bioRxiv - Plant Biology最新文献

{"title":"Foliar application of nano urea results in higher biomass, chlorophyll, and nitrogen content than equimolar bulk urea through differential gene regulation in Arabidopsis thaliana","authors":"Arpan Dey, Neelam Jangir, Devanshu Verma, Rajveer Singh Shekhawat, Pankaj Yadav, AYAN SADHUKHAN","doi":"10.1101/2024.09.03.611005","DOIUrl":"https://doi.org/10.1101/2024.09.03.611005","url":null,"abstract":"Indian Farmers Fertilizer Cooperative (IFFCO)′s liquid nano urea formulation (NUF) was applied to one-month-old <em>Arabidopsis thaliana</em> plants grown in vermiculite as a 0.4% foliar spray twice at an interval of 10 days and compared with sprays of equimolar bulk urea. NUF resulted in a 51 ± 14.9% increase in biomass, 29.5 ± 9.1% in chlorophyll, 8.4 ± 3.1% in nitrogen, and 4.5 ± 0.3% in amino acid content of the leaves, compared to bulk urea. NUF′s zeta potential of -54.7 mV and particle size of ≃27.7 nm, measured by dynamic light scattering and transmission electron microscopy, make it suitable for stomatal uptake. We conducted a differential gene expression analysis by mRNA sequencing to understand the molecular basis of the phenotypic gains under NUF rather than urea. NUF resulted in significantly higher expression levels of 211 genes (log2fold-change &gt; 0.5, FDR &lt; 0.05) involved in the biosynthesis of carbohydrates, amino acids, nucleotides, lipids, phytohormones, and secondary metabolites, cell wall biosynthesis and modification, growth and developmental processes, cell cycle, and stress response than bulk urea. On the other hand, 1,286 genes (log2fold-change &lt; -0.5) involved in cell death, abscission, senescence, nitrogen transport and metabolism, and biotic stress response showed lower expression levels upon NUF application than bulk urea. Our results suggest that although NUF foliar spray suppresses nitrogen uptake genes, possibly due to nitrogen excess, it enhances growth by up-regulating the synthesis of essential biomolecules and growth-promoting genes, compared to bulk urea.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":"125 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142183023","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mature leaves produce a multi-layered wound periderm by integrating phytohormone signaling with ATML1-mediated epidermal specification","authors":"Jung-Min Lee, Woo-Taek Jeon, Minsoo Han, Myung Kwon, Kyungyoon Kim, Hoon Jung, Geon Heo, Sujeong Je, Yasuyo Yamaoka, Yuree Lee","doi":"10.1101/2024.09.02.607870","DOIUrl":"https://doi.org/10.1101/2024.09.02.607870","url":null,"abstract":"The epidermis of plants forms a protective barrier against various stress, but how breaches in the epidermis are repaired is not well understood. Here, we investigated wound healing in the mature leaves of Arabidopsis. We discover a novel type of wound periderm comprising a multi-layered ligno-suberized barrier covered with cuticular wax, which is formed by mesophyll cells that adopt an epidermal fate. Mesophyll cells of protective layer 1 (P1), just beneath the wound, transition into epidermal cells, which seal the wound by depositing cuticle. As P1 undergoes cell death, protective layer 2 (P2), which underlies P1, takes the place of P1 and undergoes ligno-suberization. This multi-layered periderm involves integration of ethylene and jasmonic acid signaling with ATML1, a key transcription factor in epidermal specification, to coordinate cell layer-specific functions. This novel wound periderm also occurs in the leaves of tobacco and Capsella, suggesting it is a widespread phenomenon.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142183030","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evolutionary conserved RLF, a plant cytochrome b5-like heme-binding protein, is essential for organ development in Marchantia polymorpha","authors":"Kentaro P. Iwata, Takayuki Shimizu, Yuuki Sakai, Tomoyuki Furuya, Hinatamaru Fukumura, Yuki Kondo, Tatsuru Masuda, Kimitsune Ishizaki, Hidehiro Fukaki","doi":"10.1101/2024.09.02.610766","DOIUrl":"https://doi.org/10.1101/2024.09.02.610766","url":null,"abstract":"In <em>Arabidopsis thaliana</em>, REDUCED LATERAL ROOT FORMATION (RLF), a cytochrome <em>b</em>₅-like heme-binding domain (Cytb5-HBD) protein, is necessary for proper lateral root formation. Whereas the other Cytb5-HBD proteins in <em>A. thaliana</em> regulate different metabolic reactions, RLF is unique as it specifically regulates organ development. However, it remains unknown whether heme binding to RLF is necessary for its function, and whether <em>RLF</em> orthologs in different plant species also regulate organ development. We demonstrate that RLF binds to heme <em>in vitro</em> and that the two histidine residues, which are conserved among Cytb5-HBD, are crucial for both heme binding and its biological function in <em>A. thaliana</em>. In addition, Mp<em>RLF</em>, a <em>RLF</em> ortholog in <em>Marchantia polymorpha</em>, rescues the lateral root formation phenotype of the <em>A. thaliana rlf</em> mutant. Mp<em>rlf^ge</em>, the loss-of-function mutation in the Mp<em>RLF</em>, resulted in delayed thallus growth and inhibited both gemma cup and antheridiophore formation. Transcriptome analysis using Mp<em>rlf^ge</em> revealed that Mp<em>RLF</em> affects several metabolic pathways. Our findings indicate that Mp<em>RLF</em> is essential for vegetative and reproductive development in <em>M. polymorpha</em>, suggesting that the RLF-dependent redox reaction systems are evolutionarily conserved as crucial mechanisms for organ development across diverse plant species.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142183077","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative proteomic analysis of soil-grown Brassica napus responses to nutrient deficiency","authors":"Lauren E. Grubb, Sabine Scandola, Devang Mehta, Ibrahim Khodabocus, Richard Glen Uhrig","doi":"10.1101/2024.09.01.610712","DOIUrl":"https://doi.org/10.1101/2024.09.01.610712","url":null,"abstract":"Macronutrients such as nitrogen (N), phosphorus (P), potassium (K), and sulphur (S) are critical for plant growth and development. Field-grown canola (Brassica napus L.) is supplemented with fertilizers to maximize plant productivity, while deficiency in these nutrients can cause significant yield loss. A holistic understanding of the interplay between these nutrient deficiency responses in a single study and canola cultivar is thus far lacking, hindering efforts to increase the nutrient use efficiency of this important oil seed crop. To address this, we performed a comparative quantitative proteomic analysis of both shoot and root tissue harvested from soil-grown canola plants experiencing either nitrogen, phosphorus, potassium, or sulphur deficiency. Our data provide critically needed insights into the shared and distinct molecular responses to macronutrient deficiencies in canola. Importantly, we find more conserved responses to the four different nutrient deficiencies in canola roots, with more distinct proteome changes in aboveground tissue. Our results establish a foundation for a more comprehensive understanding of the shared and distinct nutrient deficiency response mechanisms of canola plants and pave the way for future breeding efforts.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142183078","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nitrogen nutrition impacts grapevine esca leaf symptom incidence, physiology and metabolism.","authors":"Ninon Dell'Acqua, Gregory A Gambetta, Gwenaelle Comont, Nathalie Ferrer, Adam Rochepeau, Pierre Petriacq, Chloe E. L. Delmas","doi":"10.1101/2024.08.31.610625","DOIUrl":"https://doi.org/10.1101/2024.08.31.610625","url":null,"abstract":"Nitrogen plays a crucial role in plant growth and defence mechanisms, yet its role in plant-pathogen interactions is complex and remains largely unexplored, especially in perennial crops. This study aimed to investigate the effects of controlled nitrogen nutrition levels on disease incidence, fungal communities, and plant physiology and metabolism. Esca is a widespread grapevine vascular disease affecting physiology, xylem integrity and metabolism. Naturally infected Vitis vinifera L. cv. 'Sauvignon blanc' were subjected to three ammonium nitrate treatments across three seasons, resulting in reduced esca incidence under nitrogen deficiency compared with medium nutrition levels, while excess nitrogen had no significant impact. Nitrogen treatments significantly impacted vine physiology and leaf metabolites but did not affect fungal wood communities. Nitrogen deficiency significantly reduced stem diameter, photosynthesis, and leaf area, likely decreasing whole-plant transpiration, while excess nitrogen increased these factors suggesting a key role of plant transpiration in esca incidence. Additionally, nitrogen deficiency led to significantly higher production of phenylpropanoids, particularly antifungal flavonoids, in leaf metabolomes compared to the medium level. These findings highlight the pivotal role of nitrogen in the development of esca through alterations in vine morphology, physiology and metabolism. Fertilization practices may be crucial in the management of vascular diseases.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":"34 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142183076","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fe deficiency causes transcriptional shift in roots leading to disruption of drought tolerance in soybean","authors":"Md Rokibul Hasan, Asha Thapa, Mohammad Golam Mostofa, Ahmad H Kabir","doi":"10.1101/2024.08.30.610582","DOIUrl":"https://doi.org/10.1101/2024.08.30.610582","url":null,"abstract":"Iron (Fe) deficiency in alkaline soils, exacerbated by drought, collectively affects soybean health. This study aimed to evaluate the physiological and transcriptional changes in Fiskeby IV, a drought-tolerant genotype that loses its tolerance when exposed to simultaneous Fe deficiency and drought. In this growth incubator study, Fe deficiency and drought stress resulted in substantial reductions in plant biomass, photosynthetic efficiency, and nutrient uptake in Fiskeby IV. Despite these disruptions, the photochemical efficiency of photosystem II remained stable, suggesting the activation of protective mechanisms to maintain essential photosynthetic functions. RNA-seq analysis highlighted a complex response, showing the upregulation of ethylene-responsive genes (Ethylene-response sensor 2, Ethylene-responsive TF018, Ethylene-responsive TF5) as well as the genes related to rhizosphere acidification (ATPase 1) and redox homeostasis (Glutaredoxin-3). It suggests that ethylene signaling and rhizosphere acidification may be responsive in coordinating Fe homeostasis and drought adaptation in soybean. On the flip side, combined stresses caused the downregulation of several genes related to nutrient uptake (nicotianamine transporter YSL1, ammonium transporter 2, sulfate transporter 3.4, and major facilitator family protein). In a targeted study, supplementation with 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, led to substantial improvements in morpho-physiological traits and Fe status under combined stress conditions. This ACC treatment enhanced root flavonoid content and rhizosphere siderophore levels accompanied by restoration of 16S and ITS microbial community under Fe deficiency and drought. It underscores the potential of targeting ethylene signaling that may facilitate Fe mobilization and microbial interactions to enhance soybean tolerance to concurrent Fe deficiency and drought. This is the first report on the transcriptional response and requirement of Fe status underlying drought tolerance, potentially guiding future strategies for improving combined stress resilience in soybean.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142183084","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"KAI2-dependent signaling controls vegetative reproduction in Marchantia polymorpha through activation of LOG-mediated cytokinin synthesis","authors":"Aino Komatsu, Mizuki Fujibayashi, Kazato Kumagai, Hidemasa Suzuki, Yuki Hata, Yumiko Takebayashi, Mikiko Kojima, Hitoshi Sakakibara, Junko Kyozuka","doi":"10.1101/2024.09.02.610783","DOIUrl":"https://doi.org/10.1101/2024.09.02.610783","url":null,"abstract":"<em>Marchantia polymorpha</em> reproduces vegetatively (asexually) by producing propagules known as gemmae within gemma cups and sexually through spores. We previously reported that KARRIKIN INSENSITIVE 2 (KAI2)-dependent signaling promotes gemma cup and gemma formation. KAI2A perceives unidentified endogenous ligand(s), tentatively referred to as KAI2 ligands (KL). Perception of KL by KAI2 triggers MAX2-dependent proteolysis of MpSMXL. In this study, we identified genes working downstream of KAI2-dependent signaling in <em>M. polymorpha</em>. We found that KAI2-dependent signaling positively controls the expression of Mp<em>LONLEY GUY</em> (Mp<em>LOG</em>), encoding a cytokinin biosynthesis enzyme. Disruption of the Mp<em>LOG</em> function decreased endogenous cytokinin levels and caused defects similar to KAI2-dependent signaling mutants. Moreover, supplying exogenous cytokinins rescued the defects of Mp<em>log</em> and KAI2-dependent signaling mutants, implying that cytokinins work downstream of KAI2-dependent signaling. Activation of Mp<em>LOG</em> by KAI2-dependent signaling occurs in a highly cell-type-specific manner, leading to cell-specific induction of <em>GEMMA CUP-ASSOCIATED MYB1</em> (<em>GCAM1</em>), the master regulator of vegetative reproduction of <em>M. polymorpha</em>. We propose a genetic cascade, starting from KAI2-dependent signaling, that promotes vegetative reproduction through the induction of Mp<em>LOG</em> and <em>GCAM1</em>. The interaction between KAI2-dependent signaling and cytokinin in <em>M. polymorpha</em> provides a novel insight into the function and evolution of KAI2-dependent signaling.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":"93 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142183075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Raffinose induces autophagy to promote plant growth","authors":"Sahar Magen, Sahar Daniel, Shahar Weiss, David J. Factor, Sergey Mursalimov, Yoram Soroka, Simon Michaeli, Tamar Avin-Wittenberg","doi":"10.1101/2024.09.02.610801","DOIUrl":"https://doi.org/10.1101/2024.09.02.610801","url":null,"abstract":"Autophagy is a vital process in eukaryotes, maintaining cellular balance by degrading and recycling cellular components. Autophagy is triggered by various nutrient-deprivation conditions and both biotic and abiotic stresses in plants. Autophagy-deficient mutants exhibit early senescence, reduced yield, and hyper-sensitivity to starvation and abiotic stress. Over-expressing autophagy-related genes in various plant species resulted in increased plant size, yield, and stress resistance. Yet, despite the considerable promise of autophagy modulation for improved plant performance, the molecular mechanisms governing its induction remain partially understood. In the current work, we identified raffinose, a sugar linked to plant stress responses, as a novel plant autophagy inducer. Raffinose treatment resulted in increased biomass and yield in an autophagy-dependent manner in several plant species. We also show that raffinose activates autophagy through the SnRK1 kinase complex, independent of TOR signaling Our findings highlight the potential of raffinose as a tool for enhancing crop resilience and productivity.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142183029","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Application of cryo-FIB-SEM for investigating organelle ultrastructure in guard cells of higher plants","authors":"Bastian L Franzisky, Xudong Zhang, Claus Jakob Burkhardt, Endre Majorovits, Eric Hummel, Andreas Schertel, Christoph-Martin Geilfus, Christian Zoerb","doi":"10.1101/2024.08.30.610476","DOIUrl":"https://doi.org/10.1101/2024.08.30.610476","url":null,"abstract":"Stomata are vital for CO2 and water vapor exchange, with guard cells' aperture and ultrastructure highly responsive to environmental cues. However, traditional methods for studying guard cell ultrastructure, which rely on chemical fixation and embedding, often distort cell morphology and compromise membrane integrity, leaving no suitable methodology until now. In contrast, plunge-freezing in liquid ethane rapidly preserves cells in a near-native vitreous state for cryogenic electron microscopy. Using this approach, we applied Cryo-Focused Ion Beam-Scanning Electron Microscopy (cryo-FIB-SEM) to study the guard cell ultrastructure of Vicia faba, a higher plant model chosen for its sensitivity to external factors and ease of epidermis isolation, advancing beyond previous cryo-FIB-SEM applications in lower plant algae. The results firstly introduced cryo-FIB-SEM volume imaging, enabling subcellular ultrastructure visualization of higher plants like V. faba in a vitrified, unaltered state. 3D models of organelles such as stromules, chloroplast protrusions, chloroplasts, starch granules, mitochondria, and vacuoles were reconstructed from cryo-FIB-SEM volumetric data, with their surface area and volume initially determined using manual segmentation. Future studies using this near-native volume imaging technique hold promise for investigating how environmental factors like drought or salinity influence stomatal behavior and the morphology of guard cells and their organelles.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":"33 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142183083","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of root exudates of black oat in the presence of interspecific weed species neighbours and intraspecific neighbours, and their effects on root traits","authors":"Cagla G. Eroglu, Alexandra A. Bennett, Teresa Steininger-Mairinger, Stephan Hann, Markus Puschenreiter, Judith Wirth, Aurelie Gfeller","doi":"10.1101/2024.09.02.610795","DOIUrl":"https://doi.org/10.1101/2024.09.02.610795","url":null,"abstract":"Root exudates are composed of primary and secondary organic compounds that function as signalling molecules and play important roles in plant-environment interactions. The quantity and composition of root exudates vary depending on the species, genotype, and environmental conditions, including the presence and identity of neighbouring plants. Although cover crops are commonly used in agricultural practices for their ecosystem services, such as weed suppression, pathogen control, and soil structure improvement, studies on their root exudates are limited. Our study provides the first characterization of the root exudates of black oat interacting with weed neighbours, redroot pigweed and blackgrass, as well as with neighbours of the same species, black oat. We investigated how these interactions influence the black oat root exudation patterns. Furthermore, we investigated the impact of black oat presence on neighbours and how exposure to black oat root exudate treatments affects the root traits of weeds. The upregulated compounds detected in root exudates in response to neighbouring plants primarily belonged to the organic oxygen compounds superclass, with most of which identified as amino acids and carbohydrates. In the presence of redroot pigweed, a general increase in root exudation was observed, with amino acids and sugar sulphates being upregulated. The presence of black oat had varying effects among the neighbouring plants. While significant decreases observed and black oat in redroot pigweed root traits, an increase was observed in blackgrass. Similarly, more pronounced effects were observed in redroot pigweed compared to blackgrass upon root exudate application. This study provides a characterization and insights into the dynamic nature of root exudates and their influence on root traits and plant interactions.","PeriodicalId":501341,"journal":{"name":"bioRxiv - Plant Biology","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142183034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}