conclude that (i) ferritin is used to buffer the iron released by degradation of the photosynthetic complexes, (ii) the physiological status of the cell determines the strategy used to overcome the impact of iron deficiency, (iii) the availability of ferritin is important for rapid degradation of PSI under iron deficiency, and (iv) ferritin plays a protective role under photo-oxidative stress conditions.     

莱茵衣藻中脂质代谢过程研究进展

微藻中脂质代谢产生的化合物,可用于生物燃料、营养品和生物药品的生产,因此具有重要的经济价值。脂质代谢贯穿微藻的全部生命过程,对微藻的生长发育和应对外界胁迫都具有重要意义。微藻与研究较清楚的真菌和陆地植物在脂质代谢过程方面具有相似性。当然,随着微藻脂质代谢相关功能基因逐渐被鉴定,人们发现微藻的脂质代谢也具有区别真菌和陆地植物的独特性,因此针对微藻脂质代谢过程的分析具有重要意义。莱茵衣藻是研究脂质代谢过程的模式生物,已经通过基因组、转录组、蛋白质组和代谢组等方法,对其质体、内质网和过氧化物酶体中进行的脂质合成和分解过程进行了研究。本文总结了近年来莱茵衣藻质体、内质网和过氧化物酶体中脂质代谢过程的研究成果,并进行综合阐述。     

Plastidial alpha-glucan phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress

To study the role of the plastidial alpha-glucan phosphorylase in starch metabolism in the leaves of Arabidopsis, two independent mutant lines containing T-DNA insertions within the phosphorylase gene were identified. Both insertions eliminate the activity of the plastidial alpha-glucan phosphorylase. Measurement of other enzymes of starch metabolism reveals only minor changes compared with the wild type. The loss of plastidial alpha-glucan phosphorylase does not cause a significant change in the total accumulation of starch during the day or its remobilization at night. Starch structure and composition are unaltered. However, mutant plants display lesions on their leaves that are not seen on wild-type plants, and mesophyll cells bordering the lesions accumulate high levels of starch. Lesion formation is abolished by growing plants under 100% humidity in still air, but subsequent transfer to circulating air with lower humidity causes extensive wilting in the mutant leaves. Wilted sectors die, causing large lesions that are bordered by starch-accumulating cells. Similar lesions are caused by the application of acute salt stress to mature plants. We conclude that plastidial phosphorylase is not required for the degradation of starch, but that it plays a role in the capacity of the leaf lamina to endure a transient water deficit.     

Calcium influx signals normal flagellar RNA induction following acid shock of Chlamydomonas reinhardtii

Acid shock of Chlamydomonas results in flagellar excision and induction of flagellar protein RNAs. The magnitude of flagellar RNA accumulations after flagellar excision by mechanical shear depends on the extracel]ular Ca2+ concentration. In this report, we demonstrate that the magnitude and duration of flagellar RNA accumulations are signaled by an acid shock-induced Ca2+ influx. RNA accumulations were greater in cells acid shocked in 500 µM CaCl2 than in 200 µM CaCl2, although the accumulation durations were similar. RNA accumulations of lower magnitude and shorter duration were observed in cells in Ca2+-containing buffer treated with CdCl2. RNA accumulations were of still lower magnitude and shorter duration in cells shocked in buffer without added CaCl2 than in cells shocked in 200 or 500 µM CaCl2 or in the presence of CdCl2. RNA accumulations similar to those in cells shocked in buffer without added CaCl2 were measured in cells following acid shock in buffer containing 200 µM CaCl2 and supplemented with neomycin, ruthenium red, or LaCl3. Acid shock of the adf-1 mutant resulted in RNA accumulations of shorter duration and lower magnitude than those measured in adf-1 cells stimulated by mechanical shear. These results are consistent with an hypothesis that acid shock generates two genetically and pharmacologically distinct signals governing flagellar RNA induction; the first signal is independent of a Ca2+ influx and flagellar excision and results in low magnitude accumulations of short duration, and the second is a consequence of a Ca2+ influx and results in accumulations of high magnitude and long duration.     

Evidence that amylose synthesis occurs within the matrix of the starch granule in potato tubers

Transgenic potatoes expressing reduced levels of granule-bound starch synthase I (GBSSI) have been used to investigate whether the synthesis of amylose occurs at the surface of the starch granule or within the matrix formed by the synthesis and organization of amylopectin. Amylose in these potatoes is wholly or largely confined to a central region of the granule. Consequently this core region stains blue with iodine whereas the peripheral zone stains red. By making extensive measurements of the relative sizes of the granules and their blue-staining cores in tubers over a range of stages of development, we have established that the blue core increases in size as the granule grows. The extent of the increase in size of the blue core is greater in potatoes with higher levels of GBSSI. These data show that amylose synthesis occurs within the matrix of the granule, and are consistent with the idea that the space available in the matrix may be an important determinant of the amylose content of storage starches.     

A mitochondrial half-size ABC transporter is involved in cadmium tolerance in Chlamydomonas reinhardtii

Five cadmium-sensitive insertional mutants, all affected at the CDS1 (' cadmium-sensitive 1 ') locus, have been previously isolated in the unicellular green alga Chlamydomonas reinhardtii . We here describe the cloning of the Cds1 gene (8314 bp with 26 introns) and the corresponding cDNA. The Cds1 gene, strongly induced by cadmium, encodes a putative protein (CrCds1) of 1062 amino acid residues that belongs to the ATM/HMT subfamily of half-size ABC transporters. This subfamily includes both vacuolar HMT-type proteins transporting phytochelatin-cadmium complexes from the cytoplasm to the vacuole and mitochondrial ATM-type proteins involved in the maturation of cytosolic Fe/S proteins. Unlike the Δsphmt1 cadmium-sensitive mutant of Schizosaccharomyces pombe that lacks a vacuolar HMT-type transporter, the cds1 mutant accumulates a high amount of phytochelatin–cadmium complexes. By epitope tagging, the CrCds1 protein was localized in the mitochondria. Even though mitochondria of cds1 do not accumulate important amounts of 'free' iron, the mutant cells are hypersensitive to high iron concentrations . Our data show for the first time that a mitochondrial ATM-like transporter plays a major role in tolerance to cadmium.     

Insertional mutagenesis to isolate acetate-requiring mutants in Chlamydomonas reinhardtii

Abstract An arg 7 mutant of the green alga Chlamydomonas reinhardtii was transformed with pARG7.8, a plasmid bearing the wild-type ARG 7 gene. Out of 4100 arg⁺ transformants selected on an arginine-free medium supplemented with acetate, nine failed to grow on acetate-free medium (ac⁻ mutants). The results of the genetic and molecular analysis of several ac⁻ mutants are in agreement with the hypothesis that they originated from insertion of the incoming plasmid into the nuclear genome. These mutants should constitute valuable tools for isolating the corresponding wild-type genes after plasmid rescue into Escherichia coli .     

Interaction between starch breakdown, acetate assimilation, and photosynthetic cyclic electron flow in Chlamydomonas reinhardtii

Spectroscopic studies on photosynthetic electron transfer generally are based upon the monitoring of dark to light changes in the electron transfer chain. These studies, which focus on the light reactions of photosynthesis, also indirectly provide information on the redox or metabolic state of the chloroplast in the dark. Here, using the unicellular microalga Chlamydomonas reinhardtii, we study the impact of heterotrophic/mixotrophic acetate feeding on chloroplast carbon metabolism by using the spectrophotometric detection of P700(+), the photooxidized primary electron donor of photosystem I. We show that, when photosynthetic linear and cyclic electron flows are blocked (DCMU inhibiting PSII and methylviologen accepting electrons from PSI), the post-illumination reduction kinetics of P700(+) directly reflect the dark metabolic production of reductants (mainly NAD(P)H) in the stroma of chloroplasts. Such results can be correlated to other metabolic studies: in the absence of acetate, for example, the P700(+) reduction rate matches the rate of starch breakdown reported previously, confirming the chloroplast localization of the upstream steps of the glycolytic pathway in Chlamydomonas. Furthermore, the question of the interplay between photosynthetic and non-photosynthetic carbon metabolism can be addressed. We show that cyclic electron flow around photosystem I is twice as fast in a starchless mutant fed with acetate than it is in the WT, and we relate how changes in the flux of electrons from carbohydrate metabolism modulate the redox poise of the plastoquinone pool in the dark through chlororespiration.