Nitrogen Starvation Treatment Research Articles

Full-length transcriptome sequencing of Arabidopsis plants provided new insights into the autophagic regulation of photosynthesis

Autophagy is a highly conserved eukaryotic pathway and plays a crucial role in cell survival under stress conditions. Here, we applied a full-length transcriptome approach to study an Arabidopsis autophagy mutant (atg5-1) subjected to nitrogen-starvation, using Oxford Nanopore Technologies. A total of 39,033 transcripts were identified, including 11,356 new transcripts. In addition, alternative splicing (AS) events and lncRNAs were also detected between Col-0 (WT) and atg5-1. Differentially expressed transcript enrichment showed that autophagy upregulates the expression of many stress-responsive genes and inhibits the transcription of photosynthesis-associated genes. The qRT-PCR results showed that the expression patterns of photosynthesis-related genes in the atg5-1 differed under the conditions of nitrogen starvation and carbon starvation. Under nitrogen starvation treatment, many genes related to photosynthesis also exhibited AS. Chlorophyll fluorescence images revealed that the Fv/Fm and ΦPSII of old atg5-1 leaves were significantly reduced after nitrogen starvation treatment, but the Y(NPQ) indices were significantly increased compared to those of the WT plants. The results of qRT-PCR suggest that autophagy appears to be involved in the degradation of genes related to photodamage repair in PSII. Taken together, the full-length transcriptiome sequencing provide new insights into how new transcripts, lncRNAs and alternative splicing (AS) are involved in plant autophagy through full-length transcriptome sequencing and suggest a new potential link between autophagy and photosynthesis.

Determination of the Effects of Nitrogen and Sulfur Starvationon Chlorophyll Content, Cell Density and Lipid Content in Chlamydomonas reinhardtii

The severe environmental impacts of the use of fossil fuels has led to the development of biofuels as an alternative source of renewable green energy. Currently, algae is being studied as a source for biofuel because it does not compete for arable land and has a fast generation time. The microalgae, Chlamydomonas reinhardtii has potential for biofuel production due to its high lipid body synthesis when subjected to nutrient deprivation. The lipid bodies, predominantly triacylglycerides (TAGs) can be converted into biodiesel which could then be used as a form of biofuel. There is still a lack of research comparing the effects of nutrient starvation in strains of C. reinhardtii. The objectives of this study are: to determine the optimum time at which C. reinhardtii produces the highest amount of lipids under nutrient starvation and to compare the effects of nitrogen, sulfur and both nitrogen sulfur starvation on cell density, chlorophyll content and lipid content of strains cc125. We hypothesize that the nitrogen starvation treatment of C. reinhardtii strain cc125 will produce higher lipid synthesis and the sulfur starvation treatment will produce the highest biomass synthesis.C. reinhardtii strains cc125 were grown to early logarithmic phase in TAP medium liquid culture under constant light on a rotary shaker. Once logarithmic phase was reached, cells were subjected to nitrogen (TAP‐N), sulfur (TAP‐S) and combined nitrogen and sulfur (TAP‐NS) deprivation over a 6 day period to promote lipid accumulation. Samples for cell density, chlorophyll content, and confocal imaging were collected for each strain and nutrient treatment every 2 days. The change in chlorophyll a, chlorophyll b, total chlorophyll and cell density at day 2, 4 and 6 from day 0 were determined. Results showed that changes in chlorophyll a, chlorophyll b and total chlorophyll among all treatments were statistically not significantly different (p>0.05). Chlorophyll a (3.02 ± 2.31), chlorophyll b (1.68 ± 0.85) and total chlorophyll (4.66 ± 2.98) were all highest in treatment TAP‐N and was lowest in TAP‐NS, (chlorophyll a, 0.34 ± 2.30; chlorophyll b, 1.12 ± 0.86; total chlorophyll, 0.6 ± 2.99). There was no statistical difference in cell density among treatments (p>0.05). There was highest increase in cell density in TAP‐S treatment while highest decrease in cell density was observed in TAP‐NS treatment. In addition, there was no statistical difference among days for all treatments in chlorophyll a, chlorophyll b, total chlorophyll and cell density. Results for biomass determination, lipid extractions and quantification; and the qualitative detection of lipid bodies by confocal microscopy will be reported. C. reinhardtii strain sta6 will also be studied. Determining which strain of C. reinhardtii and at which condition will produce high TAGs is essential for engineering and mass producing algae as an established source of biofuel production. This can lead to the adaptation of a viable fuel production system that would resolve the ever growing demand for environmentally sustainable energy resources.Support or Funding InformationACT On IDEASCollege of Arts and Sciences

HY5-HDA9 Module Transcriptionally Regulates Plant Autophagy in Response to Light-to-Dark Conversion and Nitrogen Starvation

Light is arguably one of the most important environmental factors that determines virtually all aspects of plant growth and development, but the molecular link between light signaling and the autophagy pathway has not been elucidated in plants. In this study, we demonstrate that autophagy is activated during light-to-dark conversion though transcriptional upregulation of autophagy-related genes (ATGs). We showed that depletion of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to enhanced autophagy activity and resistance to extended darkness and nitrogen starvation treatments, contributing to higher expression of ATGs. HY5 interacts with and recruits HISTONE DEACETYLASE 9 (HDA9) to ATG5 and ATG8e loci to repress their expression by deacetylation of the Lys9 and Lys27 of histone 3. Furthermore, we found that both darkness and nitrogen depletion induce the degradation of HY5 via 26S proteasome and the concomitant disassociation of HDA9 from ATG5 and ATG8e loci, leading to their depression and thereby activated autophagy. Genetic analysis further confirmed that HY5 and HDA9 act synergistically and function upstream of the autophagy pathway. Collectively, our study unveils a previously unknown transcriptional and epigenetic network that regulates autophagy in response to light-to-dark conversion and nitrogen starvation in plants.

Carbon-dependent alleviation of ammonia toxicity for algae cultivation and associated mechanisms exploration

Ammonia toxicity in wastewater is one of the factors that limit the application of algae technology in wastewater treatment. This work explored the correlation between carbon sources and ammonia assimilation and applied a glucose-assisted nitrogen starvation method to alleviate ammonia toxicity. In this study, ammonia toxicity to Chlorella sp. was observed when NH3-N concentration reached 28.03mM in artificial wastewater. Addition of alpha-ketoglutarate in wastewater promoted ammonia assimilation, but low utilization efficiency and high cost of alpha-ketoglutarate limits its application in wastewater treatment. Comparison of three common carbon sources, glucose, citric acid, and sodium bicarbonate, indicates that in terms of ammonia assimilation, glucose is the best carbon source. Experimental results suggest that organic carbon with good ability of generating energy and hydride donor may be critical to ammonia assimilation. Nitrogen starvation treatment assisted by glucose increased ammonia removal efficiencies and algal viabilities.

Genome-wide analysis of autophagy-associated genes in foxtail millet (Setaria italica L.) and characterization of the function of SiATG8a in conferring tolerance to nitrogen starvation in rice.

BackgroundAutophagy is a cellular degradation process that is highly evolutionarily-conserved in yeast, plants, and animals. In plants, autophagy plays important roles in regulating intracellular degradation and recycling of amino acids in response to nutrient starvation, senescence, and other environmental stresses. Foxtail millet (Setaria italica) has strong resistance to stresses and has been proposed as an ideal material for use in the study of the physiological mechanisms of abiotic stress tolerance in plants. Although the genome sequence of foxtail millet (Setaria italica) is available, the characteristics and functions of abiotic stress-related genes remain largely unknown for this species.ResultsA total of 37 putative ATG (autophagy-associated genes) genes in the foxtail millet genome were identified. Gene duplication analysis revealed that both segmental and tandem duplication events have played significant roles in the expansion of the ATG gene family in foxtail millet. Comparative synteny mapping between the genomes of foxtail millet and rice suggested that the ATG genes in both species have common ancestors, as their ATG genes were primarily located in similar syntenic regions. Gene expression analysis revealed the induced expression of 31 SiATG genes by one or more phytohormone treatments, 26 SiATG genes by drought, salt and cold, 24 SiATG genes by darkness and 25 SiATG genes by nitrogen starvation. Results of qRT-PCR showing that among 37 SiATG genes, the expression level of SiATG8a was the highest after nitrogen starvation treatment 24 h, suggesting its potential role in tolerance to nutrient starvation. Moreover, the heterologous expression of SiATG8a in rice improved nitrogen starvation tolerance. Compared to wild type rice, the transgenic rice performed better and had higher aboveground total nitrogen content when the plants were grown under nitrogen starvation conditions.ConclusionsOur results deepen understanding about the characteristics and functions of ATG genes in foxtail millet and also identify promising new genetic resources that should be of use in future efforts to develop varieties of foxtail millet and other crop species that have resistance to nitrogen deficiency stress.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3113-4) contains supplementary material, which is available to authorized users.

HvPap-1 C1A protease actively participates in barley proteolysis mediated by abiotic stresses

Protein breakdown and mobilization from old or stressed tissues to growing and sink organs are some of the metabolic features associated with abiotic/biotic stresses, essential for nutrient recycling. The massive degradation of proteins implies numerous proteolytic events in which cysteine-proteases are the most abundant key players. Analysing the role of barley C1A proteases in response to abiotic stresses is crucial due to their impact on plant growth and grain yield and quality. In this study, dark and nitrogen starvation treatments were selected to induce stress in barley. Results show that C1A proteases participate in the proteolytic processes triggered in leaves by both abiotic treatments, which strongly induce the expression of the HvPap-1 gene encoding a cathepsin F-like protease. Differences in biochemical parameters and C1A gene expression were found when comparing transgenic barley plants overexpressing or silencing the HvPap-1 gene and wild-type dark-treated leaves. These findings associated with morphological changes evidence a lifespan-delayed phenotype of HvPap-1 silenced lines. All these data elucidate on the role of this protease family in response to abiotic stresses and the potential of their biotechnological manipulation to control the timing of plant growth.

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.

EFFECT OF RED LIGHT-BLUE AND NITROGEN STARVATION ON GROWTH OF MICROALGAE Tetraselmis sp.

<p>Microalgae has many benefits for human being. Microalgae is potential source for biodiesel and bioethanol, but the biomass produced from the culture is relatively low. So that, the research has to be done to improve the productivity of the microalgae. Red light and blue light spectrum are known as effective spectrums for the photosynthesis process. Then nitrogen starvation medium is usually performed in order to improve the lipid content. However, nitrogen starvation treatment will decrease microalgae biomass. Therefore, this research aims to study the effect of red and blue light and nitrogen starvation on the growth of Tetraselmis sp. The parameters measured were the cells number, dry weight and chlorophyll a and b. In this study, microalgae Tetraselmis sp. was treated using red and blue light for 7 days. Then, followed by nitrogen starvation treatment with concentration 100%, 50%, and 0% of the f/2 normal medium until day 14. The number of the cells was counted every day for 14 days using a haemocytometer and dry weights were counted at day 0, 1, 5, 7 and 14. Chlorophyll contents were calculated by Jeffrey and Humphrey’s Trichromatic Equations method at absorbance 664 nm, 647 nm, 630 nm, and 750 nm. The results indicated that the red light increased the number of cells to twice as normal, while the blue light increased the number of cells to 1,5 times as normal. Furthermore, the 50% of nitrogen treatment under the red and blue light increased the dry weight to 25% and 60%. </p><p><strong>Keywords</strong>: Microalgae, Tetraselmis sp., Nitrogen Starvation, Blue and Red light</p>

Isolation and expression analyses of methyl-d-erythritol 4-phosphate (MEP) pathway genes from Haematococcus pluvialis

Haematococcus pluvialis is a green alga known to accumulate the keto-carotenoid astaxanthin under stress conditions. In H. pluvialis, carotenoids are derived from isopentenyl diphosphate (IPP), which is synthesized via the non-mevalonate methyl-d-erythritol 4-phosphate (MEP) pathway. The present study revealed that several treatments caused changes in pigment profiles and the expression levels of genes encoding MEP pathway enzymes. Additionally, photosynthesis fluorescence was monitored. Generally, under stress conditions, there was an increase in astaxanthin, along with a decrease in total chlorophyll and photo capacity. Six IPP biosynthetic genes were cloned from H. pluvialis. Expression analysis revealed that these transcripts were upregulated under stress culture conditions. However, the extent of MEP pathway gene expression varied with the stress conditions. 4-Diphosphocytidyl-2-C-methyl-d-erythritol (CDP-ME) synthase (CMS) and CDP-ME kinase (CMK) exhibited significantly higher transcriptional expression under nitrogen starvation treatments. While 1-deoxy-d-xylulose 5-phosphate (DXP) synthase (DXS), CMS, CMK 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) synthase (HDS), and HMBPP reductoisomerase (HDR) showed significant upregulation on the second day under nitrogen starvation and high light (HL-N). The enhanced expression of these genes was also observed on the third day under high light. The high expression of MEP pathway genes was correlated with the accumulation of astaxanthin under HL-N stress. This is the first report of the isolation of IPP biosynthetic genes and their differential expression in H. pluvialis under stress conditions. The present study revealed the influence of stress conditions on the expression of MEP pathway genes and changes in pigment profiles.

Wild barley: a source of genes for crop improvement in the 21st century?

The development of new barleys tolerant of abiotic and biotic stresses is an essential part of the continued improvement of the crop. The domestication of barley, as in many crops, resulted in a marked truncation of the genetical variation present in wild populations. This process is significant to agronomists and scientists because a lack of allelic variation will prevent the development of adapted cultivars and hinder the investigation of the genetic mechanisms underlying performance. Wild barley would be a useful source of new genetic variation for abiotic stress tolerance if surveys identify appropriate genetic variation and the development of marker-assisted selection allows efficient manipulation in cultivar development. There are many wild barley collections from all areas of its natural distribution, but the largest are derived from the Mediterranean region. The results of a range of assays designed to explore abiotic stress tolerance in barley are reported in this paper. The assays included; sodium chloride uptake in wild barley and a mapping population, effects for δ13C and plant dry weight in wheat aneuploids, effects of photoperiod and vernalization in wild barley, and measurements of root length in wild barley given drought and nitrogen starvation treatments in hydroponic culture. There are examples of the use of wild barley in breeding programmes, for example, as a source of new disease resistance genes, but the further exploration of the differences between wild barley and cultivars is hampered by the lack of good genetic maps. In parallel to the need for genetic studies there is also a need for the development of good physiological models of crop responses to the environment. Given these tools, wild barley offers the prospect of a ‘goldmine’ of untapped genetic reserves.

Wild barley: a source of genes for crop improvement in the 21st century?

The development of new barleys tolerant of abiotic and biotic stresses is an essential part of the continued improvement of the crop. The domestication of barley, as in many crops, resulted in a marked truncation of the genetical variation present in wild populations. This process is significant to agronomists and scientists because a lack of allelic variation will prevent the development of adapted cultivars and hinder the investigation of the genetic mechanisms underlying performance. Wild barley would be a useful source of new genetic variation for abiotic stress tolerance if surveys identify appropriate genetic variation and the development of marker-assisted selection allows efficient manipulation in cultivar development. There are many wild barley collections from all areas of its natural distribution, but the largest are derived from the Mediterranean region. The results of a range of assays designed to explore abiotic stress tolerance in barley are reported in this paper. The assays included; sodium chloride uptake in wild barley and a mapping population, effects for delta 13C and plant dry weight in wheat aneuploids, effects of photoperiod and vernalization in wild barley, and measurements of root length in wild barley given drought and nitrogen starvation treatments in hydroponic culture. There are examples of the use of wild barley in breeding programmes, for example, as a source of new disease resistance genes, but the further exploration of the differences between wild barley and cultivars is hampered by the lack of good genetic maps. In parallel to the need for genetic studies there is also a need for the development of good physiological models of crop responses to the environment. Given these tools, wild barley offers the prospect of a 'goldmine' of untapped genetic reserves.