Nitrogen-sufficient Conditions Research Articles

Combined effects of nitrogen limitation and overexpression of malic enzyme gene on lipid accumulation in Dunaliella parva

Overexpression of Dunaliella parva (D. parva) malic enzyme (ME) gene (DpME) significantly increased DpME expression and ME enzyme activity in transgenic D. parva. Nitrogen limitation had an inhibitory effect on protein content, and DpME overexpression could improve protein content. Nitrogen limitation increased carbohydrate content, and Dunaliella parva overexpressing malic enzyme gene under nitrogen limitation (DpME-N−) group showed the lowest starch content among all groups. Dunaliella parva overexpressing malic enzyme gene under nitrogen sufficient condition (DpME) and DpME-N− groups showed considerably high mRNA levels of DpME. ME activity was significantly enhanced by DpME overexpression, and nitrogen limitation caused a smaller increase. DpME overexpression and nitrogen limitation obviously enhanced lipid accumulation, and DpME overexpression had more obvious effect. Compared with control (wild type), lipid content (68.97%) obviously increased in DpME-N− group. This study indicated that the combination of DpME overexpression and nitrogen limitation was favorable to the production of microalgae biodiesel.

Bacterial endosymbionts of a nitrogen-fixing yeast Rhodotorula mucilaginosa JGTA-S1 - insights into a yet unknown micro-ecosystem.

Rhodotorula mucilaginosa JGTA-S1 is a yeast strain capable of fixing nitrogen and improving nitrogen nutrition in rice plants because of its nitrogen-fixing endobacteria, namely Stutzerimonas (Pseudomonas) stutzeri and Bradyrhizobium sp. To gain a deeper understanding of yeast endosymbionts, we conducted a whole-genome shotgun metagenomic analysis of JGTA-S1 cells grown under conditions of nitrogen sufficiency and deficiency. Our results showed that the endosymbiont population varied depending on the nitrogen regime. Upon mechanical disruption of yeast cells, we obtained endosymbionts in culturable form viz. Bacillus velezensis and Staphylococcus sp. under nitrogen-replete conditions and Lysinibacillus telephonicus., Brevibacillus sp., and Niallia circulans under nitrogen-depleted conditions. S. stutzeri and Bradyrhizobium sp. the previously reported endosymbionts remained unculturable. The culturable endosymbionts Staphylococcus sp. and Bacillus velezensis appear to possess genes for dissimilatory nitrate reduction (DNRA), an alternative pathway for ammonia synthesis. However, our findings suggest that these endosymbionts are facultative as they survive outside the host. The fitness of the yeast was not affected by curing of these microbes. Curing the yeast diazotrophic endosymbionts took a toll on its fitness. Our results also showed that the populations of S. stutzeri and B. velezensis increased significantly under nitrogen-depleted conditions compared to nitrogen-sufficient conditions. The importance of DNRA and nitrogen fixation is also reflected in the metagenomic reads of JGTA-S1.

Effects of two types of Coccomyxa sp. KJ on in vitro ruminal fermentation, methane production, and the rumen microbiota.

Coccomyxa sp. KJ is a unicellular green microalga that accumulates abundant lipids when cultured under nitrogen-deficient conditions (KJ1) and high nitrogen levels when cultured under nitrogen-sufficient conditions (KJ2). Considering the different characteristics between KJ1 and KJ2, they are expected to have different effects on rumen fermentation. This study aimed to determine the effects of KJ1 and KJ2 on in vitro ruminal fermentation, digestibility, CH4 production, and the ruminal microbiome as corn silage substrate condition. Five treatments were evaluated: substrate only (CON) and CON + 0.5% dry matter (DM) KJ1 (KJ1_L), 1.0% DM KJ1 (KJ1_H), 0.5% DM KJ2 (KJ2_L), and 1.0% DM KJ2 (KJ2_H). DM degradability-adjusted CH4 production was inhibited by 48.4 and 40.8% in KJ2_L and KJ2_H, respectively, compared with CON. The proportion of propionate was higher in the KJ1 treatments than the CON treatment and showed further increases in the KJ2 treatments. The abundances of Megasphaera, Succiniclasticum, Selenomonas, and Ruminobacter, which are related to propionate production, were higher in KJ2_H than in CON. The results suggested that the rumen microbiome was modified by the addition of 0.5-1.0% DM KJ1 and KJ2, resulting in increased propionate and reduced CH4 production. In particular, the KJ2 treatments inhibited ruminal CH4 production more than the KJ1 treatments. These findings provide important information for inhibiting ruminal CH4 emissions, which is essential for increasing animal productivity and sustaining livestock production under future population growth.

EFFECTS OF NITROGEN NUTRIENT ON THE PRODUCTION OF MYCOSPORINE-LIKE AMINO ACIDS IN NOSTOC SPECIES

Mycosporine-like amino acids MAAs belong to a family of secondary metabolites produced by a wide range of different organisms. These compounds had several potential applications in biomedical, in cosmetics and toiletries. In the present study, three Nostoc strains distributed in soil and freshwater were tested for MAA production under different nitrogen conditions. UV-B radiation significantly enhanced the production of MAAs in Nostoc linckia, Nostoc muscorum and Nostoc paludosum under nitrogen sufficient and deficient conditions. The contents of MAAs significantly increased in these three Nostoc strains with prolonged UV-B treatment under both nitrogen conditions. Both UV-B exposed N. muscorum and N. paludosum produced much less MAAs under nitrogen deficiency in comparison to culture under nitrogen sufficiency, respectively. There was just little difference of MAA contents in UV-B treated N. linckia when it was cultured under both nitrogen conditions. After separation by HPLC and characterization by in-line absorption spectra and mass spectra, one type of MAA porphyra was induced by UV-B in N. linckia under nitrogen sufficient and deficient conditions, and N. muscorum synthesized another kind of MAA shinorine under nitrogen sufficient and deficient conditions. However, UV-B induced N. paludosum to produce porphyra under nitrogen sufficient condition but shinorine under nitrogen deficient condition. These results indicated the nutrient nitrogen levels could regulate the types and amounts of MAAs in Nostoc species. These strains would be a better source for the industrial production of MAAs that can be used as organisms for studying the biosynthetic route of MAAs in nitrogen conditions.

Identification and characterization of <italic>Dunaliella salina</italic> OH214 strain newly isolated from a saltpan in Korea

Carotenoids are effective antioxidants that are found in various photosynthetic organisms. Marine microalgae are an advantageous bioresource for carotenoid production because they do not compete with other crops for freshwater and arable land. This study reports a newly isolated Dunaliella strain from the Geumhong Saltpan on Yeongjong Island, West Sea, Korea. The new strain was isolated and classified as Dunaliella salina through phylogenetic analysis and was named the OH214 strain (Deposit ID: KCTC14434BP). The newly isolated strain can survive in a wide range of NaCl concentrations (0.3−5.0 M NaCl), but grows well in 0.6 to 1.5 M NaCl culture medium. Under high-light conditions (500 ± 10 μmol photons m<sup>-2</sup> s<sup>-1</sup>), the cells accumulated three times more β-carotene than under low-light conditions (50 ± 5 μmol photons m<sup>-2</sup> s<sup>-1</sup>). The cells accumulated 2.5-fold more β-carotene under nitrogen-deficient (1 mM KNO<sub>3</sub>) conditions (3.24 ± 0.36 μg 106 cells<sup>-1</sup>) than in nitrogen-sufficient conditions (>5 mM KNO<sub>3</sub>). The lutein content under nitrogen-deficient conditions (1.73 ± 0.09 μg 106 cells<sup>-1</sup>) was more than 24% higher than that under nitrogen-sufficient conditions. Under the optimized culture condition for carotenoid induction using natural seawater, D. salina OH214 strain produced 7.97 ± 0.09 mg g DCW<sup>-1</sup> of β-carotene and 4.65 ± 0.18 mg g DCW<sup>-1</sup> of lutein, respectively. We propose that this new microalga is a promising strain for the simultaneous production of β-carotene and lutein.

Imidacloprid Disturbs the Nitrogen Metabolism and Triggers an Overall Stress Response in Maize Seedlings

Imidacloprid (IMI) protects crops from pests; however, its potential toxicity to plants and underlying mechanisms are still poorly understood. We investigated the effects of IMI on maize seedlings under different nitrogen sufficiency conditions. Our measurement of the maize seedlings’ growth traits and physiological indicators found that a 5 ppm IMI treatment stunted the maize’s growth and enhanced membrane lipid peroxidation under a nitrogen-supplied condition, but that it promoted an increase in biomass and alleviated chlorosis in maize shoots under nitrogen deficiency. These results suggest that IMI causes serious toxicity in maize seedlings under nitrogen-sufficient conditions. The content of IMI indicated that the leaf was the main site of IMI accumulation in maize, and that NO3− was beneficial for the transportation of IMI from the roots to the leaves. The three groups of seedlings, which received 0 (−N), 4 (N) or 10 mmol L−1 NO3− (NN), were either treated or not treated with 5 ppm IMI. The six sets of transcriptome profiles from the shoots and roots were compared using Illumina sequencing. Transcriptome analysis revealed that IMI treatment led to changes in the expression of the genes involved in multiple biological processes, including nitrate transporter, nitrogen assimilation, nitrogen-regulatory factors, detoxification-related genes and several antioxidant-related genes in maize roots. The above results and the data for the nitrate content, glutamine synthetase activities and nitrate reductase activities showed that IMI disturbed nitrogen absorption and metabolism in maize seedlings. Glutathione S-transferase genes, C-type ATP-binding cassette (ABC) transporter 4, anthocyanins and lignin may play an important role in the detoxification of IMI in maize. These findings have helped us to elaborate the underlying detoxification mechanisms of IMI in plants, which is highly important in the cultivation of anti-pesticide crop varieties.

The growth and lipid accumulation of Scenedesmus quadricauda under nitrogen starvation stress during xylose mixotrophic/heterotrophic cultivation.

In order to conquer the block of high cost and low yields which limit to realize the commercialization of microalgal biodiesel, the mixotrophic and heterotrophic cultivation of Scenedesmus quadricauda FACHB-1297 fed on xylose was separately studied employing six forms of media: phosphorus sufficient, phosphorus restricted, and phosphorus starvation were combined with nitrogen sufficient and nitrogen starvation conditions. The maximum lipid content (about 41% of dry weight) was obtained on the 5th day (heterotrophic cultivation) and 8th day (mixotrophic cultivation) under the nitrogen starved and phosphorus sufficient (N0&P) conditions, which was about twofold in comparison to the final lipid content on the sufficient nitrogen condition (control). Under mixotrophic and heterotrophic modes, the highest lipid production was achieved in the N0&P trial, with the value of 274.96mg/L and 193.77mg/L, respectively. Xylose utilization rate of 30-96% under heterotrophic modes was apparently higher than that of 20-50% in mixotrophic modes. In contrast, phosphorus uptake rate of 100% under mixotrophic cultivation was significantly more than that of 60-90% in heterotrophic cultivation. Furthermore, under the condition of heterotrophic cultivation using xylose as a carbon source, the phosphorus had a positive impact on microalgae cell synthesis and the lipid content enhanced with the augmentation in phosphorus concentrations. We suggested that sufficient phosphorus should be supplied for obtaining higher microalgal lipid production in the lack of nitrogen under xylose heterotrophic/mixotrophic condition. This was a highly effective way to obtain efficient microalgae lipid production.

Alanine synthesized by alanine dehydrogenase enables ammonium-tolerant nitrogen fixation in Paenibacillus sabinae T27

Most diazotrophs fix nitrogen only under nitrogen-limiting conditions, for example, in the presence of relatively low concentrations of NH4+ (0 to 2 mM). However, Paenibacillus sabinae T27 exhibits an unusual pattern of nitrogen regulation of nitrogen fixation, since although nitrogenase activities are high under nitrogen-limiting conditions (0 to 3mM NH4+) and are repressed under conditions of nitrogen sufficiency (4to 30mM NH4+), nitrogenase activity is reestablished when very high levels of NH4+ (30 to 300 mM) are present in the medium. To further understand this pattern of nitrogen fixation regulation, we carried out transcriptome analyses of P. sabinae T27 in response to increasing ammonium concentrations. As anticipated, the nif genes were highly expressed, either in the absence of fixed nitrogen or in the presence of a high concentration of NH4+ (100 mM), but were subject to negative feedback regulation at an intermediate concentration of NH4+ (10 mM). Among the differentially expressed genes, ald1, encoding alanine dehydrogenase (ADH1), was highly expressed in the presence of a high level of NH4+ (100 mM). Mutation and complementation experiments revealed that ald1 is required for nitrogen fixation at high ammonium concentrations. We demonstrate that alanine, synthesized by ADH1 from pyruvate and NH4+, inhibits GS activity, leading to a low intracellular glutamine concentration that prevents feedback inhibition of GS and mimics nitrogen limitation, enabling activation of nif transcription by the nitrogen-responsive regulator GlnR in the presence of high levels of extracellular ammonium.

Invivo detection of triacylglycerols through Nile red staining and quantification of fatty acids in hyper lipid producer Nannochloropsis sp. cultured under adequate nitrogen and deficient nitrogen condition

The green alga Nannochloropsis sp. was grown in a medium containing adequate (1.5 g/L sodium nitrate) and absence of nitrogen (0 g/L sodium nitrate) for improving the triacylglycerols (TAGs) level and fatty acid content for sustainable biodiesel production. Initially, the biomass yield of Nannochloropsis sp. was determined from the day of inoculation till 18th day and it is observed that on 14th day higher biomass yield at about 1.4 g/L was obtained. The Nile red stained cells from nitrogen sufficient and nitrogen starvation conditions observed under confocal microscopy revealed that neutral lipids exist as oil bodies were higher in nitrogen starved condition and also, the TAG content was 13.31% dryweight in nitrogen starved cells than nitrogen amended (10.46%) Nannochloropsis cells. The extracted lipids subjected for FTIR study showed the presence of TAG functional group in both condition and similarly, the FAME obtained from both condition showed the vibration at 1743 cm−1 for ester functional group. The fatty acid composition of nitrogen deplete condition revealed oleic acid methyl ester (28.7%), Behenic acid methyl ester (14.2%), cis-11, 14, 17-Eicosatrienoic acid methyl ester (8.8%) as predominant fatty acids than nitrogen supplied condition. The TAG content and FAME composition reported in this study makes Nannochloropsis sp. as plausible candidate for pilot scale biodiesel production.

Selective Feeding of a Mixotrophic Dinoflagellate (Lepidodinium sp.) in Response to Experimental Warming and Inorganic Nutrient Imbalance.

Mixotrophic protists are widely observed in the aquatic ecosystems, while how they respond to inorganic nutrient imbalance and ocean warming remains understudied. We conducted a series of experiments on a mixotrophic dinoflagellate Lepidodinium sp. isolated from subtropical coastal waters to investigate the combined effect of temperature and medium nitrate to phosphate ratio (N:P ratio) on the ingestion activities of mixotrophic protists. We found Lepidodinium sp. displayed selective feeding behaviour with a higher ingestion rate on high-N prey (N-rich Rhodomonas salina) when the ambient inorganic N:P ratio was equal to or below the Redfield ratio. The Chesson selectivity index α increased with increasing temperature, suggesting that warming exacerbated the selective feeding of Lepidodinium sp. Under inorganic nitrogen sufficient conditions (N:P ratio = 64), no selective feeding was observed at 25 and 28°C, while it occurs at 31°C, which also indicates that warming alters the feeding behaviour of Lepidodinium sp. In addition, our results revealed that the total ingestion rate of Lepidodinium sp. under the condition with normal inorganic nutrients (Redfield ratio) was significantly lower than that under nutrient-imbalanced conditions, which indicates that Lepidodinium sp. developed compensatory feeding to balance their cellular stoichiometry and satisfy their growth. Our study is the first attempt on revealing the selective feeding behaviours of mixotrophic protists on prey under different inorganic nutrient environments and rising temperatures, which will contribute to our understanding of the response of marine plankton food web to projected climate changes.

Membrane fouling in a microalgal-bacterial membrane photobioreactor: Effects of P-availability controlled by N:P ratio

Microalgal-bacterial membrane photobioreactor (MB-MPBR) is a promising technology to simultaneously remove organics and nutrients from wastewater. However, membrane fouling in MB-MPBR was seldom studied. In this study, potential effects of P-availability on biomass properties and membrane fouling in MB-MPBR were investigated. Under a nitrogen sufficient condition, a lower N:P ratio of 3.9:1 (P-rich) caused more severe membrane fouling. The dominant fouling mechanism was cake layer formation. Serial characterization showed a smaller particle size distribution (PSD), more free microalgae and significantly different surface composition of microalgal-bacterial flocs at N:P ratio of 3.9:1 compared with that of 9.7:1. The variations on PSD and surface composition were fully consistent with that of filtration resistance and thus considered as the primary contributors to the different fouling performance. The above results suggested that controlling microalgae/bacteria consortium in a good ratio by optimizing operating conditions is the key event for membrane fouling control in MB-MPBRs.

Inactivation of phosphate regulator (SphU) in cyanobacterium Synechocystis sp. 6803 directly induced acetyl phosphate pathway leading to enhanced PHB level under nitrogen-sufficient condition

Poly-β-hydroxybutyrate (PHB) is a biodegradable and biocompatible polymer that has potential in the fields of environmental, agricultural, and biomedical sciences. Cyanobacteria are considered an excellent source of PHB by bioconversion of CO2. This study aimed to prolong PHB production under nitrogen-sufficient condition in the model cyanobacterium Synechocystis sp. PCC 6803. Interestingly, the lack of phosphate regulator (SphU) enabled the mutant strain (ΔSphU) to have the ability to accumulate phosphate with higher expression of Pho regulon. When strain ΔSphU was cultured in nitrogen complete medium for 14 days, the PHB granules were more extensively accumulated in the ΔSphU strain than in the wild type. Photosynthesis activity slightly increased in ΔSphU strain, with no significant difference in chlorophyll a content between wild-type and ΔSphU strain in nitrogen-containing medium, indicating that the higher PHB content (14.57% (w/w) cell dry weight) was not influent of chlorosis. The RT-qPCR analysis revealed that genes involved in PHB biosynthesis and acetyl phosphate pathway were more upregulated in ΔSphU strain. Moreover, the level of acetate production in ΔSphU cells was higher than that in the wild type, suggesting that the deletion of the phosphate regulator could directly induce PHB metabolism by activation of the acetyl phosphate pathway. This research provides better understanding of PHB production regulation in cyanobacteria which are a promising hosts for industrial production of biodegradable plastics.

Recycled nutrients supply phosphorus and improve ryegrass yields on phosphorus-depleted soil

Recycling phosphorus (P) within the food system is fundamental to long-term sustainability. This greenhouse study compared three sources of recycled P — struvite precipitated from municipal wastewater, black soldier fly frass from food waste, and anaerobic digestate of food waste — to mono-ammonium phosphate (MAP), compost, and a control. Italian ryegrass (Lolium multiflorum) was harvested four times during a 123 d trial from P-depleted soil. In nitrogen (N) sufficient conditions, all amendments significantly increased cumulative ryegrass yields compared with the control and were not significantly different from MAP. Relative P supply was frass = MAP > struvite ≥compost ≥ digestate >> control. The recycled nutrient sources tested show promise as sustainable P sources.

Investigation of four microalgae in nitrogen deficient synthetic wastewater for biorefinery based biofuel production

Microalgae are a possible source of biofuel, but no commercial development has yet been developed. Possible approaches to improve economic feasibility involve increasing lipid content and the usage of biorefinery in microalgae. In this regard, the nitrogen-deficient-synthetic wastewater was fed to four microalgae to increase the lipid and carbohydrate content. In conjunction, changes in the amount of biorefinery components, including proteins and pigments, during nitrogen synthetic wastewater feeding have also been reported. The study revealed that both lipids and carbohydrates increased as much as 2 and 1.5 times in selected microalgae after 20 days of nitrogen-deficient-synthetic wastewater relative to nitrogen sufficient conditions, respectively. In particular, after 20 days of exposure to nitrogen deficient synthetic wastewater, the highest Lipid content of 40% was displayed by Desmodesmus sp . The same species had also shown an increased carbohydrate content of 18.3%. Carotenoids was retained in Desmodemsus sp . (0.03%) through nitrogen deficiency, but were shown to be reduced in three other algae. Analysis of the FAME profile of four nitrogen-deficient microalgae indicated that lipid was promising for biodiesel production as it showed an increase in saturated fatty acid content and a decrease in unsaturated fatty acid content. However, during nitrogen insufficiency, unavoidable side reactions involving reductions in protein levels, chlorophylls (a and b type) and biomass were observed in all four microalgae. But 23.3% protein was found in Desmodesmus sp . at end of 20 th day of nitrogen deprivation The study showed that feeding of nitrogen-deficient-synthetic wastewater raised the lipid and carbohydrate content and maintained carotenoids in selected microalgae. Above said approach is possibly a low cost economical option for biorefinery. • Lipids improved 2 fold in nitrogen deficient wastewater than control. • Carbohydrates improved 1.5 fold in nitrogen deficient wastewater than control. • Carotenoids retained in Desmodemsus sp. in nitrogen deficient wastewater. • High chlorophyll a to b ratio in N 2 lacking wastewater ensures ATP for lipid synthesis.

Biological stoichiometry and growth dynamics of a diazotrophic cyanobacteria in nitrogen sufficient and deficient conditions

The role of nitrogen (N) fixation in determining the frequency, magnitude, and extent of harmful algal blooms (HABs) has not been well studied. Dolichospermum is a common HAB species that is diazotrophic (capable of N fixation) and thus growth is often considered never to be limited by low combined N sources. However, N fixation is energetically expensive and its cost during bloom formation has not been quantified. Additionally, it is unknown how acclimation to differing nutrient ratios affects growth and cellular carbon (C):N stoichiometry. Here, we test the hypotheses that diazotrophic cyanobacteria are homeostatic for N because of their ability to fix atmospheric N2 and that previous acclimation to low N environments will result in more fixed N and lower C:N stoichiometry. Briefly, cultures that varied in resource N:phosphorus (P) ranging from 0.01 to 100 (atom), were seeded with Dolichospermum which were previously acclimated to low and high N:P conditions and then sampled temporally for growth and C:N stoichiometry. We found that Dolichospermum was not homeostatic for N and displayed classic signs of N limitation and elevated C:N stoichiometry, highlighting the necessary growth trade-off within cells when expending energy to fix N. Acclimation to N limited conditions caused differences in both C:N and fixed N at various time points in the experiment. These results highlight the importance of environmentally available N to a diazotrophic bloom, as well as how previous growth conditions can influence population growth during blooms experiencing variable N:P.

The sediment akinete bank links past and future blooms of Nostocales in a shallow lake

Abstract In this paper we analyzed how well the akinete bank in the sediments from a shallow lake reflected past blooms of planktonic Nostocales (cyanobacteria), the akinete bank’s potential for reflecting future blooms and whether different nutrient scenarios affect germination and recruitment using a laboratory experiment. Most species found in the plankton were also found in the akinete bank and in the germination experiment. Based on their shape, the most abundant viable akinetes in the sediments potentially corresponded to some of the species forming intense blooms in previous years, and coincided with the most abundant species in a 3-week germination experiment. The effects of nutrients on germination and recruitment were only observed in the early phase of the life cycle (Day 7), where nitrogen-sufficient conditions enhanced germination, while phosphorus concentration had a positive effect on the number of heterocytes. Combined approaches of morphological analysis and experimental germination seem a good method for assessing the past diversity of Nostocales and allowing predictions of the diversity of potential future blooms in shallow lakes and as a tool for water quality management, especially in water bodies lacking information on previous cyanobacteria blooms.

Intercellular and intracellular colonization of Azorhizobium caulinodans in Arabidopsis thaliana under nitrogen deficient conditions

ABSTRACT Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata. This bacterium induces nodules on the stems and roots of host plants, and fixes nitrogen in the host cells within such nodules. It is also an endophytic bacterium of some non-leguminous plants, such as Arabidopsis thaliana, rice, wheat, and tomato. It is known that this bacterium colonizes in the intercellular spaces and xylems of the roots of A. thaliana plants, but these observations were not conducted under completely nitrogen-free conditions. In this study, in which A. thaliana plants were inoculated with A. caulinodans on nitrogen-free medium, it was found that A. caulinodans promoted the growth of the A. thaliana plants, however, the promotion was not caused by nitrogen fixation. Detailed observations revealed that A. caulinodans intercellularly and intracellularly infected not only the roots but also almost the whole bodies of the A. thaliana plants, except for the stems. In the leaves, infection was frequently observed not only in the stomata and intercellular spaces of the mesophyll cells, but also in the guard cells and mesophyll cells. Interestingly, chloroplasts were also infected with A. caulinodans. In the flowers, A. caulinodans was detected in the pollen cells, in the stigma and the center of the style, and in the space around the ovule in the ovary. This suggests that A. caulinodans colonizes the inside of ovaries via pollen tubes. Taken together, it can be said that this bacterium is more likely to infect A. thaliana plants in nitrogen deficient conditions than in nitrogen sufficient conditions. The findings herein are thought to be the basis for the use of A. caulinodans inoculation for the enhancement of plant growth in oligotrophic conditions.

Sexual reproduction of the snow alga Chloromonas fukushimae (Volvocales, Chlorophyceae) induced using cultured materials.

Snow algae are microalgae, growing in melting snowpacks, and are thought to act as primary producers in the snow ecosystem. Chloromonas (Volvocales, Chlorophyceae) contains more than 15 snow-inhabiting species. Although vegetative cells and zygotes, or asexual cysts, of snow species of the genus are frequently collected in the field, sexual reproduction and zygote formation in culture have only been induced in C. tughillensis. Here we describe the sexual reproduction of another snow-inhabiting species, C. fukushimae, which was induced using both previously examined and newly established Japanese strains. Mating of isogamous gametes began after mixing two different strains, implying that C. fukushimae is an outcrossing species. Motile and nonmotile zygotes of the species were also described in this report. The nonmotile zygote of C. fukushimae was distinguishable from those of the other snow-inhabiting species of Chloromonas, based on the zygote shape and the presence of several large lipid bodies within the cell. In addition, C. fukushimae carried out sexual reproduction and produced zygotes even under the nitrogen-sufficient condition.

Early Diagnosis and Management of Nitrogen Deficiency in Plants Utilizing Raman Spectroscopy.

Nutrient deficiency alters growth and development of crop plants and compromises yield. Real-time non-invasive monitoring of the nutritional status of crops would allow timely applications of fertilizers to optimize for growth and yield at different times of the plant’s life cycle. Here, we used Raman spectroscopy to characterize Arabidopsis and two varieties of leafy vegetable crops under nitrogen sufficient and deficient conditions. We showed that the 1046 cm–1 Raman peak serves as a specific signature of nitrogen status in planta, which can be used for early diagnosis of nitrogen deficiency in plants before onset of any visible symptoms. Our research can be applied toward crop management for sustainable and precision agriculture.

Engineering the transcriptional activator NifA for the construction of Rhodobacter sphaeroides strains that produce hydrogen gas constitutively.

Purple non-sulfur photosynthetic bacteria such as Rhodobacter sphaeroides and Rhodopseudomonas palustris produce hydrogen gas (H2) via proton reduction, which is catalyzed by nitrogenase. Although the expression of nitrogenase is usually repressed under nitrogen-sufficient conditions, a partial deletion of nifA, which encodes a transcriptional activator of nitrogen-fixation genes, has been reported to enable the constitutive expression of nitrogenase in R. palustris. In this study, we evaluated the effects of a similar mutation (nifA* mutation) on H2 production during the photoheterotrophic growth of R. sphaeroides, based on the notion that H2 production by nitrogenase compensates for the loss of CO2 fixation via the Calvin cycle, thereby restoring the redox balance. The chromosomal nifA* mutation resulted in the slight restoration of the photoheterotrophic growth of a mutant strain lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the key enzyme of the Calvin cycle, when the strain was cultured in van Niel's yeast medium. In addition, the strain with the nifA* mutation produced detectable levels of H2 during photoheterotrophic growth with acetate and ammonium; however, the H2 production was considerably lower than that observed during the photoheterotrophic growth of the strain with acetate and L-glutamate, where L-glutamate serves as a poor nitrogen source, thereby causing nitrogenase derepression. On the other hand, introduction of a multicopy plasmid harboring nifA* markedly restored the photoheterotrophic growth of the RubisCO-deletion mutant in van Niel's yeast medium and resulted in efficient H2 production during the photoheterotrophic growth with acetate and ammonium.