RESSALVA Atendendo a solicitação do autor, o texto completo desta dissertação será disponibilizado somente a partir de 02/09/2024. SÃO PAULO STATE UNIVERSITY - UNESP SCHOOL OF ENGINEERING CAMPUS OF ILHA SOLTEIRA GUSTAVO DOS SANTOS COTRIM EFFECT OF POTASSIUM AVAILABILITY ON SOYBEAN METABOLISM BY INTEGRATED METABOLOMICS AND IONOMICS ANALYSIS Ilha Solteira 2022 GRADUATE PROGRAM IN AGRONOMY GUSTAVO DOS SANTOS COTRIM EFFECT OF POTASSIUM AVAILABILITY ON SOYBEAN METABOLISM BY INTEGRATED METABOLOMICS AND IONOMICS ANALYSIS Dissertation submitted to Engineering College of Ilha Solteira (FEIS - UNESP) in partial fulfillment of the requirements for the degree of Master of Science in Agronomy. Emphasis: Cropping Systems. Prof. Dr. Lucíola Santos Lannes Supervisor Prof. Dr. Clara Beatriz Hofmann Campo Co-supervisor Ilha Solteira 2022 Cotrim Effect of Potassium Availability on Soybean Metabolism by Integrated Metabolomics and Ionomics AnalysisIlha Solteira2022 65 Sim Dissertação (mestrado)Outros cursosSistemas de ProduçãoNão . . . FICHA CATALOGRÁFICA Desenvolvido pelo Serviço Técnico de Biblioteca e Documentação Cotrim, Gustavo dos Santos. Effect of potassium availability on soybean metabolism by integrated metabolomics and ionomics analysis / Gustavo dos Santos Cotrim. -- Ilha Solteira: [s.n.], 2022 65 f. : il. Dissertação (mestrado) - Universidade Estadual Paulista. Faculdade de Engenharia de Ilha Solteira. Área de conhecimento: Sistemas de Produção, 2022 Orientadora: Lucíola Santos Lannes Coorientadora: Clara Beatriz Hoffmann-Campo Inclui bibliografia 1. Glycine max. 2. Fabaceae. 3. Potassium deficiency. 4. Phytoalexins. 5. Specialised metabolism. 6. Abiotic stress. C845e UNIVERSIDADE ESTADUAL PAULISTA Câmpus de Ilha Solteira EFFECT OF POTASSIUM AVAILABILITY ON SOYBEAN METABOLISM BY INTEGRATED METABOLOMICS AND IONOMICS ANALYSIS   TÍTULO DA DISSERTAÇÃO: CERTIFICADO DE APROVAÇÃO AUTOR: GUSTAVO DOS SANTOS COTRIM ORIENTADORA: LUCÍOLA SANTOS LANNES COORIENTADORA: CLARA BEATRIZ HOFFMANN-CAMPO Aprovado como parte das exigências para obtenção do Título de Mestre em Agronomia, área: Sistemas de Produção pela Comissão Examinadora: Profa. Dra. LUCÍOLA SANTOS LANNES (Participaçao Virtual) Departamento de Biologia e Zootecnia / Faculdade de Engenharia de Ilha Solteira - UNESP Profa. Dra. CRISTIÉLE DA SILVA RIBEIRO (Participaçao Virtual) Departamento de Biologia e Zootecnia / Faculdade de Engenharia de Ilha Solteira UNESP Prof. Dr. JHONYSON ARRUDA CARVALHO GUEDES (Participaçao Virtual) Química Analítica e Físico-Química / Universidade Federal do Ceará - UFC Ilha Solteira, 02 de setembro de 2022 Faculdade de Engenharia - Câmpus de Ilha Solteira - Av. Brasil Centro, 56, 15385000 www.feis.unesp.br/#!/ppgaCNPJ: 48.031.918/0015-20. DEDICATION This work is completely dedicated to respectful memory of my grandparents Jacira Brizola dos Santos and Ranili Custodio Cotrim ACKNOWLEDGMENTS I would like to express my gratitude to: My supervisor Prof. Dr. Lucíola Santos Lannes not only enlightened me with academic knowledge but also gave me valuable advice. At the same time, my thanks for your productive discussions, encouragement, support, and opportunity conceived in the Graduate Program in Agronomy of São Paulo State University – UNESP. My co-supervisor Prof. Dr. Clara Beatriz Hoffmann Campo has generously provided me with her time, insight, continuous support, encouragement, and improving the quality of my writing. I express gratitude for the opportunity to research activities in Chemical Ecology Laboratory in Brazillian Agricultural Research Corporation – Embrapa Soybean. Dr. Adilson de Oliveira Junior, Dr. Cesar de Castro, and Dr. Guilherme Julião Zocolo for their technical support and encouragement. Dr. Deivid Metzker da Silva, Dr. José Perez da Graça, and Dr. Rejane Stubs Parpinelli for their experimental procedures support, useful discussion, suggestions, and friendship. Finally, thank you to my family and friends who have been there for me throughout graduate school. This especially includes my mother Sandra, my father Reginaldo, and my sister Ana Beatriz. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Financial Code 001. RESUMO O potássio (K+) tem funções fisiológicas vitais nas plantas e sua disponibilidade pode impactar a tolerância de plantas a condições de estresse biótico e abiótico. Anteriormente, estudos limitados investigaram o efeito da adubação potássica no metabolismo da soja. Neste trabalho, a utilização de análises ômicas integradas, metabolômica e ionômica, permitiram investigar a resposta de plantas de soja (Glycine max) em campo sob quatro níveis de disponibilidade de K+: muito baixa (KVL), baixa (KL), média (KM) e muito alta (KVH). Folhas (V7) e tecidos da vagem (R5.5) coletados foram submetidos à extração e analisados por cromatografia líquida de ultra eficiência acoplada à espectrometria de massa de alta resolução (UPLC-HRMS) e espectroscopia de emissão óptica por plasma acoplado indutivamente (ICP-OES). O emprego de modelos multivariados (PCA-X&Y e O2PLS-DA) permitiram identificar 51 compostos pertencentes a 19 vias metabólicas regulados diferencialmente nos tecidos de plantas que se desenvolveram sob condições contrastantes de disponibilidade de K+ (KVL vs. KVH). Os níveis de potássio também influenciaram o rendimento de grãos com menores níveis em KVL (2211 kg ha-1) e KL (3737 kg ha-1), diferindo dos tratamentos KM e KVH (4093 e 4096 kg ha-1, respectivamente). Sob baixíssima concentração de K+, os teores de Ca2+, Mg2+, Fe2+, Cu2+ e B aumentaram nas folhas jovens e maduras. Não somente, o conteúdo de isoflavonas, coumestanos, pterocarpanos e sojasaponinas mostraram-se positivamente regulados nas folhas severamente deficientes em K+, estando estes eventos associados a um possível estado de estresse oxidativo e fotodinâmico decorrentes da deficiência nutricional. A abordagem ômica integrada revelou que a adubação potássica é responsável por promover o aumento da biossíntese de carboidratos, galactolipídeos e flavonóis glicosídicos em folhas e vagens. No entanto, os tecidos da vagem deficientes em K+ apresentaram aumento no contéudo de aminoácidos, oligossacarídeos, derivados do ácido benzoico e isoflavonas. O aminoácido asparagina é majoritariamente acumulado nos tecidos deficientes em potássio, sinalizando como possível biomarcador para a deficiência deste macronutriente na soja. Além disso, os resultados indicam que a regulação positiva de metabólitos especializados constitutivos detectados por UPLC-HRMS não estão diretamente associados com o aumento nos níveis de K+ no solo. Em geral, a presente contribuição melhora nossa compreensão sobre como o metabolismo especializado da soja é dependente e/ou influenciado pela nutrição potássica. Palavras-chave: Glycine max; Fabaceae; Deficiência de Potássio; Fitoalexinas; Metabolismo Especializado; Estresse Abiótico; Metabolômica; Ionômica. ABSTRACT Potassium (K+) has vital physiological functions in plants and its availability can impact the tolerance of species to biotic and abiotic stress conditions. Limited studies have investigated the effect of K+ fertilization on soybean metabolism. Using integrated omics, ionomics and metabolomics, we investigated the response of field-grown soybean (Glycine max ) to four rates of soil K+ availability: very low (KVL), low (KL), medium (KM), and very high (KVH). Soybean trifoliate leaf (V7) and pod tissue (R5.5) extracts were analysed by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) and inductively coupled plasma optical emission spectroscopy (ICP-OES). Multivariate analyses showed that 51 compounds of 19 metabolic pathway maps were regulated in response to K+ availability. The soybean yield parameters also were influenced in plants under very low (KVL; 2211 kg ha-1) and low (KL; 3737 kg ha-1) differing from KM and KVH (4093 and 4096 kg ha- 1, respectively) treatments. Under very low potassium availability, soybean plants promoted the accumulation of Ca2+, Mg2+, Fe2+, Cu2+, and B in young and old leaves. Not only, isoflavones, coumestans, pterocarpans, and soyasaponins also were elicited in severely K+ deficient trifoliate leaves, which can be associated with oxidative and photodynamic stress status. Potassium fertilization upregulated carbohydrate, galactolipid, and flavonol glycoside biosynthesis in leaves and pod valves, while K+ deficient pod tissues showed increasing contents of amino acids, oligosaccharides, benzoic acid derivates, and isoflavones. Additionally, results demonstrate that asparagine content is higher in potassium deficient tissues, which suggests being a biomarker of K+ deficiency in soybean plants. These results demonstrate that potassium soil fertilization did not linearly contribute to changes in specialised constitutive metabolites of soybean. Altogether, this work provides a reference for improving the understanding of soybean metabolism as dependent on K+ availability. Keywords: Glycine max; Fabaceae; Potassium deficiency; Phytoalexins; Specialised metabolism; Abiotic stress; Metabolomics; Ionomics. TABLE OF CONTENTS 1 INTRODUCTION................................................................................................ 11 2 MATERIAL AND METHODS............................................................................. 14 2.1 GENERAL EXPERIMENTAL PROCEDURES..................................................... 14 2.2 EXPERIMENTAL DESIGN AND ASSAY............................................................ 14 2.3 PLANT MATERIALS............................................................................................. 15 2.4 SOIL CHEMICAL ANALYSIS.............................................................................. 15 2.5 PLANT EXTRACTION........................................................................................... 15 2.5.1 Extraction Process for Plant Metabolomics......................................................... 16 2.5.2 Extraction Process for Plant Ionomics................................................................. 16 2.6 INSTRUMENTAL ANALYSES............................................................................. 16 2.6.1 Chromatographic Analysis (UPLC)..................................................................... 16 2.6.2 Mass Spectrometry (ESI-QTof-MSE)................................................................... 17 2.6.3 Inductively Coupled Plasma (ICP-OES).............................................................. 17 2.7 YIELD PARAMETERS........................................................................................... 18 2.8 DATA INTERPRETATION.................................................................................... 18 2.8.1 Univariate Analysis................................................................................................ 18 2.8.2 Pre-processing and Chemometrics analysis…..................................................... 18 2.8.3 Metabolite Annotation and Molecular Networking............................................ 19 3 RESULTS AND DISCUSSION............................................................................. 21 3.1 SOYBEAN LEAF IONOMICS ANALYSIS........................................................... 21 3.2 SOYBEAN POD IONOMICS ANALYSIS............................................................. 24 3.3 YIELD PARAMETERS........................................................................................... 26 3.4 CHEMOMETRICS ANALYSIS............................................................................. 27 3.5 MOLECULAR NETWORKING AND METABOLIC PATHWAY...................... 30 3.6 SOYBEAN LEAVES UNDER K+ HOMEOSTASIS.............................................. 33 3.7 SOYBEAN POD TISSUES UNDER K+ HOMEOSTASIS.................................... 34 3.8 SOYBEAN LEAVES UNDER K+ DEFICIENCY.................................................. 37 3.9 SOYBEAN POD TISSUES UNDER K+ DEFICIENCY......................................... 42 4 CONCLUDING REMARKS............................................................................... 44 REFERENCES..................................................................................................... 45 APPENDIX A – Soil Chemical Atrributes........................................................... 57 APPENDIX B – Leaves Tukey’s t-value............................................................... 58 APPENDIX C – Leaves Growth Region Tukey’s t-value................................... 59 APPENDIX D – Pod tissues Tukey’s t-value........................................................ 60 APPENDIX E – Pod tissues Studen’t t-value....................................................... 61 APPENDIX F – Multivariate Analysis Values..................................................... 62 APPENDIX G – Putative Metabolite Identification............................................ 63 11 1 INTRODUCTION Potassium (K+) is an important macronutrient that plays a vital role in metabolic processes, growth, and adaptation to environmental stresses (HASANUZZAMAN et al., 2018; PANDEY; MAHIWAL, 2020). Many physiological and biochemical processes are dependent on K+ availability, such as cell turgidity regulation, long-distance solute transport, enzymatic regulation (AMTMANN et al., 2008; HUBER; ARNY, 1985), photosynthesis, protein synthesis, and membrane transport (HAFSI et al., 2014; MARSCHNER, 2012). Balanced K+ nutrition can promote plant tolerance to stress, as flooding (MUGNAI et al., 2011), low temperatures (DEVI et al., 2012), drought (WANG et al., 2013), and salinity (ALMEIDA et al., 2017). In contrast, plants under K+ deficiency showed oxidative stress status (HAFSI et al., 2014; HASANUZZAMAN et al., 2018; HERNANDEZ et al., 2012) modifying heavily metabolite biosynthesis (ARMENGAUD et al., 2009; CUI et al., 2019; GAALICHE et al., 2019). Potassium is responsible for activation of nearly 60 enzymes taking part in carbon and nitrogen metabolism (AMTMANN et al., 2008). Plants under adequate K+ levels synthesise large biomolecules, as cellulose, starch, and proteins (HAZANUZZAMAN et al., 2018). However, under K+ deficiency the content of small molecules, as free sugars, amino acids, organic acids, and amides increases (PRASAD et al., 2010). Also, we must not disregard that the impact of K+ supply in plant metabolism is multifaceted, tissue and species-specific, as observed in the responses to stresses in leaves and roots of tomatoes (WEINERT et al., 2021) and oil palm sapling (CUI et al., 2019). The prevalent view is that a high K+ availability decreases the severity of diseases, as caused by Cercospora kikuchii (ITO et al., 1993; MEYER; KLEPKER, 2007) and Phakopsora pachyrhizi (BALARDIN et al., 2006; PINHEIRO et al., 2011) or insect incidence in plants (PERRENOUD, 1990; SEVERTSON et al., 2016). Therefore, farmers are generally instructed to increase K+ rates to improve crop health (GAO et al., 2018; POTASH AND PHOSPHATE INSTITUTE, 1998; SEIXAS et al., 2020). In spite of the assumption that in most related cases K+ fertilization decreases the incidence of the diseases, the opposite effect is also reported (DAVIS et al., 2018; PERRENOUD, 1990), as a 63% decrease and 28% increase in insect and mite incidence in plants associated to potassium nutrition (AMTMANN et al., 2008; PERRENOUD, 1990). Potassium deficiency has also a considerable effect on plant metabolism, including alterations in the profile, concentration, and distribution of many metabolites in different tissues 12 (AMTANN et al., 2008; HAZANUZZAMAN et al., 2018). Likewise, there is a consensus that specialised metabolism is crucial for plant susceptibility and defence against insects or pathogens (AMTMANN et al., 2008; DAVIS et al., 2018; GAO et al., 2018). Nevertheless, how plants under K+ contrasting availability can cope with biotic stresses remains unclear, and according to Marschner (2012) the understanding of the impacts of K+ availability on specialised metabolism remains incipient when compared to the K+ effect on physiological and primary metabolism processes. Increase content of reducing sugars (primary metabolism) and glucosinolates (specialised metabolism) are potentially relevant to insects and/or pathogens, as detected in Arabidopsis thaliana under K+ deficiency (ARMENGAUD et al., 2009; TROUFFLARD et al., 2010). Similarly, plants of Hordeum vulgare under K+ deficiency increased jasmonic acid and other oxylipins content sensitive to the pathogen Blumeria graminis (DAVIS et al., 2018). In Glycine max (L.) Merr. (Fabaceae) field experiments with K+ fertilization revealed multiple inducing mechanisms improving plant resistance to cyst nematode Heteroda glycines via root exudation of phenolic acids and plant pathogen-related genes (GAO et al., 2018). Furthermore, the metabolomic analysis revealed differences in gentisic acid and shikimic acid content from exudates of soybean roots, when plants are grown under low K+ conditions (TANTRIANI et al., 2020). Previous studies of soybean metabolite content influenced by K+ fertilization used only methodologies that allowed the detection of few target compounds (HU et al., 2015; SEGUIN; ZHENG, 2006; VYN et al., 2002) or analytical methods covering primary metabolism (TANTRIANI et al., 2020). Certainly, these results improved our knowledge, but not enough to understand how soybean under K+ deficiency or adequate nutrition has constitutive specialised metabolites that can respond differently in plant interactions. Recently, studies comprising integrated omics greatly contributed to elucidating the influence of nutrients on plant metabolism (CUI et al., 2019; TANTRIANI et al., 2020; ZHAO et al., 2020). Ionomics and metabolomics analyses showed that K+ deficiency and waterlogging in sunflower are not simply additive stresses, but also influence respiration as well nitrogen metabolism when associated with biomarkers (CUI et al., 2019). However, it remains unknown whether K+ availability can influence the constitutive content of soybean specialised metabolites with a recognized role in biotic and abiotic interactions; thus, research methodologies based on untargeted metabolomics allow investigating of metabolome changes on a larger scale (FENG et al., 2020; YANG et al., 2018). 13 We hypothesize that K+ rates in soil fertilization can promote an increase in specialised metabolite profiles of soybeans. This work aimed to advance a step towards understanding the effect of K+ nutrition on plant interactions, and it is expected that these results can provide new insights into the importance of potassium in soybean metabolism. Therefore, we carried out untargeted metabolomics and ionomics analysis to compare the metabolic profile of trifoliate leaves and pod tissues (valves and immature seeds) of soybean influenced by K+ availability. The contributions of this work allowed us to suggest the correlation of the soybean metabolite phenotype with K+ status and list its importance previously reported in biotic and abiotic interactions. 44 4 CONCLUDING REMARKS Untargeted metabolomics reveals 51 primary and specialised metabolites that act in 19 biochemical pathways that were influenced by K+ availability. Potassium is not a structural component of plant metabolites in comparison with phosphorus and nitrogen, but under very high K+ availability, the upregulation of flavonoids, galactolipids, and carbohydrates may be resulting from enzymatic activity increasing. In contrast, the biochemical mechanisms of soybeans in response to very low K+ availability are associated to: (I) accumulation of Ca2+, Mg2+, Fe2+, Cu2+, and B in young and old leaves; (II) increase of Fe2+ and Cu2+ content in immature seeds and pod valves, respectively; (III) leaf metabolism reprogramming for the biosynthesis of phytoalexins, such as isoflavones, coumestans, pterocarpans, and soyasaponins; (IV) enhancement of the amino acids, mono and oligosaccharides, benzoic acid derivates, and isoflavones content in soybean pods (IS and PV); (V) L-asparagine accumulation in vegetative (leaves) and reproductive (pod tissues) structures. Overall, the results suggest that soybean metabolism reprogramming is associated with oxidative stress caused by low K+ availability, and shows an increased content of phytoalexins (antioxidant agents), as isoflavonoids and triterpenoid saponins. Additionally, results suggest that L-asparagine is a promising soybean biomarker under K+ deficiency. Although the literature shows that plants fertilized with K+ are more tolerant/resistant to biotic and abiotic stresses, our data reveals that increasing fertilization rates did not promote a substantial increase in the content of constitutive specialised metabolites in soybean, refuting our initial hypothesis. We understood that this interaction is more complex and thus further work using integrated omics is certainly required, to understand whether the profile of metabolites induced by biotic elicitors (e.g., insects or pathogens) may be associated to K+ content in plants. 45 REFERENCES AHMAD, P.; ASHRAF, M.; HAKEEM, K. 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