Effect Of Nitrogen Deficiency Research Articles

Transcriptome Analysis Identified PyNAC42 as a Positive Regulator of Anthocyanin Biosynthesis Induced by Nitrogen Deficiency in Pear (Pyrus spp.)

Anthocyanins are important secondary metabolites in plants, which contribute to fruit color and nutritional value. Anthocyanins can be regulated by environmental factors such as light, low temperature, water conditions, and nutrition limitations. Nitrogen (N) is an essential macroelement for plant development, its deficiency as a kind of nutrition limitation often induces anthocyanin accumulation in many plants. However, there is a lack of reports regarding the effect of nitrogen deficiency on anthocyanin biosynthesis in pears. In this study, we found that N deficiency resulted in anthocyanin accumulation in pear callus and upregulated the expression of anthocyanin biosynthesis pathway structural genes (PyPAL, PyCHS, PyCHI, PyF3H, PyDFR, PyANS, and PyUFGT) and key regulatory factors (PyMYB10, PyMYB114, and PybHLH3). Through analysis of transcriptome data of treated pear callus and RT-qPCR assay, a differentially expressed gene PyNAC42 was identified as significantly induced by the N deficiency condition. Overexpression of PyNAC42 promoted anthocyanin accumulation in “Zaosu” pear peels. Additionally, dual luciferase assay and yeast one-hybrid assay demonstrated that PyNAC42 could not directly activate the expression of PyDFR, PyANS, and PyUFGT. Furthermore, yeast two-hybrid and pull-down assays confirmed that PyNAC42 interacted with PyMYB10 both in vivo and in vitro. Co-expression of PyNAC42 and PyMYB10 significantly enhanced anthocyanin accumulation in “Zaosu” pear peels. Dual luciferase assay showed that PyNAC42 significantly enhanced the activation of PyDFR, PyANS, and PyUFGT promoters by interacting with PyMYB10, which suggests that PyNAC42 can form the PyNAC42-PyMYB10 complex to regulate anthocyanin biosynthesis in pear. Thus, the molecular mechanism underlying anthocyanin biosynthesis induced by N deficiency is preliminarily elucidated. Our finding has expanded the regulatory network of anthocyanin biosynthesis and enhanced our understanding of the mechanisms underlying nutrient deficiency modulates anthocyanin biosynthesis in pear.

Effects of nitrogen deficiency and drought stresses on miRNA expressions in Arabidopsis thaliana

MicroRNAs are now known to have an important role in regulating gene expression of eukaryotic organisms. miRNA research in plants gained importance after the discovery that several stress factors alter certain miRNA expressions, which subsequently regulate their target gene expressions and affect development and growth of plants. In this study, two of the widely studied abiotic stress conditions for plants, nitrogen deficiency and drought were used individually and as a combined stress treatment on Arabidopsis thaliana callus tissues to observe the change of expressions in certain miRNAs, when multiple stressors are encountered. Combined stress strongly inhibited callus growth compared to other conditions, while strongly altering certain miRNA expressions. Compared to control, in 7-day stress treated callus, miR165a-3p,b, miR319a,b, miR396b-5p, miR399d and miR827 showed significant downregulation for all stress treatments, while 7-day N deficiency caused miR167 upregulation. Stress treatments for 7 days mostly downregulated miR167c-5p, miR319c, miR399a, miR399e expressions except for the N deficient samples. After 14 days of stress, miR165a-3p,b, miR396a-5p, miR399b, miR399d were downregulated. During 14-day drought and combined stress, miR399a and miR396b-5p expressions were upregulated, respectively. The differences observed in this study between stress responses of 7 and 14-day-long treatments are believed to be valuable to further elucidate the associated molecular mechanisms of miRNAs, determination and validation of miRNA targets, and how plants respond to stress conditions via various genetic regulations.

Effects of Nitrogen Deficiency on Photosynthesis and Chlorophyll a Fluorescence Attributes at Two Contrasting Legume Forages

Effects of Nitrogen Deficiency on Photosynthesis and Chlorophyll a Fluorescence Attributes at Two Contrasting Legume Forages

Effects of Nitrogen Deficiency on the Metabolism of Organic Acids and Amino Acids in Oryza sativa.

Organic acids metabolism and nitrogen (N) metabolism in rice seedlings and the relationship between them are not fully understood. In this study, rice (Oryza sativa L. ssp. Indica) variety “Huanghuazhan” was used as the experimental material, and three N levels (5 mM, 1 mM, and 0 mM NH4NO3) were set by the hydroponic method for different levels of N treatment. Our results showed that the increased content of malate in rice leaves caused by reducing N level was related to the increased synthesis of malate (the activity of leaf PEPC increased)and the decreased degradation of malate (the activity of leaf NADP-ME decreased), while the increased contents of citrate and isocitrate in rice leaves caused by reducing N level might not be caused by the increased biosynthesis, but due to the decrease in degradation of citrate and isocitrate (the activities of leaf CS, ACO, and NADP-IDH decreased). The increased content of malate in rice roots caused by reducing N level might be related to the increased biosynthesis and the decreased degradation of root malate (the activities of root NAD-MDH and PEPC increased, while the activity of NADP-ME decreased). Compared to the control (5 mM NH4NO3), the increased content of citrate in rice roots caused by reducing N level might be related to the increased biosynthesis rather than the decreased degradation of citrate, due to the higher activities of CS and ACO in rice roots under 0 mM N and 1mM N treatment when compared to that of the control ones. At the same time, the increased content of isocitrate in roots was related to the increased isomerization of isocitrate (the activity of root ACO increased) and the decreased degradation of isocitrate (the activity of root NADP-IDH decreased). With the reducing N level, the activities of N metabolism-related enzymes, such as nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT), decreased in rice leaves and roots, resulting in the decreased contents of total free amino acids (TFAAs) and soluble proteins in rice seedlings, and finally led to the growth inhibition. Our results showed that the dynamics of organic acids metabolism caused by reducing N level were different in rice leaves and roots. In conclusion, there was a close correlation between organic acids metabolism and N metabolism in rice leaves and roots under N-limited conditions; furthermore, such a correlation was more obvious in rice leaves than that of roots.

RNA-Seq, physiological, and biochemical analysis of burley tobacco response to nitrogen deficiency

To explore the effects of nitrogen deficiency in burley tobacco, two varieties were cultivated and subjected to conditions of sufficient and deficient nitrogen. The natural characteristics of varieties TN90 and TN86 during tobacco cultivation were similar for nitrogen metabolism. Both carbon and nitrogen metabolism were significantly affected by reducing amounts of applied nitrogen. Under nitrogen-deficient conditions, average leaf biomass, root weight, photosynthetic rate (Pn), pigment levels, total nitrogen, and nitrate content of TN86 and TN90 were significantly decreased by 52.88%, 69.19%, 22.65%, 46.80%, 37.42%, and 79.15%, respectively (p < 0.01). Nicotine and soluble reducing sugar contents were significantly decreased by 96.67% and 95.12%, respectively, in TN86 roots (p < 0.01), which was consistent with the reductions in root surf area, average diameter, and root volume. Nitrogen deficiency induced 6318 differentially expressed genes in both TN90 and TN86, which were highly expressed. In total, 428 upregulated genes were analysed and found to be mainly enriched in the MAPK signalling pathway, sesquiterpenoid and triterpenoid biosynthesis, and arginine and proline metabolism. Meanwhile, 213 downregulated genes were analysed and found to be mainly enriched in photosynthesis, nitrogen metabolism, and amino acid biosynthesis. Reduced pigment content and Pn may result in low carbohydrate formation and decreased leaf biomass in burley tobacco under nitrogen-deficient conditions.

The Effect of Nitrogen Deficiency on the Growth and Lipid Content of Isochrysis affinis galbana in Two Photobioreactor Systems (PBR): Tubular and Flat Panel

Energy is becoming one of the most expensive production inputs nowadays. Energy reserves are starting to run out and their polluting nature has become undeniable. Therefore, there is an urgent necessity for renewable energies. One of these energy sources is algae, which are seen as promising for biofuel production. Algae can be cultured in non-agricultural land, high photosynthetic activity, harvested throughout the year high biomass production. High lipid from algae is possible by reducing some elements of growth conditions from the nutrient medium. In this study, Isochrysis affinis galbana species were cultured in two reactors; flat panel photobioreactors with different light paths (1, 3, 5, 7 and 10 cm) and tubular photobioreactors, with 50% nitrogen reduction and 20% inoculation densities. Biomass, lipid and protein ratios were determined. The highest lipid content of 33.13% was obtained from I. aff. galbana with 12.11% protein in flat panel photobioreactors with 50% nitrogen reduction and 10 cm light path, and a 0.991 g L-1 biomass rate was obtained. The highest optical density was found in the 10 cm light path flat panel photobioreactor with a 50% nitrogen reduction.

The impact of nitrogen deficiency and subsequent recovery on the photosynthetic performance of the red macroalga Gracilariopsis lemaneiformis

The effect of nitrogen deficiency and subsequent recovery on photosynthetic performance of the red macroalga Gracilariopsis lemaneiformis was measured in terms of algal growth rate, accumulation of photosynthetic pigments (i.e., phycoerythrin and chlorophyll-a), maximum effective quantum yield of photosystem II (Fv/Fm), and the transcript levels of genes related to photosynthesis and photorespiration. Nitrogen deficiency and then recovery notably promoted the growth of G. lemaneiformis, significantly inhibited the accumulation of phycoerythrin and chlorophyll-a, but had no significant influence on Fv/Fm. In addition to physiological performance of algae under nitrogen stress, the key genes encoding photorespiratory and photosynthetic enzymes (i.e., gdct, gdcp, hpr, shmt, sgat, sbp, and rub) were up-regulated which might have led to more increased in growth rate than that of control after the recovery of nitrogen. While the down-regulation of gdct, gdcp, and shmt genes at the 4th day of nitrogen deficiency might be linked to the reduced accumulation of phycoerythrin and chlorophyll-a, the up-regulation of gdct and gdcp at the beginning of nitrogen deficiency and nitrogen recovery might associate with Fv/Fm that did not change significantly. Briefly, the up- and down-regulation of these genes at different times might be due to an algal complex regulatory mechanism. Thus, the combined action of these genes allows the algae to display higher photosynthetic efficiency and better growth, eventually acclimate to the varying environmental stresses. The data provided here represent a rich source for exploring the function of genes related to photorespiration and photosynthesis as well as the mechanism of algal acclimation under environmental stress.

Effect of nitrogen deficiency on the stability of aerobic granular sludge

To assess the stability of aerobic granular sludge (AGS) under nitrogen deficiency conditions, three sequence batch reactors were operated with chemical oxygen demand (COD) to nitrogen (N) ratios of 100/5, 100/2.5, and 100/2, while COD concentration was kept consistent. AGS variations in its physicochemical characteristics, microbial community, and treatment performance were investigated. The results indicated that good treatment performance and stable AGS were achieved under nitrogen deficiency. Extracellular polymeric substances (EPS) regulating mechanism preserved AGS stability under nitrogen deficiency, especially through increased secretion of polysaccharide. In addition, members of the Anaerolineaceae were the major filamentous bacteria, which are strictly anaerobic organism, providing a possible explanation to the lack of filamentous bacteria outgrowth under N deficient condition. Insights from this study could help lower chemical costs in AGS applications for specific industrial wastewater treatments.

Improved production of jiangxienone in submerged fermentation of Cordyceps jiangxiensis under nitrogen deficiency.

Jiangxienone produced by Cordyceps jiangxiensis exhibits significant cytotoxicity and good selectivity against various human cancer cells, especially gastric cancer cells. In this work, the effect of nitrogen deficiency on the accumulation of jiangxienone and the transcription levels of jiangxienone biosynthesis genes was studied in submerged fermentation of C. jiangxiensis. Results showed that accumulation of jiangxienone was improved under nitrogen deficiency condition. A maximal jiangxienone content of 3.2 µg/g cell dry weight was reached at 5mM glutamine, and it was about 8.9-fold higher than that obtained at 60mM glutamine (control). The transcription levels of the biosynthetic pathway genes hmgr and sqs and the nitrogen regulatory gene areA were upregulated by 7-, 14-, and 28-fold, respectively, in culture with 5mM glutamine compared to the control. It was hypothesized that the jiangxienone biosynthesis may involve the mevalonate pathway in C. jiangxiensis. Taken together, our study indicated that nitrogen deficiency is an efficient strategy for enhancing jiangxienone accumulation in submerged fermentation of C. jiangxiensis, which is useful for further understanding the regulation of jiangxienone biosynthesis.

Effects of nitrogen-deficiency on efficiency of light-harvesting apparatus in radish

Nitrogen starvation has been stated to reduce chlorophyll a and accessory pigments, decrease photosynthetic efficiency, as well as modify chloroplast thylakoid membranes. However, the impact of N-deficiency on light-dependent reactions of photosynthesis has not been well understood. In this study, efficiency and structure of light-harvesting complex under N-deficiency conditions were investigated in two radish cultivars (Raphanus sativus var. sativus ‘Fluo HF1’ and ‘Suntella F1’). Light-dependent reactions of photosynthesis were investigated by measuring in vivo chlorophyll a prompt fluorescence signal. Acquired data were utilised in two ways: by plotting fast induction curves and calculating OJIP-test biophysical parameters. Detailed analysis of difference curves as well as OJIP-test results showed that major disturbances were associated with photosystem II and its subunits, including decoupling of light-harvesting complexes, dysfunction of oxygen-evolving complex, and reaction centres inactivation. The maximum quantum yield of photosystem II primary photochemistry was severely restricted, causing an inhibition in electron transport through successive protein complexes in the thylakoid membrane. Structural changes were demonstrated by recording images using Transmission Electron Microscopy (TEM). TEM investigations showed intensive starch accumulation under N-deficiency. Rare thylakoid stacks distributed in tiny layers of stroma around grains and chloroplast periphery were observed in cells of N-deficient plants. The application of principal component analysis (PCA) on OJIP-test results allowed characterizing the dynamics of stress response and separating parameters according to their influence on plants stress response. ‘Suntella F1’ genotype was found to be more sensitive to nitrogen deficiency as compared to ‘Fluo HF1’ genotype.

Effect of nitrogen deficiency on nutrients uptake and potassium uptake-related genes expression of rapeseed

Effect of nitrogen deficiency on nutrients uptake and potassium uptake-related genes expression of rapeseed

Effect of nitrogen deficiency on the ion-exchange properties of cell wall polymers from wheat roots

The ion-exchange properties of cell wall polymers isolated from the roots of wheat (Triticum aestivum L.) plants grown on either nitrate-free (N-deficient) or nitrate-containing (+N) hydroponic nutrient medium have been investigated. Irrespective of the nitrogen nutrition regimen, the studied cell walls contained four types of ion-exchange groups: primary amino groups of structural proteins (pKa < 3), carboxyl groups of polygalacturonic acid in pectin (pK a ~4.7), carboxyl groups of hydroxycinnamic acids (pK a ~7.3), and phenolic OH-groups of lignin (pKa ~10.2). The quantitative ratio between these types of ion-exchange groups, the mass fraction of cell walls in the dry weight of roots, and the swelling coefficient of cell walls depended on the nitrate presence in the growing medium. Compared to the +N variant, the N-deficient variant was characterized by a 2.4 times higher content of phenolic OH-groups in cell walls and 1.24 times higher mass fraction of cell walls; at the same time, the swelling coefficient for this variant was lower by 10%. The obtained data indicate that nitrogen deficiency results in a formation of thicker root cell walls with a higher degree of polymer cross-linking that may be caused by the increased lignin content.

Nitrogen deficiency effects on growth and photosynthesis in no-till direct seeded super hybrid rice

Field experiments were conducted in two locations in China to determine the effects of nitrogen (N) deficiency on plant growth and leaf photosynthesis of two super hybrid rice cultivars grown under no-till direct seeded conditions. Results showed that total dry mass was reduced by 39–48% in N-deficient plants. Leaf area index was decreased by 45–58% under N-deficient conditions. Less leaf number per unit land area resulting from reduced tiller number per unit area and small leaf size were responsible for the small leaf area index in N-deficient plants. N-deficient plants had 19–26% lower leaf N content and 7–12% lower relative chlorophyll content in flag leaves. In parallel with this, net photosynthetic rate and stomatal conductance were decreased by 14–24% and 14–42%, respectively, in N-deficient plants. However, the depression of stomatal conductance did not explain the lower photosynthetic capacity because intercellular CO2 concentration was not definitely reduced in N-deficient plants. Our results highlight the need for greater fundamental understanding of the effects of N deficiency on mesophyll function in no-till direct seeded super hybrid rice.

Effect of nitrogen deficiency on ascorbic acid biosynthesis and recycling pathway in cucumber seedlings

L-Ascorbic acid (AsA, ascorbate) is one of the most abundant natural antioxidants, and it is an important factor in the nutritional quality of cucumber. In this work, key enzymes involved in the ascorbic acid biosynthesis and recycling pathway in cucumber seedlings under nitrogen deficiency were investigated at the levels of transcription and enzyme activity. The activities of myo-inositol oxygenase (MIOX) and transcript levels of MIOXs increased dramatically, while the activities of ascorbate oxidase (AO) and glutathione reductase (GR) and transcript levels of AOs and GR2 decreased significantly in N-limited leaves, as did the ascorbate concentration, in nitrogen-deficient cucumber seedlings. The activities of other enzymes and transcript levels of other genes involved in the ascorbate recycling pathway and ascorbate synthesis pathways decreased or remained unchanged under nitrogen deficiency. These results indicate that nitrogen deficiency induced genes involved in the ascorbate-glutathione recycling and myo-inositol pathway in cucumber leaves. Thus, the AO, GR and MIOX involved in the pathways might play roles in AsA accumulation.

Effect of nitrogen deficiency on recombinant protein production and dimerization and growth in transgenic plants

This study investigated the effects of nitrogen (NH4+) in the soil on the expression of prostatic acid phosphatase (PAP)-IgA Fc and PAP-IgA FcK recombinant proteins in transgenic plants, and on plant growth in the greenhouse. Plant height and leaf length were greater in soil A (NH4+: 51 mg·L-1) than in soil B (NH4+: 1 mg·L-1). In soil B, the plant leaves turned yellow, with chlorophyll concentrations that were significantly lower than the chlorophyll concentration of leaves from plants grown in soil A. The expression level of the PAP-IgA Fc and PAP-IgA FcK proteins and the protein dimerization rates were lower in soil B when compared with soil A. In addition, PAP-IgA Fc and PAP-IgA FcK expression and dimerization levels were significantly lower in transgenic plants grown in soil B, when compared with transgenic plants grown in soil A. Soil characteristics such as hydrogen ion concentration (pH), electric conductivity (EC), and bulk density did not differ between soil A and B. Thus, appropriate soil NH4+ concentrations are essential for optimized plant growth and enhanced expression of dimerized PAP-IgA Fc and PAP-IgA FcK recombinant proteins in transgenic plants.

Combining nitrogen starvation with sufficient phosphorus supply for enhanced biodiesel productivity of Chlorella vulgaris fed on acetate

Heterotrophic cultivation of microalgae is attracting more attention due to its high production efficiency. In this study, Chlorella vulgaris was cultivated heterotrophically with acetate in batch systems. The effects of nitrogen deficiency and different phosphorus supply levels on biodiesel production were investigated. It was found that C. vulgaris can assimilate acetate and accumulate fatty acids simultaneously. The highest FAME (Fatty acid methyl ester) content (56%) of algae was obtained in nitrogen deficient media. Interestingly, the productivity of fatty acids under nitrogen starved conditions was three times greater than that under nitrogen sufficient conditions. Moreover, FAME productivity was further enhanced to 66mgL−1·d−1 by a sufficient phosphorus supply but there was no effect caused by phosphorus limitation. Furthermore, the conversion yields of acetate consumed to fatty acids produced (Chemical oxygen demand (COD)-based) in nitrogen starved media were also three times higher than those in nitrogen sufficient media. FAME productivity of C. vulgaris under nitrogen starvation was not improved by controlling the pH at stable values (7.5, 8.0, and 8.5), and even decreased when pH was controlled at 7.0 and 9.0.

Enhancement of FAME productivity of Scenedesmus obliquus by combining nitrogen deficiency with sufficient phosphorus supply in heterotrophic cultivation

In this study, Scenedesmus obliquus NIES-2280 was cultivated heterotrophically with acetate as the carbon source. The effects of nitrogen deficiency and different phosphorus supply levels on biodiesel production by S. obliquus were investigated. It was found that S. obliquus could make good use of assimilated acetate for fatty acid accumulation. Fatty acid contents of algae in nitrogen deficiency media increased to 38–48% after 6-day cultivation. Interestingly, the productivity of fatty acid methyl esters (FAMEs) under nitrogen starved conditions increased fourfold than that under nitrogen sufficient conditions. Moreover, FAME productivity could be further enhanced by a sufficient phosphorus supply rather than under P limitation or P deficiency conditions, and the highest FAME productivity was 55.9mgL−1d−1. Furthermore, the conversion yields of acetate to fatty acids (COD based) in nitrogen starvation media (18–28%) were much higher than those in nitrogen sufficient media (∼7%). This study indicates a great potential to combine wastewater treatment with biodiesel production via S. obliquus which can significantly improve biodiesel productivity and COD utilization under nitrogen starvation coupled with sufficient phosphorus supply.

Improved Alkane Production in Nitrogen-Fixing and Halotolerant Cyanobacteria via Abiotic Stresses and Genetic Manipulation of Alkane Synthetic Genes.

Cyanobacteria possess the unique capacity to produce alkane. In this study, effects of nitrogen deficiency and salt stress on biosynthesis of alkanes were investigated in three kinds of cyanobacteria. Intracellular alkane accumulation was increased in nitrogen-fixing cyanobacterium Anabaena sp. PCC7120, but decreased in non-diazotrophic cyanobacterium Synechococcus elongatus PCC7942 and constant in a halotolerant cyanobacterium Aphanothece halophytica under nitrogen-deficient condition. We also found that salt stress increased alkane accumulation in Anabaena sp. PCC7120 and A. halophytica. The expression levels of two alkane synthetic genes were not upregulated significantly under nitrogen deficiency or salt stress in Anabaena sp. PCC7120. The transformant Anabaena sp. PCC7120 cells with additional alkane synthetic gene set from A. halophytica increased intracellular alkane accumulation level compared to control cells. These results provide a prospect to improve bioproduction of alkanes in nitrogen-fixing halotolerant cyanobacteria via abiotic stresses and genetic engineering.

Life cycle analysis on fossil energy ratio of algal biodiesel: effects of nitrogen deficiency and oil extraction technology.

Life cycle assessment (LCA) has been widely used to analyze various pathways of biofuel preparation from “cradle to grave.” Effects of nitrogen supply for algae cultivation and technology of algal oil extraction on life cycle fossil energy ratio of biodiesel are assessed in this study. Life cycle fossil energy ratio of Chlorella vulgaris based biodiesel is improved by growing algae under nitrogen-limited conditions, while the life cycle fossil energy ratio of biodiesel production from Phaeodactylum tricornutum grown with nitrogen deprivation decreases. Compared to extraction of oil from dried algae, extraction of lipid from wet algae with subcritical cosolvents achieves a 43.83% improvement in fossil energy ratio of algal biodiesel when oilcake drying is not considered. The outcome for sensitivity analysis indicates that the algal oil conversion rate and energy content of algae are found to have the greatest effects on the LCA results of algal biodiesel production, followed by utilization ratio of algal residue, energy demand for algae drying, capacity of water mixing, and productivity of algae.

Enhancement of porosity and aerenchyma formation in nitrogen-deficient rice roots

Root aerenchyma provides oxygen from plant shoots to roots. In upland crops, aerenchyma formation is induced mainly by oxygen or nutrient deficiency. Unlike upland crops, rice forms root aerenchyma constitutively and also inductively in response to oxygen deficiency. However, the effects of nitrogen deficiency on aerenchyma formation in rice remain unknown although nitrogen deficiency is common in most of the world's soils. We aimed to clarify the spatiotemporal patterns of aerenchyma formation induced in rice roots by nitrogen deficiency upon establishment of reliable growth conditions. Rice was grown hydroponically to evaluate porosity and aerenchyma formation induced by nitrogen and oxygen deficiency. Reliable growth conditions for nitrogen and oxygen deficiency were successfully established, because seedling root elongation was significantly promoted by nitrogen deficiency and inhibited by oxygen deficiency. Porosity was higher in whole roots grown under nitrogen and oxygen deficiency than in the controls. Root aerenchyma production was induced extensively by nitrogen deficiency but partially by oxygen deficiency. Thus the physiological roles of aerenchyma induced by nitrogen deficiency likely differ from those under oxygen deficiency. It indicates a possibility that inducible aerenchyma formation in nitrogen deficiency might promote adaptation to this deficiency by reducing respiration and remobilizing nitrogen, or both.