Co-inoculation of rhizobia, azospirilla and cyanobacteria for increasing common bean production / Co-inoculação de rizobio, azospirillum e cianobactérias no aumento da produção de feijão comum

The combined inoculation of Rhizobium (R. tropici+R. freirei), Azospirillum brasilense, and Anabaena cylindrica, a diazotrophic cyanobacterium, is a technology that has not yet been tested and established in the production of the common bean (Phaseolus vulgaris). The inoculation may be a promising strategy for increasing crop productivity by combining the benefits of biological nitrogen fixation with the production of plant growth phytohormones. Therefore, the objective of this study was to evaluate the co-inoculation of Rhizobium, Azospirillum brasilense, and Anabaena cylindrica as an alternative method for optimizing the symbiotic performance and development of the common bean at greenhouse conditions. The treatments were as follows: (T1) control without N and inoculation, (T2) N addition (100kg N ha-¹), (T3) Riz (addition of R. tropici+R. freirei), (T4) Azo (Azospirillum brasilense addition), (T5)Ana (Anabaena cylindrical addition), (T6) Riz+Azo, (T7) Riz+Ana, (T8) Azo+Ana, (T9) Riz+Azo+Ana. We used a completely randomized experimental design with four replications. The co-inoculation of Riz+Azo+Ana promoted plant height, root length and volume, shoot dry matter, accumulated shoot N, number and dry matter of nodules at flowering, number of grains per pod, hundred seed weight, andgrain production of the common bean, contributing to increased yield per plant. We observed an increase in common bean grain yield ranging from 62 to 84% after double and triple co-inoculation of rhizobia with azospirilla and/or cyanobacteria, with the highest yield observed in the plants inoculated with Riz+Azo+Ana (84%), similar to those observed in plants after N addition. However, field experiments are necessary to elucidate the performances of the inoculated beneficial microorganisms.(AU)

A inoculação associada de Rhizobium, Azospirillum e uma cianobactéria diazotrófica Anabaena cylindrica é uma tecnologia que ainda não foi testada e estabelecida para o feijão comum (Phaseolus vulgaris) e pode ser uma estratégia promissora para aumentar a produtividade das culturas, combinando os benefícios da fixação biológica de nitrogênio com a produção de fitohormônios de crescimento vegetal. Portanto, o objetivo deste estudo foi avaliar a co-inoculação de Rizobium, Azospirilum brasilense e Anabaena cylindrica, como uma alternativa para otimizar o desempenho simbiótico e o desenvolvimento do feijoeiro, em casa de vegetação. Os tratamentos foram: T1-control, T2-N-mineral (100 kg N ha-¹), T3- Rhizobium (R. tropici+R. freirei), T4- A. brasilense, T5- A. cylindrica, T6- Rhizobium + A. brasilense, T7- Riz + Ana, T8- Azo + Ana, T9- Riz + Azo + Ana. O delineamento experimental foi inteiramente casualizado, com quatro repetições. A co-inoculação de Rhizobium (R.tropici+R. freirei) + Azo + Ana promoveu aumento do feijão (IPR Campos Gerais) em altura das plantas, comprimento e volume de raiz, massa seca da parte aérea, N acumulado da parte aérea, número e matéria seca dos nódulos no florescimento, número de grãos por vagem, peso de cem grãos e de grãos por planta, o que contribuiu para incremento da produção. Houve um aumento na produção de grãos de feijão com co-inoculação dupla e tripla de rizobia com azospirila e / ou cianobactérias, variando de 62a 84%, com o maior aumento de produção observado no tratamento inoculado com Riz + Azo + Ana(84%), semelhante ao tratamento com nitrogênio mineral. No entanto, experimentos de campo podem contribuir para elucidar o desempenho de microrganismos benéficos inoculados.(AU)

Co-inoculation of rhizobia, azospirilla and cyanobacteria for increasing common bean production / Co-inoculação de rizobio, azospirillum e cianobactérias no aumento da produção de feijão comum

The combined inoculation of Rhizobium (R. tropici+R. freirei), Azospirillum brasilense, and Anabaena cylindrica, a diazotrophic cyanobacterium, is a technology that has not yet been tested and established in the production of the common bean (Phaseolus vulgaris). The inoculation may be a promising strategy for increasing crop productivity by combining the benefits of biological nitrogen fixation with the production of plant growth phytohormones. Therefore, the objective of this study was to evaluate the co-inoculation of Rhizobium, Azospirillum brasilense, and Anabaena cylindrica as an alternative method for optimizing the symbiotic performance and development of the common bean at greenhouse conditions. The treatments were as follows: (T1) control without N and inoculation, (T2) N addition (100kg N ha-¹), (T3) Riz (addition of R. tropici+R. freirei), (T4) Azo (Azospirillum brasilense addition), (T5)Ana (Anabaena cylindrical addition), (T6) Riz+Azo, (T7) Riz+Ana, (T8) Azo+Ana, (T9) Riz+Azo+Ana. We used a completely randomized experimental design with four replications. The co-inoculation of Riz+Azo+Ana promoted plant height, root length and volume, shoot dry matter, accumulated shoot N, number and dry matter of nodules at flowering, number of grains per pod, hundred seed weight, andgrain production of the common bean, contributing to increased yield per plant. We observed an increase in common bean grain yield ranging from 62 to 84% after double and triple co-inoculation of rhizobia with azospirilla and/or cyanobacteria, with the highest yield observed in the plants inoculated with Riz+Azo+Ana (84%), similar to those observed in plants after N addition. However, field experiments are necessary to elucidate the performances of the inoculated beneficial microorganisms.

A inoculação associada de Rhizobium, Azospirillum e uma cianobactéria diazotrófica Anabaena cylindrica é uma tecnologia que ainda não foi testada e estabelecida para o feijão comum (Phaseolus vulgaris) e pode ser uma estratégia promissora para aumentar a produtividade das culturas, combinando os benefícios da fixação biológica de nitrogênio com a produção de fitohormônios de crescimento vegetal. Portanto, o objetivo deste estudo foi avaliar a co-inoculação de Rizobium, Azospirilum brasilense e Anabaena cylindrica, como uma alternativa para otimizar o desempenho simbiótico e o desenvolvimento do feijoeiro, em casa de vegetação. Os tratamentos foram: T1-control, T2-N-mineral (100 kg N ha-¹), T3- Rhizobium (R. tropici+R. freirei), T4- A. brasilense, T5- A. cylindrica, T6- Rhizobium + A. brasilense, T7- Riz + Ana, T8- Azo + Ana, T9- Riz + Azo + Ana. O delineamento experimental foi inteiramente casualizado, com quatro repetições. A co-inoculação de Rhizobium (R.tropici+R. freirei) + Azo + Ana promoveu aumento do feijão (IPR Campos Gerais) em altura das plantas, comprimento e volume de raiz, massa seca da parte aérea, N acumulado da parte aérea, número e matéria seca dos nódulos no florescimento, número de grãos por vagem, peso de cem grãos e de grãos por planta, o que contribuiu para incremento da produção. Houve um aumento na produção de grãos de feijão com co-inoculação dupla e tripla de rizobia com azospirila e / ou cianobactérias, variando de 62a 84%, com o maior aumento de produção observado no tratamento inoculado com Riz + Azo + Ana(84%), semelhante ao tratamento com nitrogênio mineral. No entanto, experimentos de campo podem contribuir para elucidar o desempenho de microrganismos benéficos inoculados.

Effects of monosulfuron-ester on metabolic processes of nitrogen-fixing cyanobacteria Anabaena flos-aquae and Anabaena azotica

Abstract Presence of the relatively new sulfonylurea herbicide monosulfuron-ester at 0.03-300 nmol/L affected the growth of two non-target nitrogen-fixing cyanobacteria (Anabaena flos-aquae and Anabaena azotica) and substantially inhibited in vitro Acetolactate synthase activity, with IC50 of 3.3 and 101.3 nmol/L for A. flos-aquae and A. azotica, respectively. Presenting in 30-300 nmol/L, it inhibited protein synthesis of the cyanobacteria with less amino acids produced as its concentration increased. Our findings support the view that monosulfuron-ester toxicity in both nitrogen-fixing cyanobacteria is due to its interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly Acetolactate synthase activity.

Effects of monosulfuron-ester on metabolic processes of nitrogen-fixing cyanobacteria Anabaena flos-aquae and Anabaena azotica.

Presence of the relatively new sulfonylurea herbicide monosulfuron-ester at 0.03-300nmol/L affected the growth of two non-target nitrogen-fixing cyanobacteria (Anabaena flos-aquae and Anabaena azotica) and substantially inhibited in vitro Acetolactate synthase activity, with IC50 of 3.3 and 101.3nmol/L for A. flos-aquae and A. azotica, respectively. Presenting in 30-300nmol/L, it inhibited protein synthesis of the cyanobacteria with less amino acids produced as its concentration increased. Our findings support the view that monosulfuron-ester toxicity in both nitrogen-fixing cyanobacteria is due to its interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly Acetolactate synthase activity.

Assessment of the effects of As(III) treatment on cyanobacteria lipidomic profiles by LC-MS and MCR-ALS.

Cyanobacteria are a group of photosynthetic, nitrogen-fixing bacteria present in a wide variety of habitats such as freshwater, marine, and terrestrial ecosystems. In this work, the effects of As(III), a major toxic environmental pollutant, on the lipidomic profiles of two cyanobacteria species (Anabaena and Planktothrix agardhii) were assessed by means of a recently proposed method based on the concept of regions of interest (ROI) in liquid chromatography mass spectroscopy (LC-MS) together with multivariate curve resolution alternating least squares (MCR-ALS). Cyanobacteria were exposed to two concentrations of As(III) for a week, and lipid extracts were analyzed by ultrahigh-performance liquid chromatography/time-of-flight mass spectrometry in full scan mode. The data obtained were compressed by means of the ROI strategy, and the resulting LC-MS data sets were analyzed by the MCR-ALS method. Comparison of profile peak areas resolved by MCR-ALS in control and exposed samples allowed the discrimination of lipids whose concentrations were changed due to As(III) treatment. The tentative identification of these lipids revealed an important reduction of the levels of some galactolipids such as monogalactosyldiacylglycerol, the pigment chlorophyll a and its degradation product, pheophytin a, as well as carotene compounds such as 3-hydroxycarotene and carotene-3,3'-dione, all of these compounds being essential in the photosynthetic process. These results suggested that As(III) induced important changes in the composition of lipids of cyanobacteria, which were able to compromise their energy production processes. Graphical abstract Steps of the proposed LC-MS + MCR-ALS procedure.

Antifungal compounds from cyanobacteria.

Cyanobacteria are photosynthetic prokaryotes found in a range of environments. They are infamous for the production of toxins, as well as bioactive compounds, which exhibit anticancer, antimicrobial and protease inhibition activities. Cyanobacteria produce a broad range of antifungals belonging to structural classes, such as peptides, polyketides and alkaloids. Here, we tested cyanobacteria from a wide variety of environments for antifungal activity. The potent antifungal macrolide scytophycin was detected in Anabaena sp. HAN21/1, Anabaena cf. cylindrica PH133, Nostoc sp. HAN11/1 and Scytonema sp. HAN3/2. To our knowledge, this is the first description of Anabaena strains that produce scytophycins. We detected antifungal glycolipopeptide hassallidin production in Anabaena spp. BIR JV1 and HAN7/1 and in Nostoc spp. 6sf Calc and CENA 219. These strains were isolated from brackish and freshwater samples collected in Brazil, the Czech Republic and Finland. In addition, three cyanobacterial strains, Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110 and Nostoc sp. N107.3, produced unidentified antifungal compounds that warrant further characterization. Interestingly, all of the strains shown to produce antifungal compounds in this study belong to Nostocales or Stigonematales cyanobacterial orders.

Intercellular transfer along the trichomes of the invasive terminal heterocyst forming cyanobacterium Cylindrospermopsis raciborskii CS-505.

Cylindrospermopsis raciborskii CS-505 is an invasive freshwater filamentous cyanobacterium that when grown diazotrophically may develop trichomes of up to 100 vegetative cells while differentiating only two end heterocysts, the sole sites for their N2-fixation process. We examined the diazotrophic growth and intercellular transfer mechanisms in C. raciborskii CS-505. Subjecting cultures to a combined-nitrogen-free medium to elicit N2 fixation, the trichome length remained unaffected while growth rates decreased. The structures and proteins for intercellular communication showed that while a continuous periplasmic space was apparent along the trichomes, the putative septal junction sepJ gene is divided into two open reading frames and lacks several transmembrane domains unlike the situation in Anabaena, differentiating a 5-fold higher frequency of heterocysts. FRAP analyses also showed that the dyes calcein and 5-CFDA were taken up by heterocysts and vegetative cells, and that the transfer from heterocysts and 'terminal' vegetative cells showed considerably higher transfer rates than that from vegetative cells located in the middle of the trichomes. The data suggest that C. raciborskii CS-505 compensates its low-frequency heterocyst phenotype by a highly efficient transfer of the fixed nitrogen towards cells in distal parts of the trichomes (growing rapidly) while cells in central parts suffers (slow growth).

Flavodoxin displays dose-dependent effects on photosynthesis and stress tolerance when expressed in transgenic tobacco plants.

Ferredoxins are iron-sulfur proteins involved in various one-electron transfer pathways. Ferredoxin levels decrease under adverse environmental conditions in photosynthetic organisms. In cyanobacteria, this decline is compensated by induction of flavodoxin, an isofunctional flavoprotein. Flavodoxin is not present in higher plants, but transgenic Nicotiana tabacum lines accumulating Anabaena flavodoxin in plastids display increased tolerance to different sources of environmental stress. As the degree of tolerance correlated with flavodoxin dosage in plastids of nuclear-transformed transgenic tobacco, we prepared plants expressing even higher levels of flavodoxin by direct plastid transformation. A suite of nuclear- and chloroplast-transformed lines expressing a wide range of flavodoxin levels, from 0.3 to 10.8 µmol m(-2), did not exhibit any detectable growth phenotype relative to the wild type. In the absence of stress, the contents of both chlorophyll a and carotenoids, as well as the photosynthetic performance (photosystem II maximum efficiency, photosystem II operating efficiency, electron transport rates and carbon assimilation rates), displayed a moderate increase with flavodoxin concentrations up to 1.3-2.6 µmol flavodoxin m(-2), and then declined to wild-type levels. Stress tolerance, as estimated by the damage inflicted on exposure to the pro-oxidant methyl viologen, also exhibited a bell-shaped response, with a significant, dose-dependent increase in tolerance followed by a drop in the high-expressing lines. The results indicate that optimal photosynthetic performance and stress tolerance were observed at flavodoxin levels comparable to those of endogenous ferredoxin. Further increases in flavodoxin content become detrimental to plant fitness.

Stress response of transgenic tobacco plants expressing a cyanobacterial ferredoxin in chloroplasts.

Expression of the chloroplast electron shuttle ferredoxin is induced by light through mechanisms that partially depend on sequences lying in the coding region of the gene, complicating its manipulation by promoter engineering. Ferredoxin expression is also down-regulated under virtually all stress situations, and it is unclear if light-dependent induction and stress-dependent repression proceed through the same or similar mechanisms. Previous reports have shown that expression of a cyanobacterial flavodoxin in tobacco plastids results in plants with enhanced tolerance to adverse environmental conditions such as drought, chilling and xenobiotics (Tognetti et al. in Plant Cell 18:2035-2050, 2006). The protective effect of flavodoxin was linked to functional replacement of ferredoxin, suggesting the possibility that tolerant phenotypes might be obtained by simply increasing ferredoxin contents. To bypass endogenous regulatory constraints, we transformed tobacco plants with a ferredoxin gene from Anabaena sp. PCC7120, which has only 53% identity with plant orthologs. The cyanobacterial protein was able to interact in vitro with ferredoxin-dependent plant enzymes and to mediate NADP(+) photoreduction by tobacco thylakoids. Expression of Anabaena ferredoxin was constitutive and light-independent. However, homozygous lines accumulating threefold higher ferredoxin levels than the wild-type failed to show enhanced tolerance to oxidative stress and chilling temperatures. Under these adverse conditions, Anabaena ferredoxin was down-regulated even faster than the endogenous counterparts. The results indicate that: (1) light- and stress-dependent regulations of ferredoxin expression proceed through different pathways, and (2) overexpression of ferredoxin is not an alternative to flavodoxin expression for the development of increased stress tolerance in plants.

Cyanobacterial flavodoxin complements ferredoxin deficiency in knocked-down transgenic tobacco plants.

Ferredoxins are the main electron shuttles in chloroplasts, accepting electrons from photosystem I and delivering them to essential oxido-reductive pathways in the stroma. Ferredoxin levels decrease under adverse environmental conditions in both plants and photosynthetic micro-organisms. In cyanobacteria and some algae, this decrease is compensated for by induction of flavodoxin, an isofunctional flavoprotein that can replace ferredoxin in many reactions. Flavodoxin is not present in plants, but tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin developed increased tolerance to environmental stress. Chloroplast-located flavodoxin interacts productively with endogenous ferredoxin-dependent pathways, suggesting that its protective role results from replacement of stress-labile ferredoxin. We tested this hypothesis by using RNA antisense and interference techniques to decrease ferredoxin levels in transgenic tobacco. Ferredoxin-deficient lines showed growth arrest, leaf chlorosis and decreased CO(2) assimilation. Chlorophyll fluorescence measurements indicated impaired photochemistry, over-reduction of the photosynthetic electron transport chain and enhanced non-photochemical quenching. Expression of flavodoxin from the nuclear or plastid genome restored growth, pigment contents and photosynthetic capacity, and relieved the electron pressure on the electron transport chain. Tolerance to oxidative stress also recovered. In the absence of flavodoxin, ferredoxin could not be decreased below 45% of physiological content without fatally compromising plant survival, but in its presence, lines with only 12% remaining ferredoxin could grow autotrophically, with almost wild-type phenotypes. The results indicate that the stress tolerance conferred by flavodoxin expression in plants stems largely from functional complementation of endogenous ferredoxin by the cyanobacterial flavoprotein.

Methods for detection of anatoxin-a(s) by liquid chromatography coupled to electrospray ionization-tandem mass spectrometry.

Anatoxin-a(s) is a potent irreversible inhibitor of the enzyme acetylcholinesterase with a unique N-hydroxyguanidine methylphosphate ester chemical structure. Determination of this toxin in environmental samples is hampered by the lack of specific methods for its detection. Using the toxic strain of Anabaena lemmermani PH-160 B as positive control, the fragmentation characteristics of anatoxin-a(s) under collision-induced dissociation conditions have been investigated and new LC-MS/MS methods proposed. Recommended ion transitions for correct detection of this toxin are 253>58, 253>159, 235>98 and 235>96. Chromatographic separation is better achieved under HILIC conditions employing a ZIC-HILIC column. This method was used to confirm for the first time the production of anatoxin-a(s) by strains of Anabaena oumiana ITEP-025 and ITEP-026. Considering no standard solutions are commercially available, our results will be of significant use for the correct identification of this toxin by LC-MS/MS.

The effects of Anabaena spiroides exopolysaccharides on copper accumulation in an aquatic food chain.

The influence of Anabaena spiroides exopolysaccharides (EPS) on copper speciation (total dissolved, particulate and free Cu(2+) ions) and bioavailability in aquatic organisms was investigated. Bacteria were used as the first trophic level, Paramecium caudatum (protozoan) as the second and the copepod cyclopoid Metacyclops mendocinus as the third level. The organisms were obtained from a freshwater reservoir and held under continuous laboratory controlled conditions. Freshwater media containing EPS excreted by A. spiroides (10mgL(-1)) and copper (1.0x10(-6)molL(-1)) were used for bacteria growth. Contamined bacteria were used as food source to protozoan, which was further furnished to copepods. The results showed a reduction of EPS concentration during bacteria growth and also a smaller copper accumulation by microorganisms in the presence of EPS. We concluded that A. spiroides exopolysaccharides have reduced copper entrance into the experimental aquatic microbial food chain.

Role of NtcA, a cyanobacterial global nitrogen regulator, in the regulation of sucrose metabolism gene expression in Anabaena sp. PCC 7120.

In the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120), it has been shown that spsB and susA, the genes coding for proteins related to sucrose synthesis and cleavage, respectively, exhibit converse expression regarding the nitrogen source. In the nitrogen-fixing filament, spsB expression is mostly localized to the heterocysts and susA is only expressed in vegetative cells. The aim of this work was to investigate the participation of NtcA, a global nitrogen regulator that operates in cyanobacteria, in the regulation of sucrose metabolism genes in Anabaena sp. PCC 7120. The induction of spsB expression observed in the filaments upon combined-nitrogen depletion was abolished in an NtcA-deficient mutant. In vitro experiments showed that NtcA binds specifically but with different affinities to two sites in the spsB promoter region. When susA expression was analyzed after a combined-nitrogen starvation, the levels of mRNA, polypeptide and activity increased in the mutant in comparison with the wild-type strain. Also, NtcA interacted with one site in the promoter region of susA. We conclude that sucrose metabolism is coordinated at the transcriptional level with nitrogen metabolism, suggesting a global metabolism regulating role for NtcA.

A toxic cyanobacterial bloom in an urban coastal lake, Rio Grande do Sul state, Southern Brazil / Uma floração de cianobacterias em um lago costeiro urbano, Rio Grande do Sul, Brasil

Reports of cyanobacterial blooms developing worldwide have considerably increased, and, in most cases, the predominant toxins are microcystins. The present study reports a cyanobacterial bloom in Lake Violão, Torres, Rio Grande do Sul State, in January 2005. Samples collected on January 13, 2005, were submitted to taxonomical, toxicological, and chemical studies. The taxonomical analysis showed many different species of cyanobacteria, and that Microcystis protocystis and Sphaerocavum cf. brasiliense were dominant. Besides these, Microcystis panniformis, Anabaena oumiana,Cylindrospermopsis raciborskii, and Anabaenopsis elenkinii f. circularis were also present. The toxicity of the bloom was confirmed through intraperitoneal tests in mice, and chemical analyses of bloom extracts showed that the major substance was anabaenopeptin F, followed by anabaenopeptin B, microcystin-LR, and microcystin-RR.

O número de relatos de ocorrências de florações de cianobactérias em todo o mundo vem aumentando consideravelmente e na maioria desses episódios, as toxinas dominantes são as microcistinas. O presente estudo relata a ocorrência de floração na Lagoa do Violão, município de Torres, RS, em janeiro de 2005. As amostras coletadas em 13/01/2005 foram submetidas a estudos taxonômicos, toxicológicos e químicos. O exame microscópico do fitoplancton mostrou a dominância das espécies Microcystis protocystis e Sphaerocavum cf. brasiliense; foram observadas, também, Microcystis panniformis, Anabaena oumiana,Cylindrospermopsis raciborskii e Anabaenopsis elenkinii f. circularis. A toxicidade da floração foi confirmada através de ensaio intraperitonial em camundongos e a análise química de extratos obtidos da biomassa liofilizada mostrou que a substância majoritária era a anabaenopeptina F, seguida por anabaenopeptina B, microcistina-LR e microcistina-RR.

Sucrose synthase is involved in the conversion of sucrose to polysaccharides in filamentous nitrogen-fixing cyanobacteria.

Higher plants and cyanobacteria metabolize sucrose (Suc) by a similar set of enzymes. Suc synthase (SuS, UDP-glucose: D: -fructose 2-alpha-D: -glucosyl transferase, EC 2.4.1.13) catalyses the synthesis and cleavage of Suc, and in higher plants, it plays an important role in polysaccharides biosynthesis and carbon allocation. In this work, we have studied the functional relationship between SuS and the metabolism of polysaccharides in filamentous nitrogen-fixing cyanobacteria. We show that the nitrogen and carbon sources and light regulate the expression of the SuS encoding gene (susA), in a similar way that they regulate the accumulation of polysaccharides. Furthermore, glycogen content in an Anabaena sp. mutant strain with an insertion inactivation of susA was lower than in the wild type strain under diazotrophic conditions, while both glycogen and polysaccharides levels were higher in a mutant strain constitutively overexpressing susA. We also show that there are soluble and membrane-bound forms of SuS in Anabaena. Taken together, these results strongly suggest that SuS is involved in the Suc to polysaccharides conversion according to nutritional and environmental signals in filamentous nitrogen-fixing cyanobacteria.

Enhanced plant tolerance to iron starvation by functional substitution of chloroplast ferredoxin with a bacterial flavodoxin.

Iron limitation affects one-third of the cultivable land on Earth and represents a major concern for agriculture. It causes decline of many photosynthetic components, including the Fe-S protein ferredoxin (Fd), involved in essential oxidoreductive pathways of chloroplasts. In cyanobacteria and some algae, Fd down-regulation under Fe deficit is compensated by induction of an isofunctional electron carrier, flavodoxin (Fld), a flavin mononucleotide-containing protein not found in plants. Transgenic tobacco lines expressing a cyanobacterial Fld in chloroplasts were able to grow in Fe-deficient media that severely compromised survival of WT plants. Fld expression did not improve Fe uptake or mobilization, and stressed transformants elicited a normal deficit response, including induction of ferric-chelate reductase and metal transporters. However, the presence of Fld did prevent decrease of several photosynthetic proteins (but not Fd) and partially protected photosynthesis from inactivation. It also preserved the activation state of enzymes depending on the Fd-thioredoxin pathway, which correlated with higher levels of intermediates of carbohydrate metabolism and the Calvin cycle, as well as increased contents of sucrose, glutamate, and other amino acids. These metabolic routes depend, directly or indirectly, on the provision of reduced Fd. The results indicate that Fld could compensate Fd decline during episodes of Fe deficiency by productively interacting with Fd-dependent pathways of the host, providing fresh genetic resources for the design of plants able to survive in Fe-poor lands.

Carbon cycling in Anabaena sp. PCC 7120. Sucrose synthesis in the heterocysts and possible role in nitrogen fixation.

Nitrogen (N) available to plants mostly originates from N(2) fixation carried out by prokaryotes. Certain cyanobacterial species contribute to this energetically expensive process related to carbon (C) metabolism. Several filamentous strains differentiate heterocysts, specialized N(2)-fixing cells. To understand how C and N metabolism are regulated in photodiazotrophically grown organisms, we investigated the role of sucrose (Suc) biosynthesis in N(2) fixation in Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120). The presence of two Suc-phosphate synthases (SPS), SPS-A and SPS-B, directly involved in Suc synthesis with different glucosyl donor specificity, seems to be important in the N(2)-fixing filament. Measurement of enzyme activity and polypeptide levels plus reverse transcription-polymerase chain reaction experiments showed that total SPS expression is greater in cells grown in N(2) versus combined N conditions. Only SPS-B, however, was seen to be active in the heterocyst, as confirmed by analysis of green fluorescent protein reporters. SPS-B gene expression is likely controlled at the transcriptional initiation level, probably in relation to a global N regulator. Metabolic control analysis indicated that the metabolism of glycogen and Suc is likely interconnected in N(2)-fixing filaments. These findings suggest that N(2) fixation may be spatially compatible with Suc synthesis and support the role of the disaccharide as an intermediate in the reduced C flux in heterocyst-forming cyanobacteria.

Functional replacement of ferredoxin by a cyanobacterial flavodoxin in tobacco confers broad-range stress tolerance.

Chloroplast ferredoxin (Fd) plays a pivotal role in plant cell metabolism by delivering reducing equivalents to various essential oxidoreductive pathways. Fd levels decrease under adverse environmental conditions in many microorganisms, including cyanobacteria, which share a common ancestor with chloroplasts. Conversely, stress situations induce the synthesis of flavodoxin (Fld), an electron carrier flavoprotein not found in plants, which can efficiently replace Fd in most electron transfer processes. We report here that chloroplast Fd also declined in plants exposed to oxidants or stress conditions. A purified cyanobacterial Fld was able to mediate plant Fd-dependent reactions in vitro, including NADP+ and thioredoxin reduction. Tobacco (Nicotiana tabacum) plants expressing Fld in chloroplasts displayed increased tolerance to multiple sources of stress, including redox-cycling herbicides, extreme temperatures, high irradiation, water deficit, and UV radiation. Oxidant buildup and oxidative inactivation of thioredoxin-dependent plastidic enzymes were decreased in stressed plants expressing plastid-targeted Fld, suggesting that development of the tolerant phenotype relied on productive interaction of this flavoprotein with Fd-dependent oxidoreductive pathways of the host, most remarkably, thioredoxin reduction. The use of Fld provides new tools to investigate the requirements of photosynthesis in planta and to increase plant stress tolerance based on the introduction of a cyanobacterial product that is free from endogenous regulation in higher plants.

Domain swapping between a cyanobacterial and a plant subunit ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the regulatory step in the pathway for synthesis of bacterial glycogen and starch in plants. ADP-Glc PPases from cyanobacteria (homotetramer) and from potato (Solanum tuberosum) tuber (heterotetramer) are activated by 3-phosphoglycerate and inhibited by inorganic orthophosphate. To study the function of two putative domains, chimeric enzymes were constructed. PSSANA contained the N-terminus (292 amino acids) of the potato tuber ADP-Glc PPase small subunit (PSS) and the C-terminus (159 residues) of the Anabaena PCC 7120 enzyme. ANAPSS was the inverse chimera. These constructs were expressed separately or together with the large subunit of the potato tuber ADP-Glc PPase (PLS), to obtain homo- and heterotetrameric chimeric proteins. Characterization of these forms showed that the N-terminus determines stability and regulatory redox-dependent properties. The chimeric forms exhibited intermediate 3-phosphoglycerate activation properties with respect to the wild-type homotetrameric enzymes, indicating that the interaction between the putative N- and C-domains determines the affinity for the activator. Characterization of the chimeric heterotetramers showed the functionality of the large subunit, mainly in modulating regulation of the enzyme by the coordinate action of 3-phosphoglycerate and inorganic orthophosphate.

Sucrose synthase and RuBisCo expression is similarly regulated by the nitrogen source in the nitrogen-fixing cyanobacterium Anabaena sp.

In higher plants and cyanobacteria, sucrose (Suc) metabolism is carried out by a similar set of enzymes. The function and regulation of Suc metabolism in cyanobacteria has begun to be elucidated. In strains of Anabaena sp., filamentous nitrogen-fixing cyanobacteria, Suc synthase (SuS, EC 2.4.1.13) controls Suc cell level through the cleavage of the disaccharide. The present work shows that there are two sus genes in Anabaena (Nostoc) sp. that are co-regulated regarding the nitrogen source; however, only susA accounts for the extractable SuS activity and for the control of the Suc level. Primer extension analysis has uncovered the sequence of the Anabaena susA and susB ammonium-activated putative promoters, which share a high sequence similarity with that of rbcLS encoding ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and other ammonium up-regulated genes. Moreover, susA and rbcLS expression is developmentally co-localized to the vegetative cells of the nitrogen-fixing cyanobacterial filaments. Our results strongly suggest the existence of a regulatory network that would coordinate the expression of key genes for Suc and nitrogen metabolism, carbon fixation, and development in Anabaena sp.