Nitrogen Starvation Conditions Research Articles

Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole.

Niemann-Pick type C is a storage disease caused by dysfunction of NPC proteins, which transport cholesterol from the lumen of lysosomes to the limiting membrane of that compartment. Using freeze fracture electron microscopy, we show here that the yeast NPC orthologs, Ncr1p and Npc2p, are essential for formation and expansion of raft-like domains in the vacuolar (lysosome) membrane, both in stationary phase and in acute nitrogen starvation. Moreover, the expanded raft-like domains engulf lipid droplets by a microautophagic mechanism. We also found that the multivesicular body pathway plays a crucial role in microautophagy in acute nitrogen starvation by delivering sterol to the vacuole. These data show that NPC proteins promote microautophagy in stationary phase and under nitrogen starvation conditions, likely by increasing sterol in the limiting membrane of the vacuole.

Improving carbohydrate and starch accumulation in Chlorella sp. AE10 by a novel two-stage process with cell dilution

BackgroundMicroalgae are highly efficient cellular factories that capture CO2 and are also alternative feedstock for biofuel production. Carbohydrates, proteins, and lipids are major biochemical components in microalgae. Carbohydrates or starch in microalgae are possible substrates in yeast fermentation for biofuel production. The carbon partitioning in microalgae could be regulated through environmental stresses, such as high concentration of CO2, high light intensity, and nitrogen starvation conditions. It is essential to obtain carbohydrate-rich microalgae via an optimal bioprocess strategy.ResultsThe carbohydrate accumulation in a CO2 tolerance strain, Chlorella sp. AE10, was investigated with a two-stage process. The CO2 concentration, light intensity, and initial nitrogen concentration were changed drastically in both stages. During the first stage, it was cultivated over 3 days under 1% CO2, a photon flux of 100 μmol m−2 s−1, and 1.5 g L−1 NaNO3. It was cultivated under 10% CO2, 1000 μmol m−2 s−1, and 0.375 g L−1 NaNO3 during the second stage. In addition, two operation modes were compared. At the beginning of the second stage of mode 2, cells were diluted to 0.1 g L−1 and there was no cell dilution in mode 1. The total carbohydrate productivity of mode 2 was increased about 42% compared with that of mode 1. The highest total carbohydrate content and the highest starch content of mode 2 were 77.6% (DW) and 60.3% (DW) at day 5, respectively. The starch productivity was 0.311 g L−1 day−1 and the total carbohydrate productivity was 0.421 g L−1 day−1 in 6 days.ConclusionsIn this study, a novel two-stage process was proposed for improving carbohydrate and starch accumulation in Chlorella sp. AE10. Despite cell dilution at the beginning of the second stage, environmental stress conditions of high concentration of CO2, high light intensity, and limited nitrogen concentration at the second stage were critical for carbohydrate and starch accumulation. Although the cells were diluted, the growths were not inhibited and the carbohydrate productivity was improved. These results were helpful to establish an integrated approach from CO2 capture to biofuel production by microalgae.

Proteome analysis reveals differential expression of proteins involved in triacylglycerol accumulation by Rhodococcus jostii RHA1 after addition of methyl viologen.

Rhodococcus jostii RHA1 is able to degrade toxic compounds and accumulate high amounts of triacylglycerols (TAG) upon nitrogen starvation. These NADPH-dependent processes are essential for the adaptation of rhodococci to fluctuating environmental conditions. In this study, we used an MS-based, label-free and quantitative proteomic approach to better understand the integral response of R. jostii RHA1 to the presence of methyl viologen (MV) in relation to the synthesis and accumulation of TAG. The addition of MV promoted a decrease of TAG accumulation in comparison to cells cultivated under nitrogen-limiting conditions in the absence of this pro-oxidant. Proteomic analyses revealed that the abundance of key proteins of fatty acid biosynthesis, the Kennedy pathway, glyceroneogenesis and methylmalonyl-CoA pathway, among others, decreased in the presence of MV. In contrast, some proteins involved in lipolysis and β-oxidation of fatty acids were upregulated. Some metabolic pathways linked to the synthesis of NADPH remained activated during oxidative stress as well as under nitrogen starvation conditions. Additionally, exposure to MV resulted in the activation of complete antioxidant machinery comprising superoxide dismutases, catalases, mycothiol biosynthesis, mycothione reductase and alkyl hydroperoxide reductases, among others. Our study suggests that oxidative stress response affects TAG accumulation under nitrogen-limiting conditions through programmed molecular mechanisms when both stresses occur simultaneously.

Similarities in the structure of the transcriptional repressor AmtR in two different space groups suggest a model for the interaction with GlnK.

AmtR belongs to the TetR family of transcription regulators and is a global nitrogen regulator that is induced under nitrogen-starvation conditions in Corynebacterium glutamicum. AmtR regulates the expression of transporters and enzymes for the assimilation of ammonium and alternative nitrogen sources, for example urea, amino acids etc. The recognition of operator DNA by homodimeric AmtR is not regulated by small-molecule effectors as in other TetR-family members but by a trimeric adenylylated PII-type signal transduction protein named GlnK. The crystal structure of ligand-free AmtR (AmtRorth) has been solved at a resolution of 2.1 Å in space group P21212. Comparison of its quaternary assembly with the previously solved native AmtR structure (PDB entry 5dy1) in a trigonal crystal system (AmtRtri) not only shows how a solvent-content reduction triggers a space-group switch but also suggests a model for how dimeric AmtR might stoichiometrically interact with trimeric adenylylated GlnK.

Major Lipid Body Protein: A Conserved Structural Component of Lipid Body Accumulated during Abiotic Stress in S. quadricauda CASA-CC202

Abiotic stress in oleaginous microalgae enhances lipid accumulation and is stored in a specialised organelle called lipid droplets (LDs). Both the LDs and body are enriched with major lipid droplet protein (MLDP). It serves as a major structural component and also plays a key role in recruiting other proteins and enzymes involved in lipid body maturation. In the present study, the presence of MLDP was detected in two abiotic stress condition namely nitrogen starvation and salt stress condition. Previous research reveals that nitrogen starvation enhances lipid accumulation. Therefore, the effect of salt on growth, biomass yield, and fatty acid profile is studied in detail. The specific growth rate of S. quadricauda under the salt stress of 10mM concentration is about 0.174μ and in control, the SGR is 0.241μ. An increase in the doubling time of the cells shows that the rate of cell division decreases during salt stress (2.87–5.17). The dry biomass content also decreased drastically at 50mM salt-treated cells (129mg/L) compared to control (236mg/L) on the day 20. The analysis of fatty acid composition also revealed that there is a 20% decrease in the saturated fatty acid level and 19.9% increment in monounsaturated fatty acid level, which makes salt-mediated lipid accumulation as a suitable biodiesel precursor.

Combinatorial deletions of glgC and phaCE enhance ethanol production in Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production. In the present study, a nitrogen starvation approach was applied on an ethanol producing strain for inhibiting the growth, since ethanol production competes with the cell growth. The effect of gene deletions in the glycogen and polyhydroxybutyrate (PHB) synthesis pathways was investigated. Measurements of intracellular glycogen and PHB revealed that the glycogen was accumulated under the nitrogen starvation condition and the gene deletion of glycogen synthesis pathway caused the accumulation of PHB. The ethanol producing strain harboring deletions for both the glycogen and the PHB synthesis pathways (ΔglgCΔphaCE/EtOH) produced ethanol at the specific rate of 240mgg (dry cell weight)−1 day−1 under the nitrogen starvation condition. In a high cell density culture (OD730=50) using this ΔglgCΔphaCE/EtOH strain, the ethanol production rates were 1.08 and 2.01gL−1 day−1 under light conditions of 40 and 80μmolm−2s−1, respectively.

Toward an understanding of lipid and starch accumulation in microalgae: A proteomic study of Neochloris oleoabundans cultivated under N-limited heterotrophic conditions

Neochloris oleoabundans is an oleaginous microalga of biotechnological and commercial interest. A proteomic study was performed to compare the abundance of proteins under two different culture conditions: preferential lipid accumulation and preferential carbohydrate accumulation.When N. oleoabundans was cultivated under long periods of nitrogen starvation, the acyl carrier protein and the protein biotin carboxylase from the lipid synthesis pathway were found to be upregulated. The central metabolism pathways were also found to be highly activated to redirect the carbon flow toward pyruvate dehydrogenase and ATP synthesis. Pyruvate dehydrogenase was upregulated to supply the precursors for lipid production. Furthermore, in the pentose phosphate pathway, specifically glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were upregulated to supply reducing power in the form of NADPH for lipid synthesis and inorganic nitrogen assimilation.Carbohydrate synthesis-related enzymes that channel carbon to starch and sugar synthesis, such as UDP-glucose pyrophosphorylase and starch synthase, were upregulated when short durations of nitrogen limitation were applied during N. oleoabundans cultivation. However, ADP-glucose pyrophosphorylase was upregulated under preferential lipid accumulation conditions, indicating that under prolonged nitrogen starvation conditions, this enzyme potentially hydrolyzes starch chains to channel the carbon flow to lipid synthesis, which indicates a dual function of this protein.

Genome-Scale Metabolic Model for the Green Alga Chlorella vulgaris UTEX 395 Accurately Predicts Phenotypes under Autotrophic, Heterotrophic, and Mixotrophic Growth Conditions.

The green microalga Chlorella vulgaris has been widely recognized as a promising candidate for biofuel production due to its ability to store high lipid content and its natural metabolic versatility. Compartmentalized genome-scale metabolic models constructed from genome sequences enable quantitative insight into the transport and metabolism of compounds within a target organism. These metabolic models have long been utilized to generate optimized design strategies for an improved production process. Here, we describe the reconstruction, validation, and application of a genome-scale metabolic model for C. vulgaris UTEX 395, iCZ843. The reconstruction represents the most comprehensive model for any eukaryotic photosynthetic organism to date, based on the genome size and number of genes in the reconstruction. The highly curated model accurately predicts phenotypes under photoautotrophic, heterotrophic, and mixotrophic conditions. The model was validated against experimental data and lays the foundation for model-driven strain design and medium alteration to improve yield. Calculated flux distributions under different trophic conditions show that a number of key pathways are affected by nitrogen starvation conditions, including central carbon metabolism and amino acid, nucleotide, and pigment biosynthetic pathways. Furthermore, model prediction of growth rates under various medium compositions and subsequent experimental validation showed an increased growth rate with the addition of tryptophan and methionine.

The autophagy-related gene BcATG1 is involved in fungal development and pathogenesis in Botrytis cinerea.

Autophagy, a ubiquitous intracellular degradation process, is conserved from yeasts to humans. It serves as a major survival function during nutrient depletion stress and is crucial for correct growth and differentiation. In this study, we characterized an atg1 orthologue Bcatg1 in the necrotrophic plant pathogen Botrytis cinerea. Quantitative real-time polymerase chain reaction (qRT-PCR) assays showed that the expression of BcATG1 was up-regulated under carbon or nitrogen starvation conditions. BcATG1 could functionally restore the survival defects of the yeast ATG1 mutant during nitrogen starvation. Deletion of BcATG1 (ΔBcatg1) inhibited autophagosome accumulation in the vacuoles of nitrogen-starved cells. ΔBcatg1 was dramatically impaired in vegetative growth, conidiation and sclerotial formation. In addition, most conidia of ΔBcatg1 lost the capacity to form the appressorium infection structure and failed to penetrate onion epidermis. Pathogenicity assays showed that the virulence of ΔBcatg1 on different host plant tissues was drastically impaired, which was consistent with its inability to form an appressorium. Moreover, lipid droplet accumulation was significantly reduced in the conidia of ΔBcatg1, but the glycerol content was increased. All of the defects of ΔBcatg1 were complemented by re-introduction of an intact copy of the wild-type BcATG1 into the mutant. These results indicate that BcATG1 plays a critical role in numerous developmental processes and is essential to the pathogenesis of B. cinerea.

Iron-depletion promotes mitophagy to maintain mitochondrial integrity in pathogenic yeast Candida glabrata

ABSTRACTCandida glabrata, a haploid budding yeast, is the cause of severe systemic infections in immune-compromised hosts. The amount of free iron supplied to C. glabrata cells during systemic infections is severely limited by iron-chelating proteins such as transferrin. Thus, the iron-deficiency response in C. glabrata cells is thought to play important roles in their survival inside the host's body. In this study, we found that mitophagy was induced under iron-depleted conditions, and that the disruption of a gene homologous to ATG32, which is responsible for mitophagy in Saccharomyces cerevisiae, blocked mitophagy in C. glabrata. The mitophagic activity in C. glabrata cells was not detected on short-period exposure to nitrogen-starved conditions, which is a mitophagy-inducing condition used in S. cerevisiae. The mitophagy-deficient atg32Δ mutant of C. glabrata also exhibited decreased longevity under iron-deficient conditions. The mitochondrial membrane potential in Cgatg32Δ cells was significantly lower than that in wild-type cells under iron-depleted conditions. In a mouse model of disseminated infection, the Cgatg32Δ strain resulted in significantly decreased kidney and spleen fungal burdens compared with the wild-type strain. These results indicate that mitophagy in C. glabrata occurs in an iron-poor host tissue environment, and it may contribute to the longevity of cells, mitochondrial quality control, and pathogenesis.

Assessment of triacylglycerol content in Chlorella vulgaris cultivated in a two-stage process

Chlorella vulgaris cultivation in two-stage process was applied to increase the lipid productivity without compromising the biomass productivity. At the first stage, microalgae was cultivated under nutrient sufficient conditions to obtain a maximized cell density; at the second stage, nitrate conditions are changed to trigger the accumulation of TAG. During first stage, the maximum biomass productivity (32 mg L−1 d−1) was observed after 13 days under nutrient sufficient conditions with 1.21 g L−1 NaNO3 and 0.00449 g L−1 K2HPO4. Maximum lipid content (25.4% DW), lipid productivity (7.5 mg L−1 d−1) and TAG content (41.3% in total lipids) were favored by the nitrogen starvation conditions for more 4 days, at the second stage. Oil extracted at the second stage contained lower percentage of PUFAs being more suitable for the biodiesel production when compared with the oil extracted at the first stage. This two-stage phototrophic process is promising to provide a more efficient way for on a large-scale production of algal biomass and biodiesel production.

Bacterial cellulose production by Komagataeibacter hansenii using algae-based glucose.

Bacterial cellulose (BC) is a homopolymer and it is distinguished from plant-based cellulose by its unique properties such as high purity, high crystallinity, high water-holding capacity, and good biocompatibility. Microalgae are unicellular, photosynthetic microorganisms and are known to have high protein, starch, and oil content. In this study, Chlorella vulgaris was evaluated as source of glucose for the production of BC. To increase the starch content of algae the effect of nutrient starvation (nitrogen and sulfur) and light deficiency were tested in a batch assay. The starch contents (%) were 5.27±0.04, 7.14±0.18, 5.00±0.08, and 1.35±0.04 for normal cultivation, nitrogen starvation, sulfur starvation, and dark cultivation conditions, respectively. The performance of enzymatic and acidic methods was compared for the starch hydrolysis. This study demonstrated for the first time that acid hydrolysate of algal starch can be used to substitute glucose in the fermentation medium of Komagataeibacter hansenii for BC production. Glucose was used as a control for BC production. BC production yields on dry weight basis were 1.104±0.002g/L and 1.202±0.005g/L from algae-based glucose and glucose, respectively. The characterization of both BCs produced from glucose and algae-based glucose was investigated by scanning electron microscopy and Fourier transform infrared spectroscopy. The results have shown that the structural characteristics of algae-based BC were comparable to those of glucose-based BC.

Regulation of starch, lipids and amino acids upon nitrogen sensing in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii has recently emerged as a viable alternative source of fossil fuel. However, the metabolic flow of carbon in C. reinhardtii towards making the carbon reserve is not yet understood. In addressing this issue, we have monitored the assimilation of 13C-labeled acetate by the wild-type (cw15) and the starch deficient (sta6) strains of C. reinhardtii using 13C NMR. The dynamics of starch and lipid reserves was studied under nitrogen starvation and supplement conditions. The starch was found to accumulate and mobilize faster than triacylglycerol (TAG) through the formation of CO2aq and bicarbonate. Addition of exogenous acetate significantly slowed down mobilization of both labeled starch and TAG reserves, suggesting that cells preferred incoming acetate to internal reserve as a source of carbon. In parallel, we undertook the analyses of cellular free amino acid pool changes using LC/MS to investigate how specific amino acid changes contribute towards “nitrogen sparing effect” during nitrogen starvation. Bulk of remainder amino acids do not exhibit any discernible changes in their steady state pool sizes, suggesting the possibility that carbon flow leading to starch, followed by lipid is homeostatically balanced during nitrogen starvation such that the amino acid carbon levels in the cells stay largely regulated. Taken all together, this study thus describes an excellent cellular system for probing the nitrogen starvation, nitrogen uptake and the consequent changes in carbon flow in relation to both starch/lipid accumulation and their re-mobilization and the changes in free amino acid pools in C. reinhardtii cells.

Phospholipid: diacylglycerol acyltransferase contributes to the conversion of membrane lipids into triacylglycerol in Myrmecia incisa during the nitrogen starvation stress.

In addition to the Kennedy pathway for de novo biosynthesis, triacylglycerol (TAG), the most important stock for microalgae-based biodiesel production, can be synthesized by phospholipid: diacylglycerol acyltransferase (PDAT) that transfers an acyl group from phospholipids (PLs) to diacylglycerol (DAG). This study presents a novel gene that encodes PDAT from the green microalga Myrmecia incisa Reisigl H4301 (designated MiPDAT ). MiPDAT is localized on the plasma membrane (PM) via the agroinfiltration of tobacco leaves with a green fluorescent protein-fused construct. MiPDAT synthesizes TAG based on functional complementary experiments in the mutant yeast strain H1246 and the membrane lipid phosphatidylcholine (PC) is preferentially used as substrates as revealed by in vitro enzyme activity assay. The gradually increased transcription levels of MiPDAT in M. incisa during the cultivation under nitrogen starvation conditions is proposed to be responsible for the decrease and increase of the PC and TAG levels, respectively, as detected by liquid chromatography-mass spectrometry after 4 d of nitrogen starvation. In addition, the mechanism by which MiPDAT in this microalga uses PC to yield TAG is discussed. Accordingly, it is concluded that this PM-located PDAT contributes to the conversion of membrane lipids into TAG in M. incisa during the nitrogen starvation stress.

The metabolism of the non-proteinogenic amino acid β-N-methylamino-L-alanine (BMAA) in the cyanobacterium Synechocystis PCC6803

The neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) is produced by cyanobacteria under nitrogen starvation conditions and its metabolism is closely associated with cellular nitrogen control. Very little is known regarding the metabolism or biosynthesis of this amino acid in the producing organisms and current knowledge is limited to the spontaneous formation of carbamate adducts in the presence of aqueous carbon dioxide, the rapid removal of free cellular BMAA upon the addition of ammonia to nitrogen-starved cyanobacterial cultures, and the link between cellular nitrogen status and BMAA synthesis. Data presented here show that exogenous BMAA is readily metabolised by cyanobacteria during which, the primary amino group is rapidly transferred to other cellular amino acids. Furthermore, data suggest that BMAA is metabolised in cyanobacteria via a reversible transamination reaction. This study presents novel data on BMAA metabolism in cyanobacteria and provides the first proposed biosynthetic precursor to BMAA biosynthesis in cyanobacteria.

Medium factors on anaerobic production of rhamnolipids by Pseudomonas aeruginosa SG and a simplifying medium for in situ microbial enhanced oil recovery applications.

Aerobic production of rhamnolipid by Pseudomonas aeruginosa was extensively studied. But effect of medium composition on anaerobic production of rhamnolipid by P. aeruginosa was unknown. A simplifying medium facilitating anaerobic production of rhamnolipid is urgently needed for in situ microbial enhanced oil recovery (MEOR). Medium factors affecting anaerobic production of rhamnolipid were investigated using P. aeruginosa SG (Genbank accession number KJ995745). Medium composition for anaerobic production of rhamnolipid by P. aeruginosa is different from that for aerobic production of rhamnolipid. Both hydrophobic substrate and organic nitrogen inhibited rhamnolipid production under anaerobic conditions. Glycerol and nitrate were the best carbon and nitrogen source. The commonly used N limitation under aerobic conditions was not conducive to rhamnolipid production under anaerobic conditions because the initial cell growth demanded enough nitrate for anaerobic respiration. But rhamnolipid was also fast accumulated under nitrogen starvation conditions. Sufficient phosphate was needed for anaerobic production of rhamnolipid. SO4(2-) and Mg(2+) are required for anaerobic production of rhamnolipid. Results will contribute to isolation bacteria strains which can anaerobically produce rhamnolipid and medium optimization for anaerobic production of rhamnolipid. Based on medium optimization by response surface methodology and ions composition of reservoir formation water, a simplifying medium containing 70.3 g/l glycerol, 5.25 g/l NaNO3, 5.49 g/l KH2PO4, 6.9 g/l K2HPO4·3H2O and 0.40 g/l MgSO4 was designed. Using the simplifying medium, 630 mg/l of rhamnolipid was produced by SG, and the anaerobic culture emulsified crude oil to EI24 = 82.5 %. The simplifying medium was promising for in situ MEOR applications.

Influence of tryptophan and indole-3-acetic acid on starch accumulation in the synthetic mutualistic Chlorella sorokiniana–Azospirillum brasilense system under heterotrophic conditions

This study measured the relations between tryptophan production, the phytohormone indole-3-acetic acid (IAA) and the metabolism and accumulation of starch during synthetic mutualism between the microalgae Chlorella sorokiniana and the microalgae growth-promoting bacteria Azospirillum brasilense, created by co-immobilization in alginate beads. Experiments used two wild-type A. brasilense strains (Cd and Sp6) and an IAA-attenuated mutant (SpM7918) grown under nitrogen-replete and nitrogen-starved conditions tested under dark, heterotrophic and aerobic growth conditions. Under all incubating conditions, C. sorokiniana, but not A. brasilense, produced tryptophan. A significant correlation between IAA-production by A. brasilense and starch accumulation in C. sorokiniana was found, since the IAA-attenuated mutant was not producing increased starch levels. The highest ADP-glucose pyrophosphorylase (AGPase) activity, starch content and glucose uptake were found during the interaction of A. brasilense wild type strains with the microalgae. When the microalgae were grown alone, they produced only small amounts of starch. Supplementation with synthetic IAA to C. sorokiniana grown alone enhanced the above parameters, but only transiently. Activity of α-amylase decreased under nitrogen-replete conditions, but increased under nitrogen-starved conditions. In summary, this study demonstrated that, during synthetic mutualism, the exchange of tryptophan and IAA between the partners is a mechanism that governs several changes in starch metabolism of C. sorokiniana, yielding an increase in starch content.

Proteomic analysis of mycelial proteins from Magnaporthe oryzae under nitrogen starvation.

Magnaporthe oryzae is an important model system in studies of plant pathogenic fungi, and nitrogen is a key nutrient source affecting microbial growth and development. In order to understand how nitrogen stress causes changes in mycelial proteins, we analyzed differentially expressed mycelial proteins from the M. oryzae virulent strain CH-63 using two-dimensional electrophoresis and mass spectrometry in complete medium or under nitrogen starvation conditions. A total of 975 ± 70 and 1169 ± 90 protein spots were detected in complete medium and under nitrogen starvation conditions, respectively. Forty-nine protein spots exhibited at least 2-fold up-regulation or down-regulation at the protein level according to PDQuest7.4. Moreover, 43 protein spots were successfully identified by matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry. Among these spots, 6 proteins were functionally unknown and 37 proteins were categorized into 5 groups according to their functions, including development, metabolism, biosynthesis, and biological process. These 37 proteins were further analyzed for their enriched metabolic pathways by KOBAS2.0, and 14 proteins were found to be involved in glycolysis, tricarboxylic acid cycle, and nitrogen metabolism. Taken together, the regulation of M. oryzae growth under the nitrogen starvation conditions appears to be complex because of the various proteins and enzymes involved.

Autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast.

Autophagy is a conserved intracellular degradation system, which contributes to development and differentiation of various organisms. Yeast cells undergo meiosis under nitrogen-starved conditions and require autophagy for meiosis initiation. However, the precise roles of autophagy in meiosis remain unclear. Here, we show that autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast. Autophagy-defective strains bearing a mutation in the autophagy core factor gene atg1, atg7, or atg14 exhibit deformed nuclear structures during meiosis. These mutant cells require an extracellular nitrogen supply for meiosis progression following their entry into meiosis and show delayed meiosis progression even with a nitrogen supply. In addition, they show frequent chromosome dissociation from the spindle together with spindle overextension, forming extra nuclei. Furthermore, Aurora kinase, which regulates chromosome segregation and spindle elongation, is significantly increased at the centromere and spindle in the mutant cells. Aurora kinase down-regulation eliminated delayed initiation of meiosis I and II, chromosome dissociation, and spindle overextension, indicating that increased Aurora kinase activity may cause these aberrances in the mutant cells. Our findings show a hitherto unrecognized relationship of autophagy with the nuclear structure, regulation of cell cycle progression, and chromosome segregation in meiosis.

Overexpression of the autophagy-related gene SiATG8a from foxtail millet (Setaria italica L.) confers tolerance to both nitrogen starvation and drought stress in Arabidopsis

Autophagy is an evolutionarily conserved biological process in all eukaryotes for the degradation of intracellular components for nutrient recycling. Autophagy is known to be involved in responses to low nitrogen stress in Arabidopsis. Foxtail millet has strong abiotic stress resistance to both low nutrient and drought stress. However, to date, there have only been a few genes reported to be related with abiotic stress resistance in foxtail millet. In this study, we identified an autophagy-related gene, SiATG8a, from foxtail millet. SiATG8a is mainly expressed in stems and its expression was dramatically induced by drought stress and nitrogen starvation treatments. SiATG8a was localized in the membrane and cytoplasm of foxtail millet. Overexpression of SiATG8a in Arabidopsis conferred tolerance to both nitrogen starvation and to drought stress. Under nitrogen starvation conditions, the SiATG8a transgenic plants had larger root and leaf areas and accumulated more total nitrogen than wild-type plants. The transgenic plants had lower total protein concentrations than did the WT plants. Under drought stress, the SiATG8a transgenic plants had higher survival rates, chlorophyll content, and proline content, but had lower MDA content than wild type plants. Taken together, our results represent the first identified case where overexpression of autophagy related gene can simultaneously improve plant resistance to low nitrogen and drought stresses. These findings implicate plant autophagy in plant stress responses to low nitrogen and drought and should be helpful in efforts to improve stresses resistance to nitrogen starvation and drought of crops by genetic transformation.