RAFAEL AGOSTINHO FERREIRA FROM SEEDS TO PLANTS: THE USE OF PRIMING TO IMPROVE SALT TOLERANCE IN SORGHUM LAVRAS- MG 2024 RAFAEL AGOSTINHO FERREIRA FROM SEEDS TO PLANTS: THE USE OF PRIMING TO IMPROVE SALT TOLERANCE IN SORGHUM DE SEMENTES A PLANTAS: O USO DO PRIMING PARA AUMENTAR A TOLERÂNCIA DO SORGO À SALINIDADE Tese apresentada à Universidade Federal de Lavras, como parte das exigências do Programa de Pós-Graduação em Agronomia/Fisiologia Vegetal, área de concentração em Fisiologia Vegetal, para obtenção do título de Doutor. Prof.ª Dr.ª Elisa Monteze Bicalho Orientadora PhD. Sershen Naidoo Coorientador LAVRAS - MG 2024 Ficha catalográfica elaborada pelo Sistema de Geração de Ficha Catalográfica da Biblioteca Universitária da UFLA, com dados informados pelo(a) próprio(a) autor(a). Ferreira, Rafael Agostinho. From seeds to plants: The use of priming to improve salt tolerance in sorghum / Rafael Agostinho Ferreira. - 2022 100 p. : il. Orientador(a): Elisa Monteze Bicalho. Coorientador(a): Sershen Naidoo . Tese (doutorado) - Universidade Federal de Lavras, 2022. Bibliografia. 1. oxidative stress. 2. hormopriming. 3. abiotic stress. I. Bicalho, Elisa Monteze. II. , Sershen Naidoo. III. Título. O conteúdo desta obra é de responsabilidade do(a) autor(a) e de seu orientador(a). RAFAEL AGOSTINHO FERREIRA FROM SEEDS TO PLANTS: THE USE OF PRIMING TO IMPROVE SALT TOLERANCE IN SORGHUM DE SEMENTES A PLANTAS: O USO DO PRIMING PARA AUMENTAR A TOLERÂNCIA DO SORGO À SALINIDADE Tese apresentada à Universidade Federal de Lavras, como parte das exigências do Programa de Pós-Graduação em Agronomia/Fisiologia Vegetal, área de concentração em Fisiologia Vegetal, para obtenção do título de Doutor. Aprovada em 28 de outubro de 2022. Drª. Mariana Aline Silva Artur- WUR Drª. Heloísa Oliveira dos Santos – UFLA Drª. Vanessa Cristina Stein – UFLA Dr. Eduardo Gusmão Pereira - UFV Prof.ª Dr.ª Elisa Monteze Bicalho Orientadora PhD. Sershen Naidoo Coorientador LAVRAS-MG 2024 No te des por vencido, ni aun vencido, no te sientas esclavo, ni aun esclavo; trémulo de pavor, piénsate bravo, y arremete feroz, ya mal herido. Ten el tesón del clavo enmohecido que ya viejo y ruin, vuelve a ser clavo; no la cobarde estupidez del pavo que amaina su plumaje al primer ruido. Procede como Dios que nunca llora; o como Lucifer, que nunca reza; o como el robledal, cuya grandeza necesita del agua y no la implora... Que muerda y vocifere vengadora, ya rodando en el polvo, tu cabeza! ¡PIU AVANTI! – Almafuerte AGRADECIMENTOS Ao Programa de Pós-Graduação em Fisiologia Vegetal da UFLA e seus professores, pelo acesso ao conhecimento e estrutura, tudo isso foi crucial para que este trabalho fosse executado. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Código de financiamento 001. A minha orientadora, Elisa Monteze Bicalho, pelo apoio e orientação durante todos estes anos, sempre disposta colaborar para que os resultados fossem sempre melhores do que o esperado. Ao professor João Paulo, pela recepção no programa e indicação à minha orientadora. Aos Professores Paulo Marchiori e Antonio Chalfun por permitirem acesso irrestrito aos seus laboratórios. Ao Professor Eduardo Gusmão pelo empréstimo de equipamentos sem os quais não poderia ter realizados as análises do capítulo III. Todo e qualquer agradecimento aqui contido será pouco para o que me foi proporcionado. Primeiramente, devo agradecer a minha esposa Paola, a quem dedico este trabalho. Sem seus conselhos e ombro amigo em momentos de crises não teria conseguido chegar até aqui. Sempre me apoiando e dando suporte entre todas as idas e vindas de Lavras a Juiz de Fora. Aos meus pais Elisa e Márcio, pelo apoio em todas as minhas escolhas. Aos meus avós maternos, Isaura e Sebastião Agostinho (in memoria), meus grandes incentivadores. Sempre me dando condições para que pudesse alcançar meus sonhos. Aos amigos de UFLA, Aline, Victor e Jober, por permitirem em diversas vezes que o cansaço do trabalho não fosse maior que o prazer de realizá-lo. Sem vocês este trabalho não teria sido executado da maneira que foi. Ao meu amigo Mateus, por compartilhar bons momentos durante a execução dos trabalhos, e ao chegar em casa, poder desfrutar de uma cerveja e uma boa música. Pelo apoio nas análises no laboratório até altas hora e pelas discussões dos dados. A minha cachorra Summer, que sempre esteve ao meu lado, e quando as coisas ficavam críticas estava sempre disposta a me proporcionar uma boa caminhada. A todos que de alguma forma contribuíram, meus sinceros agradecimentos. RESUMO Diversas condições ambientais podem restringir a germinação e o estabelecimento de plantas, sendo a salinidade elevada uma das mais críticas. Essa condição decorre da alta concentração de íons no solo, que provoca alterações metabólicas essenciais, afetando tanto a germinação quanto o desenvolvimento das plantas. A salinidade gera dois tipos de estresse: (i) osmótico, causado pela redução do potencial osmótico da solução do solo, e (ii) iônico, devido à dissociação de íons, que danifica membranas e outras estruturas celulares. Em regiões semiáridas e áridas, a necessidade de cultivar espécies tolerantes à salinidade é urgente, e o sorgo (Sorghum bicolor (L.) Moench), embora moderadamente tolerante, possui estágios críticos, como a germinação e o estabelecimento de plântulas, que são altamente sensíveis aos efeitos salinos. Uma alternativa promissora para mitigar esses efeitos é a técnica de priming, que melhora a germinação e o desenvolvimento das plantas. Este trabalho investigou a hipótese de que o priming com moléculas antioxidantes e fitoreguladores oferece melhores condições para a germinação e o crescimento do sorgo sob estresse salino. Para compreender os mecanismos de tolerância, foram realizados experimentos em laboratório e em casa de vegetação. No primeiro capítulo, avaliou-se a resposta de sementes de quatro variedades de sorgo submetidas ao priming com ácido ascórbico, ácido abscísico e hidropriming, expostas a diferentes concentrações de NaCl e estresse osmótico induzido por PEG-6000. Os testes analisaram parâmetros como porcentagem de germinação, índice de velocidade de germinação e tempo médio para germinação de 50% das sementes. No segundo capítulo, realizaram-se análises bioquímicas em duas cultivares contrastantes (BRS-332 e DKB 540), avaliando o metabolismo de carboidratos e aminoácidos, níveis de peróxido de hidrogênio e malondialdeído, além da atividade de enzimas antioxidantes. Já o terceiro capítulo abordou experimentos em casa de vegetação, nos quais as plantas oriundas de sementes tratadas com priming foram acompanhadas por 65 dias sob estresse salino. Parâmetros como fluorescência da clorofila a, trocas gasosas e marcadores relacionados à tolerância à salinidade identificados nas sementes foram analisados em diferentes estágios fenológicos. Os resultados evidenciaram que as cultivares possuem mecanismos distintos de tolerância ao estresse osmótico e salino. O priming promoveu efeitos positivos, especialmente pelo acúmulo de prolina e açúcares, que facilitaram o ajustamento osmótico e mitigaram o estresse oxidativo em sementes, plântulas e plantas adultas. Esses efeitos podem ser atribuídos à "memória do priming", um fenômeno que permite a recuperação de alterações metabólicas induzidas pelo tratamento inicial, resultando em melhor manutenção do metabolismo fotossintético e redução dos impactos do estresse salino ao longo dos diferentes estágios fenológicos. Dessa forma, o priming se apresenta como uma ferramenta eficaz para melhorar a tolerância do sorgo ao estresse salino, contribuindo para o desenvolvimento de estratégias sustentáveis que permitem o cultivo em áreas salinizadas, ampliando o potencial produtivo e a resiliência agrícola em regiões adversas. Palavras-chave: estresse oxidativo; mudanças climáticas; hormopriming; estresse abiótico; prolina. ABSTRACT Various environmental conditions can restrict the germination and establishment of plants, with high salinity being one of the most critical. This condition arises from the high concentration of ions in the soil, leading to essential metabolic alterations that affect both germination and plant development. Salinity induces two types of stress: (i) osmotic stress, caused by the reduction of the osmotic potential in the soil solution, and (ii) ionic stress, resulting from ion dissociation, which damages membranes and other cellular structures. In semi-arid and arid regions, there is an urgent need to cultivate species with some level of salt tolerance. Sorghum (Sorghum bicolor (L.) Moench), although moderately tolerant, exhibits critical stages such as germination and seedling establishment, which are highly sensitive to salinity effects. A promising approach to mitigating these effects is the priming technique, which enhances germination and plant development. This study investigated the hypothesis that priming with antioxidant molecules and growth regulators provides better conditions for sorghum germination and growth under salt stress. To understand the tolerance mechanisms, experiments were conducted both in laboratories and greenhouse conditions. In the first chapter, the response of seeds from four sorghum varieties subjected to priming with ascorbic acid, abscisic acid, and hydropriming was evaluated under different NaCl concentrations and osmotic stress induced by PEG-6000. Tests analyzed parameters such as germination percentage, germination speed index, and the mean time for 50% seed germination. In the second chapter, biochemical analyses were performed on two contrasting cultivars (BRS-332 and DKB 540), assessing carbohydrate and amino acid metabolism, hydrogen peroxide and malondialdehyde levels, and antioxidant enzyme activity. The third chapter involved greenhouse experiments in which plants grown from seeds treated with priming were monitored over 65 days under salt stress. Parameters such as chlorophyll a fluorescence, gas exchange, and markers related to salt tolerance identified in the seeds were analyzed during different phenological stages. The results showed that the cultivars exhibit distinct tolerance mechanisms to osmotic and salt stress. Priming induced positive effects, particularly through the accumulation of proline and sugars, which facilitated osmotic adjustment and mitigated oxidative stress in seeds, seedlings, and adult plants. These effects can be attributed to "priming memory," a phenomenon that allows the recovery of metabolic alterations induced by the initial treatment, resulting in better maintenance of photosynthetic metabolism and reduced impacts of salt stress across different phenological stages. In conclusion, priming is an effective tool for enhancing sorghum tolerance to salt stress, contributing to the development of sustainable strategies that enable cultivation in saline areas. This approach broadens productive potential and agricultural resilience in adverse regions, making it a valuable technique for ensuring food security and sustainable agricultural practices in the face of environmental challenges. Key words: oxidative stress; climate change; hormopriming; abiotic stress, proline. INDICADORES DE IMPACTO A aplicação do priming em sementes de sorgo (Sorghum bicolor (L.) Moench) sob condições de estresse salino trouxe impactos notáveis em diversas dimensões: social, tecnológica, econômica e cultural. Socialmente, a técnica contribui para a segurança alimentar em regiões áridas e semiáridas, onde a salinização do solo é uma barreira significativa para a produção agrícola. Ao permitir o cultivo de uma cultura amplamente utilizada como o sorgo, especialmente em comunidades dependentes da agricultura para subsistência, o priming ajuda a mitigar os efeitos das mudanças climáticas e das condições adversas do solo. Além disso, ao aumentar a resiliência das plantas a ambientes desafiadores, essa abordagem promove o fortalecimento de pequenos agricultores, especialmente em áreas vulneráveis, promovendo maior autonomia e estabilidade. Do ponto de vista tecnológico, o priming representa um avanço significativo nas práticas de tratamento de sementes. Ao utilizar moléculas antioxidantes, como o ácido ascórbico, e fitoreguladores, como o ácido abscísico, a técnica possibilita melhorias no processo germinativo e na tolerância das plantas durante diferentes estágios fenológicos. A memória metabólica induzida pelo priming, evidenciada pelo acúmulo de prolina e açúcares, destaca-se como um mecanismo inovador que permite às plantas ajustarem seu metabolismo osmótico, reduzindo os danos causados pelo estresse salino e oxidativo. Essa abordagem abre caminhos para novos estudos e implementações em outras culturas agrícolas, ampliando o escopo de aplicação da tecnologia. Economicamente, os benefícios são expressivos. O aumento na germinação e no vigor inicial das plântulas gera plantas mais saudáveis e produtivas, resultando em maior rendimento por área cultivada. Esse fator é crucial em regiões onde a disponibilidade de recursos é limitada e a maximização da produtividade se torna indispensável. Além disso, a utilização do priming pode reduzir a necessidade de insumos químicos, como fertilizantes e defensivos, contribuindo para a diminuição de custos de produção. A longo prazo, a técnica também favorece a sustentabilidade econômica, pois culturas mais tolerantes ao estresse salino demandam menos recursos hídricos de alta qualidade, otimizando o uso da água em regiões com escassez hídrica. Culturalmente, o priming desempenha um papel importante ao integrar práticas agrícolas sustentáveis e tecnológicas nas rotinas de agricultores locais. Em regiões onde a agricultura é mais do que uma atividade econômica, mas parte da identidade cultural, o uso de métodos como o priming pode impulsionar uma transição para práticas mais resilientes e conscientes. Além disso, o sucesso da técnica reforça a valorização de culturas adaptadas a condições adversas, como o sorgo, que possuem relevância histórica e cultural em diversas comunidades. Em suma, a aplicação do priming em sementes de sorgo vai além do aumento da tolerância ao estresse salino, gerando impactos positivos em diversas dimensões da sociedade. Ao promover inovação tecnológica, garantir maior segurança alimentar, reduzir custos de produção e valorizar práticas agrícolas tradicionais adaptadas a novas realidades, a técnica se consolida como uma solução sustentável e transformadora, alinhada às necessidades de um mundo em constante mudança. IMPACT INDEX The application of priming in sorghum seeds (Sorghum bicolor (L.) Moench) under salt stress conditions has brought notable impacts across various dimensions: social, technological, economic, and cultural. Socially, the technique contributes to food security in arid and semi- arid regions, where soil salinization poses a significant barrier to agricultural production. By enabling the cultivation of a widely used crop like sorghum, especially in communities reliant on agriculture for subsistence, priming helps mitigate the effects of climate change and adverse soil conditions. Furthermore, by enhancing plant resilience in challenging environments, this approach empowers small-scale farmers, particularly in vulnerable areas, fostering greater autonomy and stability. From a technological perspective, priming represents a significant advancement in seed treatment practices. By using antioxidant molecules, such as ascorbic acid, and growth regulators, such as abscisic acid, the technique enables improvements in germination processes and plant tolerance during different phenological stages. The metabolic memory induced by priming, evidenced by the accumulation of proline and sugars, stands out as an innovative mechanism that allows plants to adjust their osmotic metabolism, reducing damage caused by salt and oxidative stress. This approach paves the way for further studies and applications in other agricultural crops, broadening the scope of this technology. Economically, the benefits are substantial. Increased germination and initial seedling vigor lead to healthier and more productive plants, resulting in higher yields per cultivated area. This is crucial in regions where resources are limited, and maximizing productivity is essential. Additionally, priming can reduce the need for chemical inputs, such as fertilizers and pesticides, contributing to lower production costs. In the long term, the technique also favors economic sustainability, as crops more tolerant to salt stress require fewer high-quality water resources, optimizing water usage in regions with water scarcity. Culturally, priming plays an important role in integrating sustainable and technological agricultural practices into the routines of local farmers. In regions where agriculture is more than just an economic activity but a part of cultural identity, using methods like priming can drive a transition toward more resilient and conscientious practices. Furthermore, the success of the technique reinforces the value of crops adapted to adverse conditions, such as sorghum, which holds historical and cultural relevance in many communities. In summary, the application of priming in sorghum seeds goes beyond increasing tolerance to salt stress, generating positive impacts across various dimensions of society. By promoting technological innovation, ensuring greater food security, reducing production costs, and valuing traditional agricultural practices adapted to new realities, the technique establishes itself as a sustainable and transformative solution aligned with the needs of a constantly changing world. SUMMARY 1 INTRODUCTION ............................................................................................................. 15 2 THEORETICAL FRAMEWORK................................................................................... 17 2.1 Seed germination and seedling growth – implications for crops´ establishment ........ 17 2.1.1 Seed priming: is it just a technique to synchronize germination? .................... 18 2.1.2 Salinity and its effects on germination, seedling establishment and plant growth ..... 19 2.1.3 Sorghum Varieties: socio-economic importance and stress tolerance .............. 21 REFERENCES ...................................................................................................... 23 CHAPTER I - PRIMING ON SORGHUM SEEDS UNDER SALINITY AND OSMOTIC TREATMENTS: ARE THE SEEDS CAPABLE TO GERMINATE UNDER HARSH CONDITIONS? ........................ 25 1 INTRODUCTION ............................................................................................................ 26 2 MATERIAL AND METHODS ........................................................................................ 27 2.1 Plant material ......................................................................................................... 27 2.1.1 Priming procedure ................................................................................................. 27 2.1.2 Experimental conditions ....................................................................................... 28 2.1.3 Germination parameters ....................................................................................... 28 2.1.4 Statistical analysis .................................................................................................. 28 3 RESULTS ........................................................................................................................... 29 4 DISCUSSION..................................................................................................................... 41 5 CONCLUSIONS ................................................................................................................ 43 6 ACKNOWLEDGEMENTS .............................................................................................. 44 REFERENCES .................................................................................................................. 45 CHAPTER II - PRIMING TECHNIQUES: IMPROVING GERMINATION THROUGH DIFFERENT MECHANISMS ....................................... 50 1 INTRODUCTION ................................................................................................. 51 2 MATERIAL AND METHODS ........................................................................................ 53 2.1 Plant material ......................................................................................................... 53 2.1.1 Experimental conditions ....................................................................................... 53 2.1.2 Priming procedure ................................................................................................. 53 2.1.3 Germination test .................................................................................................... 54 2.1.4 Sampling ................................................................................................................. 54 2.1.5 Antioxidant enzymatic assays ............................................................................... 54 2.1.6 Proline content ....................................................................................................... 55 2.1.7 Total soluble sugars ............................................................................................... 55 2.1.8 Reducing sugars ..................................................................................................... 55 2.1.9 Quantification of total soluble amino acids ......................................................... 56 2.1.10 Statistical analysis .................................................................................................. 56 3 RESULTS ............................................................................................................... 56 4 DISCUSSION ......................................................................................................... 61 5 CONCLUSIONS ................................................................................................................ 64 6 ACKNOWLEDGEMENTS .............................................................................................. 65 REFERENCES .................................................................................................................. 66 CHAPTER III - THE MEMORY REMAINS: HOW PRIMING PROMOTE SORGHUM SEEDS GERMINATION UNDER OSMOTIC AND SALINITY CONDITION? ……………………………………..71 1 INTRODUCTION ............................................................................................................. 72 2 MATERIAL AND METHODS ........................................................................................ 74 2.1 Plant material and Experimental conditions ...................................................... 74 2.1.1 Sampling ................................................................................................................. 74 2.1.2 Evaluation of photosynthetic responses ............................................................... 74 2.1.3 Chlorophyll a fluorescence .................................................................................... 75 2.1.4 Photosynthetic Pigments ....................................................................................... 75 2.1.5 Lipid peroxidation and hydrogen peroxide concentration measurements ...... 75 2.1.6 Total soluble sugars ............................................................................................... 75 2.1.7 Reducing sugars ..................................................................................................... 75 2.1.8 Proline quantification ............................................................................................ 76 2.1.9 Statistical analysis .................................................................................................. 76 3 RESULTS ............................................................................................................... 77 4 DISCUSSION ......................................................................................................... 82 5 CONCLUSIONS ................................................................................................................ 84 6 AKNOWLEDGMENTS ................................................................................................... 85 7 SUPPLEMENTARY MATERIAL .................................................................................. 86 8 ACKNOWLEDGEMENTS .............................................................................................. 95 REFERENCES .................................................................................................................. 96 1 CONCLUDING REMARKS ............................................................................................ 98 15 1. INTRODUCTION Germination is an event that occurs essentially in the presence of water, which plays a key role in providing hydration of tissues, through imbibition, and leads to the recovery of metabolism culminating in the protrusion of the radicle, ending the germination event (Bewley and Black., 2013; Bewley and Black., 2004). However, because this occurs strictly in the presence of water, several factors can affect germination, which can slow down this process or even inhibit it permanently by the time the embryo dies (Çakmakçi et al., 2019). Once it occurs successfully, germination will give rise to a seedling that will colonize the environment and ensure reproductive success. This stage also presents fragility due to stressful environmental factors, such as drought stress, salinity, high radiation and temperature, leading to limitations in the establishment of this seedling that may culminate in lower production yields. Thus, the role of germination is of extreme importance and highly affected by external and internal factors (Ceritoglu et al., 2020). Several technologies are adopted to improve seedlings tolerance, all aiming to provide conditions for germination and post-germination events to occur adequately, resulting in healthy plants. Among these techniques, seed priming emerges as an efficient and low cost one. Priming consists in the imbibition of seeds, which is interrupted before the protrusion of the radicle (phase II of imbibition curve), followed by drying (preferably fast, to avoid the consumption of reserves) and subsequent storage (Singh et al., 2018; Noreen et al., 2020). This technique can be performed using distinct molecules or solutions. Some time ago, the seed priming was used just for increasing the speed and synchronizing of germination for agronomic purposes. Nowadays, seed priming is being used to recruit a metabolism imposed by exposure to the seeds to a stress simulation. In other words, the priming application in seeds build up a ‘stress memory’, through enhancing different physiological and biochemical mechanisms that can be recruited when they (seeds, seedlings or plants) are exposed to adverse conditions (Aziz et al., 2021). Therefore, the priming is being used for increasing stress tolerance in plants. One of the main concerned abiotic stressful conditions around the world is the salinity. This condition occurs when there is an increase in the availability of salts (NaCl, KCl and others) in the environment. The increase of these ions in the environment can lead to severe impacts on germination and establishment of seedlings and plant growth and development. The negative impacts of high salinity occur mostly in the dissociation of ions, which may compromise the uptake of water and nutrients by seeds and plants. The impacts imposed by this 16 condition will be guided by (i) the occurrence of ion dissociation, leading to changes in osmotic potential, and consequently water absorption, or (ii) the cytotoxic effects of the accumulation of these ions. Due to the occurrence of salt stress, there are delays in seed germination, reductions in seedlings establishment, and plant´s physiology, i. e, water relations, photosynthesis and nutrition. The salinity culminates, in field conditions, in a poor plant stand and low agronomic indices (Rajabi Dehnavi et al. 2020). The causes of the salinity are both natural or by anthropomorphic interferences, such as the use of wastewater, increasing agrochemicals using, and misuse of the land. Regions with low rainfall (e.g. semi-arid regions) need not only to deal with drought, but also with the effects of salinity. In these regions the use of crops that better tolerate these conditions is carried out in order to promote food and feed supply, as well as feedstock for fuel or energy production (Nimir et al., 2020). Therefore, the knowing of which crops can stand salinity around the world is essential. and One of these widely used crops is sorghum (Sorghum bicolor Moench (L)). Sorghum is a high nutritional species that shows moderate to high tolerance to drought, depending on the phenological stage (McCann et al. 2015; Impa et al. 2019). This crop is usually cultivated in areas where salinity is a hindrance to food production, such as part of the African continent, Asia, and the Brazilian semiarid (Ibrahim 2016). In these regions, there is a search for genetic materials or techniques that show or increase better responses to salinity and drought. In this way, the use of the priming technique in seeds can be a powerful tool to increase crop´s tolerance to stressful conditions. The priming can also reduce the development time and resources of generating new tolerant cultivars, emerging as a parallel and cheap technique that provides conditions for better establishment of crops, achieving better agricultural results (Shakeri et al., 2017; Çakmakçı and Dallar, 2019). Thus, this work was conducted based on the hypothesis that the application of priming in sorghum seeds (with water or solutions with hormone or antioxidant) improve seed germination, seedling establishment, and plant growth under salinity conditions through in antioxidant and osmotic adjustments. Three manuscripts (chapters), which the main objectives are described, as follows, compose this work. In the chapter 1, we investigate how the application of priming promotes improvements in the germination of sorghum seeds exposed to high salinity and osmotic stress conditions, using germination parameters for contrasting cultivars for drought tolerance. In the chapter 2, we aimed to identify by which mechanisms the priming improved germination comparing the most discrepant cultivars (higher sensitivity and higher tolerance) to salinity and osmotic conditions. Finally, in the chapter 3, there were 17 evaluated the effect of different types of priming on amino acid, sugar and photosynthetic metabolism of sorghum seedlings and plants grown under doses of NaCl. 2. THEORETICAL FRAMEWORK 2.1. Seed germination and seedling growth – implications for crops´ establishment Seed germination is a physiological process that begins with the absorption of water by the seed and ends with the protrusion of the radicle (Bewley et al., 2013). Water soaking can be observed as an increase in seed fresh weight due to an increase in water content by the end of germination. Seed water soaking follows a pattern proposed by Bewley and Black (1994) where there are three distinct times following a three-phase pattern. This triphasic pattern is defined as the weight/water content of the seeds varies. In the work of Bewley et al. (2013) there is a definition of these three phases during germination. In the first stage (I) there is the absorption of water by the seed, mainly due to the potential differences between the seed and the germination medium. The third phase (III) is the moment when there is the protrusion of the radicle and an increase in seed weight. Although initially this process is governed by the difference between the osmotic potentials of the soil and seed, there are intrinsic and abiotic factors that can compromise germination or even cause seed death. The factors that may cause a delay in germination, conditioned to the hormonal balance of orthodox seeds, are initially caused by dormancy, primary or secondary dormancy, and the environmental factors are related to the osmotic potential. The main environmental factors are drought, which can cause an irregular imbibition, that is, the seed starts soaking, however there are no conditions for it to occur properly, or factors that can cause changes in the osmotic potential of the soil solution, caused mainly by ions that compromise the absorption of water by seeds. We must also consider that until phase II of the soaking curve, seed desiccation can occur without compromising the resumption of soaking. However, if this occurs at the end of phase III, where there is protrusion of the radicle, the seed is unable to undergo desiccation, resulting in embryo death and consequently non-germination of the seed. Among the factors that most impact the establishment of crops, we can highlight that the abiotic factors are the most detrimental. 18 2.1.1. Seed priming: is it just a technique to synchronize germination? Seed priming is a worldwide and millenary technique used for promoting improvements and synchronizing germination Sharma et al., 2014. The priming consists in imbibe the seeds until phase II of germination, drying them, storing, following by rehydration (Lutts 2016). Seed priming interferes on duration of germination phases and may cause an anticipation of radicle protrusion, through the regulation of metabolic processes that are responsible for the initial phases of germination (Ibrahim et al., 2016. This technique leads to several improvements, especially in reducing the imbibition time, leading to increases in the production of metabolites, such as amino acids, and synthesis of enzymes and proteins that may promote DNA repair or regulation of redox metabolism (Wardah et al., 2019). The application of this technique has shown to be very efficient in promoting tolerance to unfavorable environmental conditions to germination, such as drought, salt and heat stress. In addition, the use of priming in seeds, besides being safe, can present itself as a low-cost alternative for crop production (Ibrahim, 2016). This technique results in increasing speed and uniformity of germination as well as improve seed vigor and seedling establishment under stressful conditions (Gupta et al., 2008, Sharma et al., 2014; Patade et al., 2009, Bewley et al., 2013). The application of the technique is usually in aqueous solutions, followed by the rapid drying of these seeds soon after exposure to rehydration. It is important to note that although it is performed with the use of aqueous solutions there are techniques that use solid substances to perform the technique. The type of priming to be applied is an important step to obtain effective responses to the various unfavorable environmental conditions faced by seeds and plants. The most commonly used techniques are the hydropriming (water as a medium of imbibition), halopriming (salt solutions such as NaCl or MgCl), osmopriming (use of solutions with different osmotic potentials, usually induced by polyethylene glycol - PEG) or hormopriming (use of growth regulators) (Karadag et al., 2017; Hassini et al., 2017; Roychoudhuryet al., 2016; Salama et al., 2015; Nakaune et al., 2012). The effects of priming can be observed during all phenological stages of plants (Huang et al., 2016). In seeds, the effects may be observed that will promote faster imbibition, ensuring the integrity of the macrostructures and membranes, preventing damage that compromises germination success. The application of priming allows a rapid absorption of water, ensuring greater uniformity of germination and also the recovery of metabolism more efficiently, mitigating deleterious effects such as the formation of reactive oxygen species (ROS) that may 19 compromise the germination and generation of normal seedlings (Talukdar, 2012; Kumar et al., 2014). Thus, when primed, seeds may exhibit conditions that trigger modifications of metabolism pathways, making them more able to maintain their development during unfavorable conditions. In the primed state, seeds can exhibit multiple epigenetic changes, especially up-regulation of stress-responsive transcription factors, thereby improving germination and seedling establishment compared to untreated seeds and promoting increases in productivity (Sharma et al., 2014; Jisha et al., 2013; Farooqet al., 2009; Bruce et al., 2007). In seedlings and plants, the effects can be observed when analyzing the benefits in maintaining redox homeostasis and in improvements in photosynthetic metabolism (Fujita et al., 2013). During these phenological stages, it can be observed that there are improvements that go through the inhibition of cytotoxic effects, besides the maintenance in the repair of photosystems and regulation of stomatal opening. The application of the priming technique besides these adjustments, also promotes the de novo synthesis of proteins and also the accumulation of hormones in Vigna radiata and Arabidopsis (Jisha et al., 2013; Rajjou et al., 2012; Zhou et al., 2012), a higher content of sugars and proline has been observed in rice by Karalija and Selovic (2018) and Gul et al. (2020) and Nouairi (2019) have observed an improvement in photosynthetic rate in rice and fava beans. All these findings leading us to believe that the application of priming could be highly effective in promote the benefits expected. 2.1.2. Salinity and its effects on germination, seedling establishment and plant growth Most of the agricultural areas are subject to salinization that is conceptualized by the excessive deposition and accumulation of mineral salts in the soil (FAO, 2011; Fedoroff et al., 2010). Salinity is a current problem, in a growing scenario, and that demands high investments in its repair. The deposition of ions in the soil can occur in natural ways, due to leaching of rocks or by the indiscriminate use of fertilizers and wastewater containing high concentrations of ions such as KCl or NaCl (Fedoroff et al., 2010). The effects of salinity can occur in two ways, (i) by the osmotic effect, caused by the decrease in osmotic potential; and (ii) by the ionic effect, due to the dissociation of ions leading to severe cytotoxic effects (Li et al., 2020). Under stressful environmental conditions, such as drought and salinity, there is the perception of these conditions mainly by systemic signaling, and one of the first responses is 20 the accumulation of the phytohormone abscisic acid (ABA). In seeds, this accumulation may inhibit germination, due to the imposition of a secondary dormancy, under salinity conditions in the soil, germination is compromised as the deposition of salts advances. Low concentrations of salts in the soil tend to induce a state of dormancy in the seed. Whereas if an increase in the concentration of salts in the soil solution occurs, germination is inhibited, culminating in a lower percentage of germination by healthy seeds (Khan et al., 2006; Thiam et al., 2013). In seedlings and plants, this may lead to a decrease in stomatal conductance, which is one of the conditions responsible for the increased production of ROS. Depending on the extent and duration of the stress condition, the increasing ROS formation may cause oxidative stress, that is measured by the oxidative damage (Sharma et al., 2012; Huang et al., 2016). In salinity conditions, it is possible to observe in plants an accumulation of Na+ in leaves. The accumulation of this ion leads to severe damage that compromises the development and yield of crops. The main damage that can be observed and the most deleterious could be observed in seeds, seedlings and full growth plants. Seeds are affected by salinity through the decrease in the osmotic potential, delaying the imbibition of water, inhibiting the germination, or even by the damage caused by the dissociation of ion, these conditions take place cause these phenomena causes toxicity and could lead the seeds to death (Li et al., 2020). In seedlings and plant, since growth are dependent of photosynthesis, the effects are related by the alteration in water and nutrients uptake, and gas exchanges and oxidative damage. In plants are a row of effects that comprises the metabolism, that culminated in reduction of biomass and metabolic impairments, the main observed effect are i- dehydration of membranes reducing the CO2 permeability, ii-the effects in water and nutrient uptakes, that could impact the photosynthesis and other metabolic processes, iii- reduction in the CO2 supply by the stomatal closure, this condition lead to an condition of oxidative damage, since there is an excess of energy captured by the light harvest complex that was not conducted to the carboxylation process, this energy could interact with oxygen and generate reactive oxygen species. These deleterious effects on are related to photosynthetic efficiency (decrease in the efficiency of FSII), and decrease in the quantum efficiency of FSII (Fv/Fm), as well as photoinhibition. An increase in ROS formation due to the detour of electrons from the electron transport chain (ETC), the formation of these molecules when above levels that allow their elimination can lead to structural damage (membrane lipid peroxidation by H2O2) and DNA damage (Yang et al., 2014; Murata et al., 2007). 21 The application of priming is able to promote modifications in the way plants circumvent this scenario. Firstly, there is a decrease in imbibition and germination time, this effect associated with priming allows the rapid emergence of the seedlings allows for a rapid perception of the environment, leading to metabolic changes and adjustments that will make it more able to pass through the imposed adverse condition (Wardah et al., 2019). Among these, one can point out improvements through morphological modifications, (phenotypic plasticity), physiological (such as membrane stability through better osmotic adjustment), in addition to changes in biochemical and molecular mechanisms (accumulation of proline, auxins, ABA and ethylene, stress-sensitive proteins, transcription factors and secondary messengers). All these changes that lead to plant adaptation are objectives in breeding programs, and when they can be observed through the application of priming, this makes the technique even more relevant, since there is a considerable decrease in the time required in breeding through the application of this technique (Langeroodi and Noora, 2017; Mohammadi et al., 2014). Due to application of the technique, there is a phenomenon called priming memory, this type of memory is a set of modification imposed by the priming procedure that allow the seeds and plants to recovery a arrange of metabolic modifications, induced by the priming, when they face stress conditions. The evocation of this memory enable the seeds to acclimatize to harsh condition to germination, by recruiting osmoprotective molecules, as well as antioxidants. Any plants can “access” this memory by recovering biochemical and physiological adaptative process to overcome the stressful conditions (Hameed et al., 2021; Valivand et al., 2019). 2.1.3. Sorghum Varieties: socio-economic importance and stress tolerance Sorghum (Sorghum bicolor (L.) Moench), the family Poaceae, subfamily Panicoidea, is the fifth largest grain crop on the world and due to its economic importance, sorghum is grown worldwide, especially in tropical regions, both for human and animal food, as well as for the production of biomass for biofuels (Awika et al., 2004; Queiroz et al., 2011; Fita et al., 2015). The sorghum culture is divided into two areas; one area is concentrated in African and Asian countries, where production occurs in a traditional way, as subsistence agriculture, without technological apparatus, having low productivity. In this region, the production is focused on human food. The second region where the crop prevails includes some developed countries in America, where a large-scale mechanized system is employed, and reaching high productivity. 22 In these regions, the production is destined for animal feed. The largest producers of sorghum are the United States, Mexico and Nigeria and the main exporters are the United States, Australia and Argentina (FAOSTAT, 2014; Sanders et al., 2019). Due to high versatility of growing, the main cultivation regions of the species are in tropical and subtropical areas. It is liable, this way, to find this crop in some cultivation areas in which the environmental and soil conditions are often not profitable to seed germination, seedling establishment, and growth and development, which can culminate in loss of productivity (Fita et al., 2015; Kebede et al, 2001). Therefore, the species could be considered tolerant to many abiotic stressful conditions. Although sorghum is noted as a crop with moderate tolerance to salinity, germination and seedling establishment are severely affected by the excess of inorganic ions in the soil (Dias et al., 2010). This crop is an important source of supplies in regions where there are high concentrations of salts in the soil, as well as the practice of irrigation with water from salt- contaminated reservoirs is an available resource as in semiarid regions of the African continent and in Brazil (Hassanein et al., 2010; El Naim et al., 2012, Sun et al 2014; Guimaraes et al., 2016). Even among the varieties considered tolerant to drought, it is important to investigate the degree of tolerance to salinity, once sorghum is usually cultivated in regions with high concentrations of salt. In this work we used the varieties of sorghum, the adoption of these cultivars is based on their drought tolerance or sensibility. We selected two drought-tolerant cultivars (BRS-310 and DKB 540) and two cultivars susceptible to drought (DOW 50A10 and BRS-332) to evaluate the distinct effects of salinity. The cultivars BRS-310 and 332 are developed by the Embrapa, both are highly cultivated in Brazil. The cultivars DOW 50A10 and DKB 540 have been developed by Dow and Dekalb respectively. The selection of these cultivars are based on the necessity of cultivars that provide to us biochemical markers that allow us to access how different varieties responds to the effects drought and salinity stress. 23 REFERENCES FAROOQ, M., BARSA, S., & WAHID, A. Priming of field-sown rice seed enhances germination, seedling establishment, allometry and yield. Plant growth regulation, 49(2), 285-294. 2006. FAROOQ, M., BASRA, S. M. A., WAHID, A., AHMAD, N.,SALEEM, B. A. Improving the drought tolerance in rice (Oryza sativa L.) by exogenous application of salicylic acid. Journal of Agronomy and Crop Science, v. 195, n. 4, p. 237-246, 2009. GUL, F., ARFAN, M., SHAHBAZ, M., & BASRA, S. Salicylic acid seed priming modulates morphology, nutrient relations and photosynthetic attributes of wheat grown under cadmium stress. Int. J. Agric. Biol, 23, 197-204. 2020. HUBER, A. E., & BAUERLE, T. L. Long-distance plant signaling pathways in response to multiple stressors: the gap in knowledge. Journal of Experimental Botany, 67(7), 2063- 2079. 2016. HUSSAIN, M., FAROOQ, M., & LEE, D. J. Evaluating the role of seed priming in improving drought tolerance of pigmented and non‐pigmented rice. Journal of Agronomy and Crop Science, 203(4), 269-276. 2017. IBRAHIM, EHAB A. Seed priming to alleviate salinity stress in germinating seeds. Journal of Plant Physiology, v. 192, p. 38-46, 2016. JISHA, K. C., & PUTHUR, J. T. Seed priming with BABA (β-amino butyric acid): a cost- effective method of abiotic stress tolerance in Vigna radiata (L.) Wilczek. Protoplasma, 253(2), 277-289. 2016 JISHA, K. C., VIJAYAKUMARI, K., & PUTHUR, J. T. Seed priming for abiotic stress tolerance: an overview. Acta Physiologiae Plantarum, 35(5), 1381-1396. 2013. KARALIJA, E., & SELOVIĆ, A. The effect of hydro and proline seed priming on growth, proline and sugar content, and antioxidant activity of maize under cadmium stress. Environmental Science and Pollution Research, 25(33), 33370-33380. 2018. LANGEROODI, A. R. S., & NOORA, R. Seed priming improves the germination and field performance of soybean under drought stress. Journal of animal and plant sciences, 27(5), 1611-1620. 2017. MOHAMMADI, G. R., KOOHI, Y., GHOBADI, M., & NAJAPHY, A. Effects of seed priming, planting density and row spacing on seedling emergence and some phenological indices of corn (Zea mays L.). Philippine Agricultural Scientist, 97(3), 300-306. 2014. MURATA, N., TAKAHASHI, S., NISHIYAMA, Y., & ALLAKHVERDIEV, S. I. Photoinhibition of photosystem II under environmental stress. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1767(6), 414-421. 2007. 24 MUSTAFA, H. S. B., MAHMOOD, T., ULLAH, A., SHARIF, A., BHATTI, A. N., NADEEM, M., & ALI, R. Role of seed priming to enhance growth and development of crop plants against biotic and abiotic stresses. Section Plant Sciences, 2(2), 1-11. 2017. NOUAIRI, I., JALALI, K., ZRIBI, F., BARHOUMI, F., ZRIBI, K., & MHADHBI, H. Seed priming with calcium chloride improves the photosynthesis performance of faba bean plants subjected to cadmium stress. Photosynthetica, 57(2), 438-445. 2019. RAJJOU L, DUVAL M, GALLARDO K, CATUSSE J, BALLY J, JOB C, JOB D. Seed germination and vigor. Annu Rev Plant Biol 63:507–533. 2012. ROYCHOUDHURY, A; BANERJEE, A. Endogenous glycine betaine accumulation mediates abiotic stress tolerance in plants. Trop Plant Res, v. 3, p. 105-111, 2016. TABASSUM, T., FAROOQ, M., AHMAD, R., ZOHAIB, A., WAHID, A., & SHAHID, M. Terminal drought and seed priming improves drought tolerance in wheat. Physiology and Molecular Biology of Plants, 24(5), 845-856. 2018. TALUKDAR, D. Ascorbate deficient semi-dwarf asfL1 mutant of Lathyrussativus exhibits alterations in antioxidant defense. BiologiaPlantarum, v. 56, n. 4, p. 675-682, 2012. WAQAS, M., KORRES, N. E., KHAN, M. D., NIZAMI, A. S., DEEBA, F., ALI, I., & HUSSAIN, H. Advances in the concept and methods of seed priming. In Priming and pretreatment of seeds and seedlings (pp. 11-41). Springer, Singapore. 2019. WARDAH, M., RIAZ, A., MUZZAMMIL, H., MUHAMMAD, F., & ABDUL, W. Influence of seed priming and sowing methods on growth and productivity of wheat cultivars differing in seed size in rice-wheat cropping system of Punjab, Pakistan. International Journal of Agriculture and Biology, 21(4), 803-809. 2019. YANG, C., ZHANG, Z. S., GAO, H. Y., FAN, X. L., LIU, M. J., & LI, X. D. The mechanism by which NaCl treatment alleviates PSI photoinhibition under chilling-light treatment. Journal of Photochemistry and Photobiology B: Biology, 140, 286-291. 2014. ZHOU, Y., CHU, P., CHEN, H., LI, Y., LIU, J., DING, Y., ... & HUANG, S. Overexpression of Nelumbo nucifera metallothioneins 2a and 3 enhances seed germination vigor in Arabidopsis. Planta, 235(3), 523-537. 2012. 25 CHAPTER I PRIMING ON SORGHUM SEEDS UNDER SALINITY AND OSMOTIC TREATMENTS: ARE THE SEEDS CAPABLE TO GERMINATE UNDER HARSH CONDITIONS? ABSTRACT Seed priming is a widely used technique to improve seed germination. However, it has been used for increasing seed tolerance to harsh conditions. Seeds are highly susceptible to variations in osmotic potential, since imbibition is a crucial process for germination. If the osmotic potential changes, severe damage can occur. Osmotic and salt stresses present as a major problem, since they cause serious losses in crops´ establishment. This work aimed to investigate the efficiency of hydropriming and priming with different molecules (ascorbic acid (AsA) and abscisic acid (ABA)) in varieties of sorghum seeds after priming application and under salinity and osmotic conditions. The main question addressed is if the type of priming can improve germination in drought-tolerant and sensitive varieties of sorghum under salt and osmotic conditions. Some of the observed changes are related to the application of priming, it is possible to observe that there are benefits from the application of priming, especially with the molecules AsA and ABA, in the seeds of the sensitive cultivar (BRS-332). This benefit can be found for both osmotic and salt stress conditions. The tolerant cultivars (BRS-310 and DKB 540) were efficient under both conditions when submitted to priming and hydropriming, germination values were high even under the highest NaCl doses and lowest osmotic potentials. Seeds of the cultivar DOW 50A10 (sensitive the lowest germination rate, and oscillations in the T50 and GSI parameters, indicating compromises that could not be bypassed by priming. Overall, the results are optimistic in clarifying the effects of different types of priming on germination, since the results obtained especially for the sensitive cultivar, which had a similar performance to tolerant cultivars when subjected to priming. Key words: Ascorbic acid; Abscisic acid; germination; Sorghum bicolor; PEG6000; NaCl. 26 1. INTRODUCTION The impacts of salinity in crops is worrying even in high tolerant species like sorghum (Krishnamurthy et al. 2007), the world's fifth largest cereal crop, present in the dietary of more than 500 million people (FAOSTAT, 2014). Sorghum is an important staple crop for more than half a billion people, mainly in developing countries in the semi-arid and arid tropics, which are prone to water scarcity. It provides nutrition that is rich in protein, fiber, and gluten- free (McCann et al. 2015; Impa et al. 2019). In addition to human nutrition, it is being used as a source of feedstock for bioethanol production (Mathur et al. 2017). Many studies reveal that the tolerance of sorghum genotypes to salinity must be monitored since seed germination and seedling establishment (Krishnamurthy et al. 2007). Since high salinity and osmotic stress can lead to severe impacts that negatively affect crop production, several efforts have been made to improve low-cost techniques to achieve efficient results in promoting better seed germination. One of these techniques is the seed priming, a low cost and ‘ecologically friendly’ technique to improve seed germination (Ibrahim 2016). During seed priming, several physicochemical alterations can occur that can modify the characteristics of the plant, improving the physiological activity of the embryo and the future plant (Kaur and Gupta 2018). The incorporation of growth regulators and hormones in the priming technique is a promising alternative, since it is possible inducing remodeling pathways towards salinity tolerance. Two molecules that, when applied as conditioners, show promising results are ascorbic acid (AsA) and abscisic acid (ABA). AsA is a potent antioxidant, and has an efficient effect in combating stress damage, and some studies using exogenous applications of AsA have suggested that it can counteract the adverse effects of salinity (Singh et al., 2018; Noreen et al., 2020). Under salinity conditions, ABA has been shown to play an important role in improving salinity tolerance through enhanced germination and growth performance of different crops (Gurmani et al., 2011). The metabolic and molecular changes by the ABA and AsA signaling lead to an accumulation of osmolytes and expression of transcription factors that allowed the seeds to circumvent the unfavorable condition (Nadarajah et al., 2020; Alam et al., 2019). However, besides being promising, the use of AsA and ABA to induce cross-tolerance in seeds by priming application is still underexploited and, consequently, poorly understood. This way, we investigate in this work the effects of primed-sorghum seeds (with water – hydropriming; and with ascorbic acid (AsA) or ABA) subjected to salinity and simulated 27 osmotic stress. We hypothesize that priming with AsA and ABA, can improve seed germination and uniformity even in high concentration of salt, in relation to non-primed seeds. It is also expected that contrasting drought-tolerant sorghum varieties can germinate differently on salinity or osmotic conditions. It could give to us cues about the improvements on germination capacity upon priming treatments when exposed to osmotic stressful conditions. 2. MATERIAL AND METHODS 2.1. Plant material Seeds of four Sorghum bicolor (L.) Moench cultivars were used in this experiment. They differ in tolerance and susceptibility to water deficit: BRS 310 and DKB 540 are drought-tolerant; BRS 332 and DOW 50A10 are sensitive. All material was provided by Embrapa Maize and Sorghum, Brazil. 2.1.1. Priming procedure The priming technique was set according to Ibrahim (2016), considering a imbibition step until phase II (with water or solutions), a drying step to reach the initial weight, a short- time storing step, followed by germination. The conditioners concentrations were previously determined according to pre-tests and based on doses found in the literature. The ABA solution was performed following Vieira et al. (2017). ABA was diluted in 1N KOH solution (the content used to the complete dissolution of ABA was approximately 0,2ml), in dark conditions. The volume was adjusted to 400ml and the pH was adjusted for 7.0, if needed. The AsA solution was performed in the dark, with dilution in deionized water. The seeds were imbibed using solutions containing the phytoregulator ABA or AsA, or just water, for a period of 8 hours. The time of 8h-imbibition was set in a previous experiment to estimate the imbibition curve and matched the half of phase II. The doses of hormones and ascorbic acid were set at 100 µM, according to the results obtained in the pre-test (the concentration that did not reduce seed germination percentage and velocity). Then the seeds were dried in a forced circulation oven for 12 hours at 30°C, until achieve the initial water content. After this, the seeds were stored for 3-7 days maximum at room-controlled temperature (25 ºC) until the germination experiments. 28 2.1.2. Experimental conditions The experiment was conducted in the Laboratory of Plant Growth and Development (LCDP) of the Federal University of Lavras (Lavras-MG, Brazil). An individual experiment was conducted for each cultivar, in each experimental condition (priming treatments vs salinity and osmotic conditions). The seeds were exposed to priming with the signaling molecules (ABA and AsA, as well as to hydropriming (for insulating priming effects without conditioners) in the times of imbibition and drying as mentioned. After priming procedure, the seeds were exposed to the experimental conditions: five NaCl doses (0, 60, 120, 180 and 240) and the respective osmotic potentials (-2.93, -5.86, -8.8, and -11.73 MPa), induced with PEG6000 (polyethylene glycol). The osmotic potential of the solutions was calculated based on equation of Van´t Hoff (Van´t Hoff 1887). Thus, the experiment was designed in a 5x4 factorial scheme (5 doses of NaCl or PEG6000 vs 4 priming conditions) for each cultivar. 2.1.3. Germination parameters To obtain the germination parameters, the seeds were placed in Petri dishes between a double layer of germination paper, moistened with 10ml of solution containing NaCl or PEG- 6000. There were used 4 repetitions with 25 seeds in a germination chamber at 12h photoperiod at 40µmol photons m-2 s-1 at 25º C. Germination was observed every eight hours for a period of 48 hours and the criterion was 2mm of radicle protrusion. The germination percentage (G) was used to build the cumulative germination curve. The germination speed index (GVI) was calculated according to Maguire (1962). The median germination time (T50), that corresponds to the time required for 50% of the seed lot to germinate, was calculated according to Farooq et al., (2005). 2.1.4. Statistical analysis The statistical analysis was performed with the software Rbio (Bhering, 2017). Data was subjected to two-way ANOVA, the Tukey means test was performed in case of normal distribution. The Principal Components Analysis was performed using the software R (stats and factoextra packages). 29 For a better understanding of the effect of the conditions, doses and osmotic potentials on the analyzed variables we performed principal component analysis (PCA) comparing the cultivars and the conditions, and comparing the effects of NaCl doses and osmotic potentials simulated by PEG into the cultivars. The variables analyzed were germination percentage for the cultivars, and the effects of the doses and potentials within the variable G and T50. 3. RESULTS The seeds of the cultivar BRS-310 showed higher germination when subjected to hydropriming or AsA in comparison to the dry seeds. It is possible to observe (Fig. 1.1) that the primed seeds presented enhanced germinability when submitted to NaCl than the PEG6000 solutions, even at lower osmotic potentials. Primed seeds presented higher germination during the first 8 hours of evaluation, with the lowest percentages of germination at this period being observed at the highest doses of salt (180 and 240 mM respectively). 30 Figure 1.1 - Accumulated germination of the cultivar BRS-310 that was subjected to PEG- 6000 solutions at respective osmotic potentials -2.93, -5.86, -8.8, and -11.73, and to solutions of water with 60, 120, 180, and 240 mM NaCl. Values represents means ±Standard error (n=4) Asterisks represents the statistical differences at P<0.05 (Tukey test) at final germination percentage. Source: Ferreira (2022) Under osmotic conditions, BRS-332 presented a higher sensitivity at PEG6000 for germination when compared to salt in the period between 8h and 16h of evaluation (Fig. 1.2). The primed seeds, besides increased germinability than the dry seeds, still presented a lower germination at lower osmotic potentials. However, under saline conditions, the hydropriming was able to provide a similar germinative percentage up to 180mM. 31 Figure 1.2 - Accumulated germination of the cultivar BRS-332 that was subjected to PEG- 6000 solutions at respective osmotic potentials -2.93, -5.86, -8.8, and -11.73, and to solutions of water with 60, 120, 180, and 240 mM NaCl. Values represents means ±Standard error (n=4) Asterisks represents the statistical differences at P<0.05 (Tukey test) at final germination percentage. Source: Ferreira (2022) Similar to BRS-310 and BRS-332, the seeds of the cultivar DKB 540 had higher germination during the initial periods of evaluation when submitted to priming and at saline conditions (Fig. 1.3). When compared to PEG, the seeds submitted to salt solutions exhibited enhanced germinability between 8h and 16h, however, at 8h the cumulative germination of the seeds submitted to priming was higher than the dry seeds. 32 Figure 1.3 - Accumulated germination of the cultivar DKB 540 that was subjected to PEG- 6000 solutions at respective osmotic potentials -2.93, -5.86, -8.8, and -11.73, and to solutions of water with 60, 120, 180, and 240 mM NaCl. Values represents means ±Standard error (n=4) Asterisks represents the statistical differences at P<0.05 (Tukey test) at final germination percentage. Source: Ferreira (2022) Under both conditions (PEG and NaCl), the seeds of the cultivar DOW50A10 presented asynchrony of germination when compared to the other cultivars, achieving approximately 50% of germination at 24-32 hours and lowest germination among the evaluated cultivars, even when submitted to priming (Fig. 1.4). For all cultivars, maximum germination was reached close to 32 hours after evaluation, timing in which BRS-310, BRS- 332 and DKB 540 even in NaCl or PEG treatments had the germination percentage stabilization. 33 Figure 1.4- Accumulated germination of the cultivar DOW 50A10 that was subjected to PEG- 6000 solutions at respective osmotic potentials -2.93, -5.86, -8.8, and -11.73, and to solutions of water with 60, 120, 180, and 240 mM NaCl. Values represents means ±Standard error (n=4) Asterisks represents the statistical differences at P<0.05 (Tukey test) at final germination percentage. Source: Ferreira (2022) When subjected to PEG, except for DOW 50A10, which showed uninform germination and the lowest and GSI under the control conditions (0 mM). In the lowest osmotic potentials, the two cultivars BRS (332 and 310) and DKB 540, showed similar GSI values. The lowest values for GSI were observed for the lowest osmotic potentials, even within the conditioners, with BRS-332 showing the lowest values when subjected to the - 11.73 potential (Fig. 1.5). 34 Figure 1.5- Germination Speed Index of four Sorghum cultivars (BRS-310, BRS-332, DKB 450 and DOW 50A10) subjected to PEG-6000 at respective osmotic potentials - 2.93, -5.86, -8.8, and -11.73, and water solutions with 60, 120, 180, and 240 mM NaCl. Comparison by upper case Source: Ferreira (2022) letter of doses within conditioning, comparison by lower case letter of doses within conditioning or osmotic potential. Values represents means ±Standard error (n=4) Asterisks represents the statistical difference at P<0.05 (Tukey) The cultivar DOW 50A10 showed similar responses to the BRS-332 when subjected to salt doses. BRS-332 the drought sensitive cultivar, presented a better GSI when compared to the osmotic stress condition simulated by PEG. The other cultivars did not differ statistically, although it is possible to observe a higher GSI for BRS-310 and DKB 540 when submitted to salinity conditions. The average germination time (T50) was negatively affected in seeds subjected to PEG-6000. In this condition the drought susceptible cultivars took longer to establish 50% of germination, with BRS-332 showing an average time of approximately 5h and DOW 50A10 an average time of 4.5h. Meanwhile, BRS-310 and DKB 540 in these same conditions showed similar T50, around 2h at higher water potentials and values close to 3h at lower potentials (Fig. 1.6). 35 Figure 1.6- Mean germination time (T50) of four Sorghum cultivars (BRS-310, BRS-332, DKB 450 and DOW 50A10) subjected to PEG-6000 at respective osmotic potentials -2.93, -5.86, -8.8, and -11.73, and water solutions with 60, 120, 180, and 240 mM NaCl. Comparison by upper case letter of doses within conditioning, comparison by lower case letter of doses within conditioning or osmotic potential. Values represents means ±Standard error (n=4) Asterisks represents the statistical difference at P<0.05 (Tukey) Source: Ferreira (2022) When analyzing these cultivars under salinity conditions, DKB showed a lower T50, reaching it around 3h in all treatments. The drought susceptible cultivars had a T50 near 4h in the lower doses of salt, however, when the dose was increased this T50 reached near 6h, both for BRS-332 and DOW50A10. Non-primed seeds are more dispersal then the seeds subjected to priming, regardless the type of priming, indicating that the use of priming are responsible to promote a better grouping close to the arrow who evaluate the final germination (Fig. 1.7 and 1.8). This distance of the germination value and the proximity of T50 arrow indicate that these parameters are highly affected by the use of priming, even for the salinity conditions or the simulated osmotic stress through PEG-6000. For the cultivars BRS-332 and BRS-310, the hydropriming and the priming with ABA showed higher correlation. It is possible to observe that the cultivar BRS-332 had a negative correlation between G% and T50; this indicates that there is a lower germination and a considerable increase in T50, because this parameter has increased due to a longer period to occur the germination of 36 50% of the seeds, explained by axis 1 (82.2%). Hydropriming and ABA had closer proximity to axis 1. The DOW cultivar also showed a negative correlation between germination percentage and T50, with lower values being observed for the G variable. ASA and ABA have a higher proximity to axis 1 (83.9%) and are the priming types that best represent the results. For the water deficit tolerant cultivars (BRS-310 and DKB 540), it could be observed that BRS-310 has the greater results for ASA and ABA, although they also had a negative correlation, these priming types are closer to axis 1 (85.1% of the data). Similar to BRS-310, DKB 540 showed a negative correlation between T50 and germination percentage; the primed seeds had higher values for G and lower values for T50. 37 Figure 1.7- Principal components analyses of four cultivars of sorghum (BRS-310, BRS-332, DKB 450 and DOW50A10) primed with 100µM of AsA, ABA, hydroprimed and non-primed subjected to water solutions contend 60, 120, 180, and 240 mM NaCl. Source: Ferreira (2022) Under the treatments with PEG6000 (Fig. 8), it is possible to observe the representations for the different cultivars. The cultivars 332 and DOW showed a negative correlation between the variables evaluated under osmotic stress condition, occurring negative effects on germination, leading to the occurrence of an increase in the variable T50. For both cultivars, it can be observed that there is a greater delay in germination for non-primed seeds. The cultivars BRS-310 and DKB showed a similar response. The priming with ASA and ABA, as well as hydropriming promoted a higher G. It is possible to observe higher values for T50 in the non-primed seeds of both cultivars. 38 Figure 1.8- Principal components analyses of the effects of PEG-6000 simulated osmotic potentials (-2.93, -5.86, -8.8, and -11.73) on T50 and germination percentage of four cultivars of sorghum (BRS-310, BRS-332, DKB 450 and DOW50A10) primed with 100µM of AsA, ABA, hydroprimed and non-primed. Source: Ferreira (2022) Figures 9 and 10 show the principal component analysis of the effects of salt doses and the osmotic potentials for sorghum cultivars regarding the variables G and T50. In general, the higher doses were responsible for providing a greater proximity of the T50 values.For the cultivars, when submitted to salt stress (Fig. 1.9), it was possible to observe the lowest values for germination percentage and a significant increase for the cultivars BRS-332 and DOW, especially above the 120mM NaCl dose, negatively affecting these two cultivars. DKB and BRS-310 showed an increase in T50 for the dose of 240mM of NaCl, and there were better responses for the variable germination percentage for the doses up to 180mM, especially for the DKB540. 39 Figure 1.9- Principal component analysis of the effects of water solutions contend 60, 120, 180, and 240 mM NaCl on four sorghum cultivars (BRS-310, BRS-332, DKB 450 and DOW 50A10) primed with 100µM of AsA, ABA, hydroprimed and non-primed. Source: Ferreira (2022) 40 There was a negative correlation between the variables for the cultivars BRS-332 and DOW under the condition of osmotic stress simulated by PEG-6000 (Fig. 1.10). It is possible to observe a gradual distancing of axis 1 (81.6%) as the water potential increased. At the lowest potential, there was a greater proximity to the variable T50, that was negatively affected (longer time to reach 50% germinated seeds). Similarly, the cv. DOW50A10 showed a negative correlation, however, even at the highest potentials, it was possible to observe negative changes in the G. The cv. BRS-310 exhibited effects of the higher potentials due to a greater proximity to the axis of the T50 variable, although until the potential of -8.8 MPa there was still a considerably higher germination. DKB showed high germination percentage at potentials closer to -8.8 MPa, being negatively affected only at the highest potential. Figure 1.10- Principal component analysis four sorghum cultivars (BRS-310, BRS-332, DKB 450 and DOW 50A10) subjected PEG-6000 simulated osmotic potentials effects (-2.93, -5.86, -8.8, and -11.73) Source: Ferreira (2022) 41 4. DISCUSSION Although the known tolerance of sorghum plants to drought, in this work the seeds were negatively affected by salinity and osmotic conditions, significantly reducing seed germination in distinct levels regarding the cultivar evaluated. Even tolerant cultivars, i.e. BRS310, showed sensibility to salt and osmotic stressful conditions. On the other hand, BRS332 demonstrated relative sensitivity and more tolerant than DOW50A10 cultivar. The dissociation of ions in the aqueous solution of soil can cause both osmotic stress and ionic stress due to ions imbalance and its interferences on water potential (Ceritoglu et al., 2020). Moreover, regions that face problems due to soil salinity usually also face drought stress conditions, considerably affecting crop establishment, mainly regarding the early stages of plant life cycle (Ibrahim et al., 2016). Therefore, the use of priming technique with known conditioners could provide better acclimation and tolerance for sorghum seeds cultivated in saline soils. Ascorbic acid is one of the most important antioxidants found in plants, avoiding damage losses, increasing germination timing (Kumar et al., 2013). In this work, the application of priming with AsA was able to promote benefits to seed germination. Under the different stress conditions, the priming was able to provide an increase in germination, especially at higher potentials and at higher doses of NaCl. Similarly, the priming with ABA was also efficient in promoting benefits in germination. Similar effects were observed in experiments conducted by Bahrabadi et al. (2022) when corn seedlings were primed with ABA and other hormones, and by Shah et al. (2019) when wheat seeds were subjected to doses of AsA. Even the hydropriming, as showed here, was able to standardize seed germination under stressful conditions at the lower concentrations in the tolerant and one sensitive cultivar. It indicates that the priming technique per se is able to improve germinability and tolerance against osmotic stress in sorghum seeds. In this work, we verified that salinity and osmotic stress caused changes in the germination of all cultivars, even those tolerant to water stress. BRS-310 and DKB 540 had reductions in germination, however, the most affected cultivars were those susceptible to drought (BRS-332 and DOW 50A10), which affected germination even at lower NaCl doses and higher osmotic potentials. Similar to these results, Dehnavi et al. (2020) observed a decrease in germination of sorghum cultivars according to increasing NaCl concentrations. However, under the lower NaCl doses, an increase in germination can be observed, Nimir at al. (2017) obtained interesting results with sorghum seeds showing an improvement in germination 42 subjected to doses of 50mM, thus a type of priming can be associated with these low doses, promoting a better germination in a short period. The germination speed index showed changes especially at higher doses (180 and 240mM). At these doses, even when primed, a decrease in this index could be observed, indicating that germination was compromised, especially in cultivars susceptible to water deficit (BRS-332 and DOW 50A10) exposed to drought simulated by PEG-6000. The cultivar DKB performed similar in both conditions (salinity and osmotic stress), however, BRS-310, also tolerant to water stress, showed differences between the two conditions. We can assume, therefore, that the lower osmotic potential caused a delay in germination. It is worth noting that increasing the salt dose can lead to an increase in the time it takes for germination to occur, or even dormancy (Chen et al, 2020; Rajabi Dehnavi et al., 2020). The average germination time (T50) reflects the germination speed index; with this parameter, it is possible to stipulate the effect of the two conditions on seed germination. It was observed that there was an increase in the T50 of seeds from the susceptible cultivars BRS-332 and DOW 50A10 also from the tolerant BRS-310. As T50 increases, we can start to see some delays in germination as NaCl doses increase, these changes are observed by several authors in experiments with other plant species (Önal Aşçı and Üney, 2016; Chen et al.,2020). The mean germination time (T50) also reflects the speed of seed germination (Bijanzadeh and Egan, 2018). However, in this study, it was verified that the time to reach 50% of seed germination (T50) was extended due to increasing salt content for all sorghum cultivars. In other studies, T50 was also significantly affected it was reported that salt dosages extended germination time and there were differences between cultivars in MGT due to this fact, we can infer that the higher salt concentrations were responsible for influencing lower germination of these materials by significantly delaying germination. Interestingly, in this study, it was observed that the effects of PEG6000 were more effective to separate the germination parameters of all cultivars, mainly those susceptible. In fact, the cultivars studied here are known to be tolerant or sensible to drought and less information is available of them regarding salinity. This way, probably the lower osmotic potential was more effective on reducing germination percentage and velocity in sorghum seeds studied here. Although salinity and drought stress are physiologically related and the tolerance mechanisms overlap, some aspect of seed physiology and metabolism may differ when the plant experiments single saline and water stresses simultaneously (Sucre and Suárez 2011). In this work we clearly verified the differences on germinability and velocity index of four sorghum cultivars in salt and osmotic conditions. Both tolerant cultivars showed higher improvement in 43 germination parameters when the seeds were primed, independent of the kind of conditioner. For both susceptible cultivars, the priming effects were not able to reduce the restrictions imposed by the treatments, decreasing germinability. However, for either tolerant or susceptible cultivars it was possible to verify that were differences on the levels of tolerance or sensibility. The cultivar BRS332, even characterized as sensitive, demonstrate huge capacity to germinate in concentrations below 180mM of NaCl regardless of the kind of priming treatment. It suggests that the use of seed priming in this cultivar can be an interesting option for moderate salt soils. On the other hand, almost no differences were verified on germination of the cultivar DBK540 independent of the concentration of salt or water potential. This cultivar, definitely, presents extreme tolerance to salinity and drought. This way, the decreases in germination remains that different damages may be responsible for this impairment, as observed in some studies, including damage to membranes and other structures, as well as damage at molecular levels (Zhang et al., 2020; Huang., 2018). Therefore, further experiments comparing mechanisms and strategies of both tolerant and susceptible cultivars are invited. Evaluating germination parameters in the laboratory is a sure way to ensure that environmental conditions will not be limiting factors for germination and consequent establishment of the crop. All the parameters evaluated in this work are efficient indicators to demonstrate a predictability of what to expect for the seeds in the field, and thus, to adopt measures that can circumvent the deleterious effects that may occur. Therefore, breeding programs as tools for phenotyping could opportunely, incorporate the prediction results with seeds in the laboratory conditions. 5. CONCLUSIONS Even under high salinity and high osmotic potential conditions, we could observe a beneficial effect of the priming application on the seeds, even though there was a delay in germination, the priming was efficient in promoting germination at higher salt doses and lower osmotic potentials. In general, the priming was efficient and brought benefits to the germination, mainly for the susceptible to water deficit cultivars, which showed a performance similar of the tolerant cultivars. It is still necessary to understand the metabolic effects provided by priming with ascorbic acid and ABA, since the results coming from their application could be used as guidelines for the development of cultivars that adapt to the various realities faced by breeders of this species. Studies that also point out if these benefits are desirable to be 44 performed in seedlings and plants are necessary in order to fill an existing gap in the works carried out using the priming technique. 6. 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First off the mark: early seed germination. Journal of experimental botany, 62(10), 3289-3309. 2011. YEMM, E. W., & WILLIS, A. The estimation of carbohydrates in plant extracts by anthrone. Biochemical journal, 57(3), 508. 1954. ZHANG, F., SAPKOTA, S., NEUPANE, A., YU, J., WANG, Y., ZHU, K., ... & ZOU, J. Effect of salt stress on growth and physiological parameters of sorghum genotypes at an early growth stage. 2020. ZHU, G., AN, L., JIAO, X., CHEN, X., ZHOU, G., & MCLAUGHLIN, N. Effects of gibberellic acid on water uptake and germination of sweet sorghum seeds under salinity stress. Chilean journal of agricultural research, 79(3), 415-424. 2019. 50 CHAPTER II PRIMING TECHNIQUES: IMPROVING GERMINATION THROUGH DIFFERENT MECHANISMS ABSTRACT Affecting crop production, salinity and osmotic stress can promote various impacts on plants. These conditions directly affect seed metabolism and fitness, causing lower germination through the imbalance of osmotic potentials. In the scenario where global changes are increasing, irregular precipitation leads to higher ion accumulation in the soil, as well as the impact of drought technique that can promote better germination and seedling establishment are the focus of the seed scientist, as these processes are highly affected by these conditions. Seed priming appears as a promising technique with lower costs and high efficiency. The use of different molecules in this procedure is investigated in promoting a more efficient antioxidant enzyme metabolism and the accumulation of osmoprotectants. In our work, we observed that the application of the priming technique with ascorbic acid (AsA) and abscisic acid (ABA) increased antioxidant metabolism and proline accumulation in sorghum plants, as well as carbohydrates, promoting an adjustment of seed homeostasis. It is possible to observe in our work that there was a symmetry in the germination percentage (%G) of the cultivar DKB 540, as an increase in the concentration of NaCl and also a decrease in the osmotic potential, however, even under the application of priming this symmetry could not be observed in the cultivar BRS-332. The concentration of MDA and H2O2 was lower in the cultivar BRS-332, either under osmotic stress or exposed to higher doses of NaCl, causing it to behave in the same way as the cultivar DKB 540 when submitted to osmotic stress. The enzymes of antioxidant metabolism showed no statistical difference, being the antioxidant potential of amino acids such as proline possibly responsible for maintaining the redox and osmoprotective functions of the seeds submitted to stressful conditions. Key words: oxidative stress; climate change; hormopriming; abiotic stress, proline. 51 1. INTRODUCTION Improving seed germination and quality is a constant pursuit in agronomy in the present days. Several environmental conditions significantly affect crop establishment, and controlling their effects is a way to achieve better yields in the field and promote food security (Ashraf et al., 2018). Among these limiting factors, the imposition of the osmotic and salinity stress stand out, since these conditions are closely linked and impose restrictions on the germination process (Ibrahim, 2013; Molazem et al., 2015, Corwin, 2021). Osmotic and saline stress are considered one of the world's most detrimental abiotic stresses and impacts agricultural yields significantly (Nimir et al., 2020). The insecure global climate with irregular rainfall patterns are the major causes of the recurrent onset of water stress worldwide (Moussa and Hassan, 2016). Salt and wa