SÃO PAULO STATE UNIVERSITY - UNESP CAMPUS OF JABOTICABAL SELENIUM AND SULPHUR: MITIGATION IN PLANT STRESSES Leonardo Warzea Lima Biologist 2016 II SÃO PAULO STATE UNIVERSITY - UNESP CAMPUS OF JABOTICABAL SELENIUM AND SULPHUR: MITIGATION IN PLANT STRESSES Leonardo Warzea Lima Advisor: Dr. Priscila Lupino Gratão Co-Advisor: Dr. André R. dos Reis Dissertation submitted to the College of Agricultural and Veterinary Sciences – UNESP, Campus of Jaboticabal, in partial fulfillment of the requirements for the degree of Master of Science in Agronomy (Crop production). 2016 III Lima, Leonardo Warzea L732s Selenium and Sulfur: Mitigation in plant stresses / Leonardo Warzea Lima. – – Jaboticabal, 2016 xi, 97 f. : il. ; 28 cm Dissertação (mestrado) – Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, 2016 Orientador: Priscila Lupino Gratão Co-orientador: André Rodrigues dos Reis Banca examinadora: Tiago Tezzoto, Tiago Santana Balbuena Bibliografia 1. Estresse oxidativo. 2. Cádmio. 3. Selênio. 4. Enxofre. I. Título. II. Jaboticabal-Faculdade de Ciências Agrárias e Veterinárias. CDU 635.64:546.4 Ficha catalográfica elaborada pela Seção Técnica de Aquisição e Tratamento da Informação – Serviço Técnico de Biblioteca e Documentação - UNESP,Câmpus de Jaboticabal IV V AUTHOR’S CURRICULUM INFORMATION Leonardo Warzea Lima was born on March 01, 1987 in São Paulo, Brazil. His parents Eliana Regina Warzea Lima and Wladimir Godoy Lima. In 2005 he finished the high school in São Paulo. In 2010 his career path was clear and started to study for his bachelor degree in Biology at the São Paulo State University (UNESP), campus of Jaboticabal, which provided a rich and solid background for his research career. Among all the experiences on different departments, the one year training at the Plant Physiology Laboratory in 2012, working on the area of abiotic stress, was a watershed experience. Under the supervision of the Dr. Priscila Lupino Gratão, he could be surrounded by the routine of a laboratory with basic training and the specific methodology used to measure the level of the oxidative stress on different vegetable tissues. At the same year, he started his Undergraduate Research Mentorship Program, when received a scholarship to develop the project called “Selenium antioxidant effects on fruits of mutant tomato in response to the exposure to Cadmium”. After one year studying Selenium (Se) and the heavy metal contamination, the interest in using science to minimize vegetable stress and environmental impact of different pollutants started to bloom. In 2013 he got involved with different academic activities at the university, occupying the position of public relations at the Government of Students Union and the student’s representative to the Board of the Department of Biology, furthermore he created the Biological Sciences Academic Journal called “Conexão (Connection)”, which became a powerful way to disseminate information and ideas through interviews and articles among students. As a curious Biologist, passionate about plant physiology, Leonardo was approved in 2014 to continue his studies on a Master of Science degree program in Agronomy, at the same University, in order to prepare for the long term goal of pursuing a career of teaching and research. As a Master’s student, he became more independent, creative and proactive, able to conduct the research on the analysis of the antioxidants effects of Se and also to look forward to work on related areas of research. In March 2016, Leonardo was approved to continue his studies on a PhD program in Botany at the Colorado State University, Fort Collins-USA, under the supervision of the Dr. Elizabeth A. H. Pilon-Smits. VI “Discovery consists of seeing what everybody has seen, and thinking what nobody has thought”. Albert von Szent-Györgyi VII To my parents, Wladimir and Eliana, for the unconditional love and support. VIII SUMMARY Pages ABSTRACT ...................................................................................................... 11 CHAPTER 1 – GENERAL CONSIDERATIONS .............................................. 12 1. Introduction ................................................................................................ 12 2. Literature Review ....................................................................................... 13 2.1 Antioxidant defense mechanisms ........................................................ 13 2.2 Enzymatic defense mechanisms ......................................................... 13 2.2.1 Superoxide dismutase (SOD, EC 1.15.1.1) .................................. 13 2.2.2 Catalase (CAT, EC 1.11.1.6) ........................................................ 14 2.2.3 Glutathione (GSH) ........................................................................ 17 2.3 Non-enzymatic defense mechanisms ................................................. 19 2.4 Cadmium contamination and the induced stress ................................. 20 2.5 Selenium as a stress alleviation strategy in plants .............................. 22 3. References ................................................................................................ 23 CHAPTER 2 - SULFUR METABOLISM AND STRESS DEFENSE RESPONSES IN PLANTS ............................................................................... 27 Abstract ......................................................................................................... 27 Keywords ...................................................................................................... 27 Introduction ................................................................................................... 27 Sulfur uptake and assimilation ...................................................................... 29 Phytohormones in S assimilation .................................................................. 35 Sulfur oxidation states in the cell ................................................................... 37 Methionine biosynthesis ................................................................................ 39 Sulfur compounds related to plant defense ................................................... 41 Glutathione (GSH), metallothioneins (MTs) and phytochelatins (PCs) .......... 45 Glucosinolates............................................................................................... 46 Final Considerations ..................................................................................... 47 Acknowledgments ......................................................................................... 49 References .................................................................................................... 49 CHAPTER 3 - SELENIUM AND HEAVY METALS .......................................... 65 1. Selenium: a powerful antioxidant ............................................................... 65 2. Antioxidant defense mechanisms .............................................................. 65 3. Role of selenium against heavy metal stress in plants............................... 66 IX 3.1 Cadmium (Cd) ..................................................................................... 66 3.2 Arsenic (As) ......................................................................................... 69 3.3 Lead (Pb) ............................................................................................ 70 3.4 Other heavy metals ............................................................................. 70 4. Final considerations ................................................................................... 71 5. References ................................................................................................ 72 CHAPTER 4 - SELENIUM ALLEVIATES CADMIUM TOXICITY IN TOMATO FRUITS ............................................................................................................ 74 Abstract. ........................................................................................................... 74 Keywords. ......................................................................................................... 74 1. Introduction ................................................................................................ 75 2. Material and methods ................................................................................ 76 2.1 Plant material and experimental treatments ........................................ 76 2.2 Fruits dry weight .................................................................................. 77 2.3 Fruits concentration of chlorophyll and carotenoids ................................ 77 2.4 Fruits proline concentration ..................................................................... 78 2.5 Fruits Cd2+ and Se quantification and nutritional analyses ...................... 78 2.6 Cd2+ and Se Translocation index ......................................................... 79 2.7 Statistical analyses .............................................................................. 79 3. Results and Discussion.............................................................................. 80 3.1 Nutritional analyses. ............................................................................ 80 3.2 Cd2+ and Se concentration and translocation index ............................ 83 3.3 Fruit dry weight .................................................................................... 85 3.4 Chlorophyll and carotenoids concentration ......................................... 87 3.5 Proline concentration ........................................................................... 90 4. Conclusions ............................................................................................... 92 5. Acknowledgements .................................................................................... 92 6. References ................................................................................................ 92 X LIST OF FIGURES Pages Chapter 1 – General Considerations Figure 1. Dismutation reaction of the O2 -• by the SOD metalloenzyme…….....14 Figure 2. Haber-Weiss reaction……………………………………………………14 Figure 3. Chemical structure of Catalase (CAT)…………………………………15 Figure 4. Simplified scheme of the H2O2 degradation by CAT…………………16 Figure 5. Ascorbate-glutathione cycle (Halliwell-Asada)………………………..18 Figure 6. GSH and Phytochelatins synthesis in plants………………………….19 Chapter 2 - Sulfur metabolism and stress defense responses in plants Figure 1. Biosynthetic pathways for S-containing amino acids and their derivatives………………………………………………………………..34 Figure 2. Sulfur-containing defense compounds………………………………...38 Figure 3. Sulfur assimilation and its physiological functions……………………48 Chapter 3 - Selenium and heavy metals Figure 1. Selenium effects against the oxidative stress…………………………68 Chapter 4 - Selenium alleviates cadmium toxicity in tomato fruits Figure 1. Fruits dry weight………………………………………………………….86 Figure 2. Fruits pigments concentration…………………………………………..89 Figure 3. Fruits proline concentration…………………………………………..…91 LIST OF TABLES Pages Table 1. Nutrients concentration in fruits………………………………………….82 Table 2. Selenium (Se) and Cadmium (Cd2+) concentration in fruits, leaves, roots and translocation index TI)………………………………………..84 XI SELENIUM AND SULPHUR: MITIGATION IN PLANT STRESSES ABSTRACT - Plants do not have specific defense mechanisms to counteract the diverse range of abiotic stresses and pollutants into the environment, and its survival depends on the flexibility and adaptability of its own natural defense mechanisms. Furthermore, the maintenance of cellular homeostasis depends on several interlinked and complex mechanisms, while the cellular defense system does not follow a specific pattern of action and may differ due to various factors such as plant species, exposure time to the stress, plant developmental stage, different organs and tissues analyzed. In the light of these considerations, this dissertation aimed to highlight and investigate the role of Sulfur and Selenium against different plant stresses, through the enzymatic and non-enzymatic plant responses and other related defense mechanisms. In the first chapter the author characterize the general biochemical mechanisms of the antioxidant cell defense, specifically the reactive oxygen species (EROs) formation and its chemical singularities and the induced oxidative stress, the enzymatic antioxidant defense system, specifically the superoxide dismutase (SOD) and Catalase (CAT) enzymes, the non-enzymatic mechanisms against the stress, including the Ascorbate-Glutathione cycle, the GSH (reduced glutathione), the phytochelatins and also proline formation. The plant nutritional status during the stress is crucial in order to maintain a proper defense response. In view of this, the chapter two is a published review about the participation of Sulfur (S) on the stress defense. This nutrient has a role in fundamental processes such as electron transport, structure, regulation and it is also associated with photosynthetic oxygen production, abiotic and biotic stress resistance and secondary metabolism. Moreover, few chemical elements are considered benefic to plants, while Selenium (Se) is the most relevant. In the chapter three the author describes the role of Se to detoxify the stress induced by heavy metal contamination, its powerful antioxidant characteristics and the improvement of the antioxidant enzymes activity and overall defense mechanisms. The chapter four consists of a scientific project conducted by the author. The aim of this study was to investigate whether Selenium, under the form of selenite (Na2SeO3), may avoid the uptake, translocation and concentration of Cadmium (CdCl2), in different tomato tissues, indicating possible mechanisms to counteract the stress, as well as to analyze the fruits overall status through the nutritional analyses, dry weight, pigments and proline concentration. The results demonstrate that alleviating effect of Se in tomato under Cd contamination could be related to restriction of Cd2+ uptake and translocation, enhancing micronutrient concentration in fruits and, finally, enhancing fruit proline concentration. Key-Words: Heavy metal, acclimation, stress adaptations, oxidative stress 12 *Published. Journal: Tropical Plant Biology (2015) 8:60–73. DOI 10.1007/s12042-015-9152-1 CHAPTER 1 – GENERAL CONSIDERATIONS 1. Introduction In order to facilitate the understanding and comprehension of the degenerative mechanisms triggered by different sources of abiotic stresses, the meaning of the word “stress” must be primarily defined under the vegetal perspective. Plants maintain a very intimate and necessary contact with the environment in which they are located, through the water and minerals absorption from the soil as well as the capture of the light energy from the sun, among other processes. Therefore, it is easy to assume that the environment exerts an important role to the physiology, anatomy and the plants biochemistry, influencing systemically their growth and development. Consequently, any alterations of the adequate and specific environmental conditions for each vegetable species to develop accordingly and healthy can disrupt physical and chemical changes in the plant as a result of a primary reaction to the stress, such as water deficiency or excess of salts in the soil, which can cause the stomatal closure and an increased production of compatible osmolytes, like proline and glycine-betaine, for example (BANU, et al., 2009; SHEVYAKOVA et al., 2013). Such modifications are reversible and all plant metabolism regulates after the environmental conditions returns to the regular state. However, when the stress is strongly severe or persists for a long time an imbalance in the cellular redox state occurs, mainly due to the overproduction of reactive oxygen species (ROS) above the cellular antioxidant capacity, leading to the destruction of membrane lipids, proteins, nucleic acids and other cellular components, resulting in a secondary stress called oxidative (OPDENAKKER et al., 2012, IRFAN et al., 2013). These ROS are unstable and partially reduced forms of the atmospheric oxygen (O2), formed during the aerobic cellular metabolism in all cell organelles that have the electron transport chain (the oxygen acts as the final electron acceptor in the chain) or a highly oxidized metabolic rate, such as mitochondria and peroxisomes (CUYPERS, et al., 2010). These ROS results from the transfer of one, two or three electrons to the O2 molecule, forming respectively the superoxide radical (O2 -•), the hydrogen peroxide (H2O2) or the hydroxyl 13 radical (OH•) and also by the O2 excitation processes which generates the “Singlet” oxygen (SHIEBER and CHANDEL, 2014). The O2 is a stable and not reactive molecule but, contrarily, these ROS are able to oxidize and denature many other molecules and structures due to the unpaired number of electrons in their final outer shell, leading to the cell destruction and thus to the death of tissues and organs such as roots, leaves, fruits or seeds. Consequently, the oxidative stress occurs when there is a serious imbalance between the ROS production and the antioxidant defense system, in any cellular compartment. 2. Literature Review 2.1 Antioxidant defense mechanisms The cellular defense mechanisms corresponds to the enzymatic and non- enzymatic antioxidant responses capable to directly denature the ROS, such as the superoxide radical (O2 -•) or the hydrogen peroxide (H2O2), and also neutralize the stressor which is in excess in the cell environment (normally a metal ion), thus neutralizing the deleterious effects of the reactive processes. 2.2 Enzymatic defense mechanisms 2.2.1 Superoxide dismutase (SOD, EC 1.15.1.1) The superoxide dismutases (SODs) are metalloenzymes that constitute the first enzymatic barrier against the induced oxidative stress, catalyzing the dismutation reaction of the O2 -•, forming O2 (cell oxygen) and H2O2 (hydrogen peroxide) (SHIEBER and CHANDEL, 2014) (Figure 1). It is important to mention that the antioxidant process triggered by the SODs can be considered as the primary defense mechanism of the plant cells against the ROS, mainly because it interferes negatively and directly in the Haber-Weiss reaction (Figure 2), which produces the highly reactive and not destructible hydroxyl radical (OH•), from the combination of O2 • and H2O2 (CUYPERS et al., 2010). 14 Figure 1. Dismutation reaction of the O2 -•, forming O2 (cell oxygen) and H2O2 (hydrogen peroxide), by the superoxide dismutases (SODs) metalloenzymes. Figure 2. Haber-Weiss reaction: hydroxyl radical (OH•) formation from the combination of O2 • and H2O2. These SODs enzymes are present in different plant tissues and are mainly found in chloroplasts, the mitochondrial matrix and the cytoplasm of cells. Moreover, three different isoforms of this metalloenzymes are known, differing by the metal ion present in the molecule active site and also by the action location in the cells, while the most abundant isoform in vegetables is the SOD which have copper (Cu) and zinc (Zn) in its active site (Cu / Zn - SODs), found mainly in the stroma of chloroplasts and also in the cytosol. Another important isoform are those SODs which have manganese (Mn) on the active site, present in the mitochondrial matrix. On the other hand, the SOD which have Fe (iron) in its active site are rarely found in plants, however this isoform can be associated with chloroplasts (KUMAR et al., 2014). These SOD isoforms are very similar when are compared among different plant species, so that the differences are associated with the isoform found and the concentration in the tissues. 2.2.2 Catalase (CAT, EC 1.11.1.6) The hydrogen peroxide (H2O2) is an unstable and highly reactive molecule and its concentration generally becomes higher in the cellular environment during a stressful condition and also as a result of the enzymatic reaction of the SOD, which can induce the oxidative damage of other molecules and tissues. Consequently, the H2O2 can be quickly converted into H2O (water) and O2 (cell oxygen) by the specific action of enzymes such as catalase (CAT, 15 EC 1.11.1.6) and other peroxidases in different cellular compartments (ROYCHOUDHURY et al. 2012). Catalase was the first antioxidant enzyme discovered and characterized, and basically consists of a polypeptide with an approximate weight of 70 kDa, arranged in a tetrameric molecule, so that each monomer contains a prosthetic heme group with a central atom of Fe, as shown in figure 3 (MHANDI et al., 2010). Figure 3. Chemical structure of Catalase (CAT). This enzyme can be found in all living organisms and is the main route of H2O2 degradation in order to form H2O and O2, according to the reaction shown in Figure 4. The dismutation reaction occurs in four distinct steps, so that the O2 molecule formed at the end of the reaction (step 4) is derived from a single molecule of H2O2, while the two H2O molecules formed during the process (steps 2 and 4) are derived from the combination of the two H2O2 substrates (Figure 4). Additionally, both CAT and other peroxidases primarily reduces the H2O2 molecule by breaking the O2 binding site, forming a molecule of water and one intermediate molecule called compound I, where the central atom of Fe (from the CAT enzyme) covalently binds to the liberated oxygen after the H2O2 reduction (step 2). The CAT specific activity consists of the oxidation of a second H2O2 molecule to form O2 and H2O (step 4), while Fe is reduced again 16 to its original oxidation state and the process restarts (step 4 and 1). Finally, it is important to mention that CAT is unique among all the other enzymes responsible for the H2O2 degradation (peroxidases), mainly because it does not use any equivalent reducing agent, acting directly and efficiently during the entire process (Figure 4). Figure 4. Simplified scheme of the H2O2 degradation catalyzed by the CAT enzyme, resulting in H2O (water) and O2 (oxygen) formation. Both H2O2 molecules used in the process as well as their products from their degradation (steps 2 and 4) are represented in the same color. Roman numbers represent the oxidation state of the Fe atom. Adapted from Mhamdi et al., 2010. Furthermore, CAT has three different types of isoenzymes referred as CAT1, responsible for 80% of the total H2O2 degradation in the cell (specifically in peroxisomes), formed during the photorespiration process in chloroplasts. CAT2, mainly found in vascular tissues and CAT3, located in the mesophyll of leaves (MHAMDI et al., 2010). This enzyme is essential for the H2O2 denaturation process in plants that have higher rates of photorespiration, such as all the C3 plants. Other enzymes are also responsible for the H2O2 degradation in the cell environment. Peroxidases are hemeproteins (have a heme group in the 17 molecule, characterized as a central iron atom in an organic porphyrin ring) responsible for the H2O2 molecule reduction concomitantly with the oxidation of a specific substrate, as the ascorbate peroxidase (APX, EC 1.11.1.11), glutathione peroxidase (GPX, EC 1.11.1.9) and guaiacol peroxidase (GPOX activity, EC 1.11.1.7), for example. These peroxidases participate in many essential metabolic processes such as the cell growth regulation, lignification, phenolic oxidation, defense against pathogens, antioxidant defense and protection against various stresses. The guaiacol peroxidase (GPOX, EC 1.11.1.7) enzyme, for example, is also part of this group and presents basics and acids isoforms in plants. The acid isoform is directly related to the processes involved in the cell wall biosynthesis, including the lignin formation, while its basic isoform participates in the regulation of the AIA (indolylacetic acid, auxins) degradation. In vitro experiments the GPOX catalyze the hydrogen donors oxidation, in the absence of a specific substrate, however, in vivo, the detoxification can become the primary function of certain isoforms. 2.2.3 Glutathione (GSH) Glutathione (tripeptide formed from the amino acids glutamate, cysteine and glycine) is a major metabolite in the defense system against ROS and its degenerative effects during the enzymatic antioxidant defense, participating along with the ascorbate peroxidase (APX) enzyme in the Halliwell-Asada cycle or ascorbate-glutathione cycle (Figure 5), being necessary to the proper functioning of this cycle. This metabolite can be found in the plant under the oxidized form (GSSG) or also the reduced form (GSH), which is important for non-enzymatic antioxidant defense (see section 2.3), so that the system always prioritize an increased concentration of GSH over the GSSG in the cellular environment. 18 Figure 5. Ascorbate-Glutathione cycle (Halliwell-Asada). Enzymes: ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), glutathione reductase (GR, EC 1.6.4.2). Other compounds: hydrogen peroxide (H2O2), water (H2O), ascorbate (ASC), dehydroascorbate (DHA), monodehydroascorbate (MDHA), glutathione (GSH), oxidized glutathione (GSSG). Adapted from Inzé and Montago (1995). In this cycle, the antioxidant action occurs by the conversion of the H2O2 molecule into H2O through the APX enzyme; however, the process is completely dependent upon the glutathione conversion cycle (Figure 5). The enzyme glutathione reductase (GR, EC 1.6.4.2) is responsible for the conversion of GSSG into GSH using as an electron donor the NADPH, while the formed GSH corresponds to the substrate used by the dehydroascorbate reductase (DHAR) enzyme in the ascorbate (ASC) formation pathway, which is used as a substrate for the APX enzyme antioxidant activity (Figure 5) (INZÉ and MONTAGO, 1995). The glutathione reductase (GR) enzyme is almost of universal occurrence, being found in eukaryotes and in prokaryotes, from heterotrophic and photosynthetic bacteria to higher plants. This enzyme has the prosthetic group FAD (flavin adenine dinucleotide), responsible for catalyzing the electron transfer reaction from NADPH to GSSG, forming the GSH (VOET and VOET, 1995). 19 2.3 Non-enzymatic defense mechanisms The non-enzymatic mechanisms for cellular detoxification against the ROS are important and act jointly with the enzymatic antioxidant system in order to maintain the cellular redox state. Participate in these processes the phytochelatins (PCs), proline, flavonoids, alkaloids and carotenoids, among others (FOYER and NOCTOR, 2012). The phytochelatins represent the most important route against the metals, semimetals and heavy metals contamination, since these molecules have the ability to complex and inactivate these compounds, storing them into vacuoles in the cell. The glutathione (GSH) plays an essential role in both the enzymatic antioxidant defense against ROS and the PCs formation, and determining a pathway of action will result in the inactivation of the other corresponding via (ROYCHOUDHURY et al., 2012). The GSH synthesis process involves glutamate, glycine and cysteine, and the PCs are synthesized from the GSH already formed, as shown in Figure 6. The GSH formation consists of two reactions triggered by γGlutamate-cysteine ligase (EC 6.3 .2.2) and GSH synthase (EC 6.3.2.3) enzymes, while the conversion of GSH in phytochelatins occurs by the specific action of the glutathione-γGlutamyl-cysteinyl-transferase (EC 2.3.2.15) enzyme or PC synthase (Figure 6). Figure 6. GSH and Phytochelatin synthesis in plants. Adapted from Inouhe, 2005. The amino acid proline is part of the compatible osmolytes group, which is responsible for the cell osmotic adjustment maintenance during the water and salt stress and also by the excess of nutrients in the soil, with an important role in plant protection against other different abiotic stresses, such as the exposure 20 to low temperatures, soil acidity and the heavy metals exposure (SHEVYAKOVA et al., 2013). Furthermore, different studies demonstrate that this osmolyte is able to actively clean the ROS from the cell during the oxidative stress, besides conferring protection and stabilization of some cellular structures such as membranes, proteins and also enzymes during severe stresses (BANU et al., 2009). It is important to note that the products resulting from the proline molecule catabolism, after the alleviation of the stress condition, will be used in the oxidative phosphorylation process in mitochondria, generating molecules of ATP (adenosine 5'-triphosphate), which is important in the cellular recovery processes (ASHRAF and FOOLAD, 2007). Additionally, the proline synthesis in plants occurs in chloroplasts and also in the cytoplasm, with the glutamic acid as a precursor. This acid is converted to glutamate γ-semialdehyde acid (GSA) by the Δ1-pyrroline-5-carboxylate synthetase (P5CS) enzyme, which in turn is spontaneously converted to the Δ1- pyrroline-5-carboxylate (P5C), which it is then reduced to proline by the P5C reductase enzyme (VERBRUGGEN and HERMANS, 2008). In summary, the non-enzymatic antioxidant system operates through different mechanisms responsible for the direct elimination of the stressor, cellular homeostasis regulation or even with molecules that acts directly as antioxidants, such as flavonoids, which inactivate the ROS, and alkaloids or carotenoids which are capable to donate H+, which mitigates the negative effects of the oxidation processes caused by the reactive oxygen species. 2.4 Cadmium contamination and the induced stress Industrial activities involved in metal smelting and the increasing in mining processes on a global scale can be considered as the major sources of environmental contamination. Heavy metals are among the main pollutants generated by these sources and represents a major threat to living organisms because of its high toxicity, persistence in the environment and bioaccumulation (LIU et al., 2010, 2011). However, several other anthropogenic activities are responsible to release into the environment a large amount of these pollutants, which accumulates in 21 the soil and limit the plant productivity (ZHOU et al., 2013). According to Xu et al. (2013), the presence of heavy metals in agricultural soil, such as Cadmium (Cd), occurs as a consequence to the excessive use of phosphate fertilizers and pesticides, which allow its assimilation in crops and the consequent transfer through the food chain, representing a great danger to human and animal health. Cadmium is the most toxic of all heavy metals. It is responsible to cause numerous health problems to humans (XU et al., 2013), because of its accumulation in different organs such as kidneys, liver and central nervous system, with a half-life that can reach fifteen to thirty years in the organism (GONÇALVES et al., 2012). Due to its strong global demand, approximately 30000 tons of Cd are released into the atmosphere every year, while 4000 to 14000 tons of this total amount comes only from industrial activities, such as the rechargeable batteries, alloys for welding, pigments and paints industry (ATSDR , 2012). The presence of Cd in high concentration in the soil inhibits the plant growth (CHENN et al., 2011; IRFAN et al., 2013) causes a decrease in the chlorophyll and carotenoids content (LIU et al., 2010, 2011), affect the proper function of chloroplasts and the CO2 fixation, induces the antioxidant enzymes inactivation and the ROS overproduction, leading to the lipid peroxidation process and the consequent oxidative stress (XU et al., 2013). It is important to mention that Cd is not capable to induce the ROS overproduction directly but reacts with the Sulphur present in the thiol group in the cysteine and other different proteins, which results in a lower GSH concentration in the cell environment. This process results to a deflected defense system against the oxidative stress (CUYPERS et al., 2010). Furthermore, the increased concentration of iron (Fe) ions in the cellular environment, due to its replacement by Cd in different proteins, may also induce the production of OH- radicals, which are highly reactive and resistant to cellular defense mechanisms, resulting in a severe oxidative stress (DORTA et al., 2003). Great number of proteins and enzymes have metals in their active sites, the capacity of Cd to replace them due to chemical similarities molecules it is notable. Consequently, the antioxidant system metalloenzymes, such as the 22 SOD, can be inactivated by this replacement (GONÇALVES et al., 2008). According to Guimarães et al. (2008) CAT levels in peroxisomes are also drastically reduced in the presence of 50 mM of Cd due to its oxidation, which also impairs the oxidative stress management. Leaf chlorosis is also a regular symptom of the Cd induced stress, which demonstrates that the cellular photosynthetic apparatus is severely affected by this heavy metal. The molecular structure of chlorophyll is characterized by the presence of a central Magnesium (Mg) atom and its replacement by Cd alters its stability, decreasing the absorption of the light energy and the consequent production of carbohydrates (CHENN, et al., 2011; XU et al., 2013). The leaf chlorosis appearance can also be explained by the decrease in the amount and replication of chloroplasts in the cell, deficiency of phosphorus (P), manganese (Mn) and a negative influence on the activity of the enzymes related to the production of chlorophyll (GUIMARÃES et al., 2008). 2.5 Selenium as a stress alleviation strategy in plants Selenium (Se) is an essential micronutrient for humans and animals and exerts important roles in the organism, participating on the antioxidant defense system (CATANIA et al., 2009). The absence or inadequate ingestion of this nutrient in the diet can cause health problems related to malnutrition. In vegetables, Se in low concentrations exerts positive physiological effects, such as growth improvement, reduction of ROS concentration and lipid peroxidation, improves the accumulation of starch and sugars and provides a delayed senescence (XUE et al, 2001; FENG and WEI, 2012). Despite not been recognized as a nutrient, some studies demonstrate the beneficial effects to the plants, such as the increased antioxidant capacity after Se application (XUE et al., 2001; ZEMBALA et al, 2010) and also its ability to reduce the heavy metals availability while alleviate its toxic effects (MUÑOZ et al., 2007) (see chapters 3 and 4 for more detailed information regarding selenium and heavy metals). This mineral can be found under different forms in the soil, such as selenite (SeO3 2-), selenate (SeO4 2-) and also under its organic molecules like SeCys (selenium atom bonded to a cysteine molecule) and SeMet (selenium 23 atom bonded to a methionine molecule), which are assimilated by the roots (NOWAK, 2013). Furthermore, the presence of Se in the intracellular environment induces an increased levels and activity of different antioxidant enzymes, thereby regulating the concentration of ROS (see chapter 3). The glutathione peroxidase (GSH-Px) enzyme, for example, demonstrates an enhanced activity after the Se application, eliminating effectively the H2O2 and alleviating the oxidative stress (ZEMBALA et al, 2010; FENG and WEI, 2012). According to Feng and Wei (2012), Se can also stimulate the spontaneous dismutation of O2 - into H2O2 radical, without the catalytic process exerted by the SOD enzyme. However, these authors report that excessive Se concentrations can lead to an imbalance in the levels of GSH, thiol radicals (-SH) and also the NADPH in the cell, which are important for the ROS elimination and stress defense mechanisms. 3. References ASHRAF, M.; FOOLAD, M.R. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ. Exp. Bot. v. 59, p. 206–216. 2007. ATSDR; Public Health Statement, Agency for Toxic Substances and Disease Registry, 2012. BANU, M.N.A.; HOQUE, M.A.; SUGIMOTO, M.W.; MATSUOKA, K.; NAKAMURA, Y.; SHIMOISHI Y.; MURATA, Y. Proline and glycinebetaine induce antioxidant defense gene expression and suppress cell death in cultured tobacco cells under salt stress. J Plant Physiol. v. 166, p. 146-156. 2009. CATANIA, A. S.; BARROS, C. R.; FERREIRA S. R. G. Vitaminas e minerais com propriedades antioxidantes e risco cardiometabólico: Controvérsias e perspectivas, Arq Bras Endocrinol Metab. 2009. 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Safety, v. 95, p. 130-136, 2013. 27 *Published. Journal: Tropical Plant Biology (2015) 8:60–73. DOI 10.1007/s12042-015-9152-1 CHAPTER 2 - SULFUR METABOLISM AND STRESS DEFENSE RESPONSES IN PLANTS* Abstract Sulfur management is an important issue in crop plant nutrition. Sulfur has a role in fundamental processes such as electron transport, structure and regulation. It is also associated with photosynthetic oxygen production, abiotic and biotic stress resistance and secondary metabolism. Sulfate uptake, reductive assimilation and integration into cysteine and methionine are the central processes that direct oxidized and reduced forms of organically bound S into their various functions. Sulfur-containing defense compounds that are crucial for plant survival during biotic and abiotic stress include elemental sulfur, hydrogen sulfide, glutathione, phytochelatins, S-rich proteins and various secondary metabolites. Formation of these compounds in plants is closely related to the supply, demand, uptake and assimilation of S. This review will highlight the role of S during the stress response in plants and the relationship between S metabolism and primary S nutrition. Keywords Abiotic stress, Antioxidants, Oxidative stress, Plant nutrition, Sulfur uptake and metabolism Introduction Environmental variation triggers plant acclimation, adaptation or death. Natural and anthropogenic activities induce biotic and abiotic stresses during agricultural and forestry operations. Plant metabolism is often damaged by toxic compounds and hazardous chemicals present in soils and air (Su et al. 2014; Iannone et al. 2015). Thus, acclimation and adaptation processes are crucial to plant survival, and the identification and understanding of plant tolerance mechanisms are of major importance. Stress can be defined as any alteration in normal plant growth conditions (Boaretto et al. 2014). Some of these alterations 28 are related to temperature, salinity, water supply, ozone, soil acidification and heavy metal toxicity, among others (Azevedo et al. 1998; Monteiro et al. 2011; Bulbovas et al. 2014; Nogueirol et al. 2015). Studies of plants have been conducted to evaluate the effects of these changes on growth and development. Plants possess very efficient defense pathways that allow the scavenging of reactive oxygen species (ROS), protecting the cells from oxidative damage (Gratão et al. 2005). The primary function of regulatory mechanisms is to manage fluxes of sulfur (S) in response to developmental and environmental changing conditions. The goal for the plant is to optimize the use of available S to match the demands for growth and development, and resistance to stress (Hawkesford 2012). Sulfur assimilation starts from the uptake of external sulfate by the activity of sulfate transporter (SULTR) in roots. On the other hand, plants are able to use foliar absorbed H2S as S source for growth, especially under conditions where the S to the roots is limited (Koralewska et al. 2008). Sulfate is activated by Adenosine-5´-triphosphate sulfurylase (ATPS, EC 2.7.7.4) and then catalyzed by Adenylyl-sulfate reductase (APR, EC 1.8.99.2) and sulfite reductase (EC 1.8.7.1) to produce sulfide. Regarding the primary metabolism in plants, nitrate and sulfate need to be reduced prior to their incorporation into various essential organic nitrogen (N) and S compounds. The uptake and assimilation of S and N are strongly interrelated, since the major proportion of the reduced N and S in plants is incorporated into amino acids and subsequently into proteins (Stulen and De Kok 2012). An important coordination with C/N metabolism occurs at the level of cysteine and methionine biosynthesis, with the cysteine synthase complex (serine acetyltransferase (SAT, EC 2.3.1.3) and O-acetylserine(thiol)lyase (OASTL, EC 4.2.99.8)) acting as both a sensor and a regulator, mediated by a reversible association/dissociation of the complex. SAT is active when associated with OASTL, but inactive when dissociated. As the dissociation is promoted by excess OAS, the complex effectively senses both OAS and S availability and self regulates further OAS production accordingly (Hawkesford 2012). Thus, OAS is a signal mediating between substrate availability and flux. OASTL, which is in excess, will always catalyze synthesis of cysteine given availability of OAS and sulfide. The formation of these compounds is closely related to the 29 supply, demand, uptake and assimilation of S in plants. In this review, we provide essential information about the role of these S-containing compounds, particularly with regard to abiotic stress acclimation and plant tolerance adaptations. Sulfur uptake and assimilation Most soils currently used for agricultural and forest crops are naturally low in fertility, and chemical fertilization should be implemented to provide the crop requirements for essential nutrients such as nitrogen (N), phosphorus (P), potassium (K) and S. S uptake is directly driven by demand. Inadequate S nutrition can cause the inefficient use of other nutrients, such as carbon (C) and N, leading to deficiencies and decreases in protein biosynthesis, chlorophyll content and eventually crop yield (Lunde et al. 2008; Mazid et al. 2011; Iqbal et al. 2013). On the other hand, environmental pollution from sulfur dioxide (SO2), H2S, sulfite (SO3 2-) and sulfate (SO4 2-) is a serious global problem and can be toxic to plants (Krischan et al. 2012). S is a component of proteins, the amino acids cysteine (Cys) and methionine (Met), vitamins (biotin and thiamin), cofactors (Co-A and S-adenosyl methionine, SAM) and a range of secondary metabolites (Mazid et al. 2011). S is an essential macronutrient for living organisms and has multiple roles in plant development, including catalytic, regulatory and structural functions (such as in protein disulfide bonds, cellular membrane SO4 2- esters and electron transport through Fe-S groups). S-containing compounds such as PCS and GSH also have a role in trace element homeostasis (Na and Salt 2011). S is an important substrate/reductant in reactions during abiotic stress processes; GSH, a major antioxidant in plant stress defense and the major non-protein S source in plants (Kopriva and Rennenberg 2004; Ghelfi et al. 2011; Rennenberg and Herschbach 2012; Seth et al. 2012) is present in all root and leaf cell compartments, with the exception of the apoplast in the absence of stress (Josefczak et al. 2012). S is taken up from the soil solution predominantly as SO4 2- in an energy- dependent process mediated by specific membrane-bound SO4 - transporters (Buchner et al. 2004; Davidian and Kopriva 2010). Plants can also obtain 30 organic forms of S, such as S-containing amino acids, organic SO4 2- and elemental S, from the soil solution. Although of less significance, S in the form of atmospheric SO2 can be absorbed by plant leaves and fruits (Mazid et al. 2011), and atmospheric H2S can be absorbed through leaf stomata (Riemenschneider et al. 2005). The translocation of SO4 - into plastids for assimilation, storage in vacuoles, and long-distance transport among organs requires specific transporters; the mechanism of plasma membrane transport is proton-coupled co-transport (Buchner et al. 2004). There are approximately 12 to 16 reported genes encoding SO4 2- transporters (SULTR) in plant species. SULTR proteins can be classified according to their protein sequence similarities into SULTR 1 to 5 (for a review: Buchner et al. 2004; Davidian and Kopriva 2010). These transporters can move SO4 2- into the plant when soils are deficient in S; SULTR1;1 (skilled in trace SO4 2- uptake) and SULTR1;2 (major component) have been identified on root hairs and root epidermal and cortical cells of knockout mutants of Arabidopsis (Takahashi et al. 2011). From the structural perspective, SULTR are members of a family of membrane-bound solute transporters provided to 12 domains that cross the plasma membrane (Takahashi et al. 2012). SULTR2:1 is a low-affinity SULTR that appears to be involved in SO4 2- translocation from roots to shoots. The initial uptake by the root epidermis and cortical cells depends on high-affinity transport, as well as on the displacement of SO4 2- into tissues and/or organs, which can be very specialized. Low-affinity transporters (in Arabidopsis: AtSULTR2;1 and AtSULTR2;2) were shown to be more related to vascular transport of SO4 2- and to the regulation of the cytoplasmic SO4 2- concentration during accumulation in the vacuole (Buchner et al. 2004; Davidian and Kopriva 2010). The coordination and the dynamics between the pathways of short- and long-distance transporters require specific signaling mechanisms to control and regulate a range of genes encoding specific proteins involved in S uptake, transport and assimilation (Davidian and Kopriva 2010). Expression of these transporters is regulated by internal and external sulfate signals and by the N, C and S reductive assimilation pathways, including phytohormones and variable metabolites (Gojon et al. 2009; Davidian and Kopriva 2010). 31 After uptake, SO4 2- is assimilated into Cys, an amino acid at the intersection of primary metabolism, protein synthesis and the formation of low molecular weight S-containing defense compounds (Rausch and Wachter 2005; Gotor et al. 2014). Excess SO4 2- transported to leaves is stored in vacuoles and constitutes a large S reserve for plant metabolism (Iqbal et al. 2013). Cys synthesis is required for GSH biosynthesis and occurs in plastids, mitochondria and the cytosol. Activated SO4 2- also forms 3′-phosphoadenosine 5′-phosphosulfate (PAPS, EC 1.8.99.2), the S-donor for sulfonation, sulfation or sulfuryl-transfer reactions; the reaction is catalyzed by sulfotransferases (STs) that play important roles in cell communication, plant development and defense (Negishi et al. 2001). In parallel with S assimilation, some ‘dissimilatory’ reactions, such as the release of H2S from Cys 1 and 2, might contribute to defense processes (Rausch and Wachter 2005; Lisjak et al. 2011; for more information on H2S see Lisjak et al. 2010 and Lisjak et al. 2013). SO4 2- assimilation is critical for providing reduced S for various cellular redox processes (for review: Jacob and Anwar 2008; Takahashi et al. 2011) and for the synthesis of GSH (Kopriva and Rennenberg 2004; Ghelfi et al. 2011; Seth et al. 2012). GSH has many distinct functions in plant cell metabolism, including controlling gene expression linked to the redox state of cells or subcellular compartments; being an important reducing cofactor of many enzymes related to ROS detoxification (for review: Noctor 2006; Foyer and Shigeoka 2011); and directly controlling the S assimilation pathway. Reduced forms of S decrease significantly during S uptake and assimilation (Kopriva 2006; Chan et al. 2013). Many studies have emphasized the importance of N in S assimilation and plant stress defenses (Kopriva and Rennemberg 2004; Siddiqui et al. 2008; 2012; Salvagiotti et al. 2009; Carfagna et al. 2011). SO4 2- assimilation declines under nitrate (NO3 -) deficiency, and the capacity to reduce NO3 - and the activity of nitrate reductase (NR, EC 1.6.6.1-3) are diminished in plants that are starved for SO4 2- (Kopriva and Rennenberg 2004; De Bona et al. 2011). In tobacco plants, for example, SO4 2- uptake by the roots was drastically reduced when NR was inactivated (Kruse et al. 2007; Siddiqui et al. 2012). Moreover, in N-starved plants, the activities of enzymes responsible for S assimilation and the mRNA levels associated with related genes decreased, but the addition of two distinct http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD1-4H21KMG-2&_user=5674931&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000049650&_version=1&_urlVersion=0&_userid=5674931&md5=d908ebee09a77ab069b9ec25bb8ada74#bib1#bib1 http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD1-4H21KMG-2&_user=5674931&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000049650&_version=1&_urlVersion=0&_userid=5674931&md5=d908ebee09a77ab069b9ec25bb8ada74#bib2#bib2 32 N sources (NO3 -and ammonium, NH4 +) quickly restored the enzymatic function (Koprivova et al. 2000). In cereal plant species, increases in S fertilization can enhance the efficiency of N uptake and use because S is a constituent of some enzymes involved in N metabolism (Salvagiotti et al. 2009; De Bona et al. 2011). Wheat plants exposed to distinct N and S levels revealed an important relationship between N and S. When N was less limiting, N uptake was high at the highest S concentration. This increase in uptake was more directly correlated with recovery efficiency than with internal use efficiency (Salvagiotti et al. 2009). Nonetheless, it has been demonstrated that adenosine 5' phosphosulfate reductase (APR, EC 1.8.99.2), the key enzyme for SO4 2- assimilation, is regulated by carbohydrates (Lewandowka and Sirko 2008; Chan et al. 2013). The availability of Cys is another crucial factor in GSH synthesis, but an adequate supply of glutamate and glycine is also important (Kopriva and Rennenberg 2004). The activity and expression of SO4 2- transporters and APR in plants are modulated by their S status and the demand for growth (Koralewska et al. 2008). A key enzyme of plant S metabolism, O-acetylserine(thiol) lyase (OAS- TL, EC 2.5.1.47, also named cysteine synthase), catalyzes the formation of Cys from the sulfide ion (S2-) and O-acetylserine, as illustrated in Fig. 1 (Youssefian et al. 2001). Cys biosynthesis can be regarded as the exclusive function of S reduction in plants and is a key limiting step in the production of GSH and in tolerance to biotic and abiotic stresses (Youssefian et al. 2001; Mera et al. 2014). OAS-TL plays a key role in the synthesis of Cys and GSH, which are required for regulation of plant responses in response to oxidative stress (Youssefian et al. 2001; Gotor et al. 2014). Studies of barley plants demonstrated that N or S deficiency altered GSH levels in leaves. In N- and S-starved plants, GSH levels doubled, and the Cys concentration was shown to increase by 50% (Carfagna et al. 2011). In Brassica juncea, N and S enhanced the activity of adenosine triphosphate- sulfurylase (ATP-S, EC 2.7.7.4), a key enzyme in the S assimilation pathway, which activates SO4 2- via an ATP-dependent reaction. In response to an environmental N deficit, the addition of NO3 - or NH4 + rapidly improved ATP-S and OASTL function (Siddiqui et al. 2012). N addition positively affected OASTL http://apps.isiknowledge.com/DaisyOneClickSearch.do?product=WOS&search_mode=DaisyOneClickSearch&doc=1&db_id=&SID=U1imC78Fbha46n9IAEJ&name=Koralewska%20A&ut=000257196300007&pos=1&cacheurlFromRightClick=no http://apps.isiknowledge.com/DaisyOneClickSearch.do?product=WOS&search_mode=DaisyOneClickSearch&doc=6&db_id=&SID=U1imC78Fbha46n9IAEJ&name=Youssefian%20S&ut=000169884200013&pos=1&cacheurlFromRightClick=no http://apps.isiknowledge.com/DaisyOneClickSearch.do?product=WOS&search_mode=DaisyOneClickSearch&doc=6&db_id=&SID=U1imC78Fbha46n9IAEJ&name=Youssefian%20S&ut=000169884200013&pos=1&cacheurlFromRightClick=no 33 activity in plant roots, and a precise sequence of N metabolism and S assimilation is necessary to provide the N precursors for Cys biosynthesis (Carfagna et al. 2011). Thus, S assimilation is significantly related to assimilation of NO3 - and C (Yoshimoto et al. 2007). Some transcriptional factors responsible for SO4 2- uptake and assimilation have been identified, demonstrating a relationship between mRNA levels (to APR and ATP-S), protein biosynthesis and enzyme activity (Koprivova et al 2000; Hesse et al. 2003; Davidian and Kopriva 2010). 34 Figure 1. Biosynthetic pathways for S-containing amino acids and their derivatives. A key enzyme of plant S metabolism, OAS-TL, also named cysteine synthase, catalyzes the formation of Cys from the sulfide ion (S2-) and O-acetylserine. APS: adenosine-5’- phosphosulfate; PAPS: 3’-phosphoadenosine-5’-phosphosulfate; γ- EC: γ-glutamyl-cysteine; OAS: O-acetylserine; CoA: acetyl coenzyme A; SAM: S-adenosylmethionine (S-AdoMet); SMM: S- methylmethionine (modified from Hawkesford 2005; Koprivova and Kopriva 2014). 35 Phytohormones in S assimilation Some studies have reviewed the importance of the relationship between S assimilation and phytohormones (Maruyama-Nakashita et al. 2004; Maruyama-Nakashita et al. 2005; Kopriva 2006; Khan et al. 2013). Phytohormones are essential for plant acclimation and adaptation to environmental changes (Peleg and Blumwald 2011). The signaling pathway of phytohormones is linked to efficient nutrient use, plant defense pathways and plant developmental processes and metabolism (Fatma et al. 2012). Phytohormones such as cytokinins (CK), gibberellins (GA), auxins (AU), ethylene (ET), jasmonates (JA) and salicylic acid (SA) can interact with mineral nutrients under both normal and stress conditions, playing an essential role in salt stress control and affecting plant growth recovery, cell division, germination and seed production, even when applied exogenously (Fatma et al. 2012). CK is known to be related to the N cycle and metabolism, being involved in N and P assimilation. Therefore, it seems that CK has a general role in the assimilation of nutrients, including S (Kopriva 2006). The expression of indole-3-acetic-acid-amido synthetase (IAA-amido synthetase, EC 6.3.2.-) in rice seedlings was correlated with an increase in expression of LEA (late embryogenesis abundant) genes, which have been shown to promote drought stress tolerance (Zhang et al. 2009). The expression of many other genes related to auxin synthesis and to enzyme biosynthesis, transporters and activity can also be regulated by ET, whilst auxin seems to affect ET biosynthesis (Peleg and Blumwald 2011). ET has an important role in improving N use efficiency, photosynthetic rates and plant growth in N-optimal and N-deficient Brassica juncea plants (Khan et al. 2008). ET signaling increases with SA and/or JA, resulting in expression of a wide range of genes related to plant defense. GA enhances the effects of salt stress in soybean, perhaps by regulating the availability of other phytohormones. Abscisic acid (ABA) reduced Na+ and Cl- content and, consequently, the Na+/K+ ratio and increased the Ca2+, K+, soluble sugar and proline contents in rice crops (Khorshidi et al. 2009; Iqbal et al. 2013). Phytohormones can alleviate the effects of salt stress and improve 36 plant tolerance by influencing proline metabolism. N and/or Ca2+ accumulation are altered, and a link between Ca2+ signaling and SA content improves proline content (Du et al. 2009; Al-Whaibi et al. 2012). Proline accumulation seems to be regulated by ABA-dependent and ABA-independent pathways (Iqbal et al. 2013). ABA appears to be the phytohormone that responds most rapidly to plant stress. ABA synthesis and expression of ABA-inducible genes cause stomatal closure in plants under drought stress (Peleg and Blumwald 2011). Many genes associated with ABA biosynthesis and ABA receptors were identified in Arabidopsis (Brocard-Gifford et al. 2004) and maize (Peleg and Blumwald 2011). Plants exposed to SA and ABA exhibited higher GSH concentrations and glutathione reductase activity (GR, EC 1.8.1.7, also named glutathione-disulfide reductase, GSR) (Kopriva 2006; Nazar et al. 2011; Pál et al. 2014). This result confirms the relationship among GSH content, S assimilation and stress defense. ABA is related to environmental stress adaptation and SA plays a key role in plant stress tolerance, expression of genes that encode chaperones, heat-shock proteins and antioxidants, and genes related to secondary metabolites (Kopriva 2006; Pál et al. 2014; Peleg and Blumwald 2011; for a review on the role of phytohormones in stress tolerance, see Carvalho et al. 2011). ABA increases the levels of mRNA encoding cytosolic OAS-TL, a key enzyme in S assimilation and metabolism (Kopriva 2006). The increased demand for GSH can be met by activation of pathways involved in S assimilation and Cys biosynthesis. Microarray analyses have indicated that the messenger ribonucleic acid (mRNA) transcript level of OAS- TL was upregulated in response to zinc stress in A. thaliana (Becher et al. 2004) and was constitutively elevated in its metal-tolerant relative, A. halleri (Weber et al. 2004). Many studies address the production and influence of GSH and Cys in relieving biotic and abiotic stress in plants, such as in the protective response to oxidative stress resulting from various factors (Ruiz and Blumwald 2002; Rahoui et al. 2014; Zhang et al. 2014). Under abiotic stress, GSH demand increases (to promote stress tolerance), activating enzymes in the S assimilation pathway. Enzymatic activity, genetic manipulation of enzymes involved in S assimilation http://www.ncbi.nlm.nih.gov/pubmed?term=Brocard-Gifford%20I%5BAuthor%5D&cauthor=true&cauthor_uid=14742875 37 and external S supply can lead to abiotic stress tolerance in plants (Rennemberg et al. 2007; Nazar et al. 2011). Cys content and GSH content and biosynthesis at the transcriptional level are regulated by JA (known to participate in the transduction of stress response) and methyl jasmonate (MeJA) (Shan and Liang 2010; Gfeller et al. 2011). Treatment of Arabidopsis with MeJa increased the mRNA levels corresponding to many genes involved in S assimilation and GSH synthesis, without affecting the content of S metabolites or of mRNA levels associated with SO4 2- transporters (Kopriva 2006). These signaling compounds also increased the accumulation of mRNA associated with genes that are involved in S metabolism (Takahashi et al. 2011) and associated with S deficiency. This suggests that JA has a signaling role for inducing S assimilation under S deficiency (Sasaki-Sekimoto et al. 2005; Srivastava et al. 2013). Several limitations of the suggested stress response have to be emphasized because of the importance of the GSH system with respect to other components of the photoprotective and antioxidative defense systems. The roles of JA, SA, GA and ABA in regulating S assimilation enzymes are crucial for acquired abiotic stress tolerance in plants. Sulfur oxidation states in the cell Under stressful conditions, the induction or increase in S compounds related to plant defense is also crucial for detoxification of excessive ROS (Noctor et al. 2012), resulting in diverse modes of action for S-containing secondary metabolites (Fig. 2). In the cell, S can be found in several oxidation states mediated by different enzyme families. For example, the sulfotransferase protein family (SOT, EC 2.8.2) catalyzes the transfer of sulfonate molecules in the highest oxidation state to an appropriate hydroxyl group of many substrates using 3’-phosphoadenosine 5’-phosphosulfate (PAPS) as the sulfuryl donor. The SOT also catalyzes the sulfonation of a wide range of compounds and produces SO4 2- esters and conjugates (Klein and Papenbrock 2004). 38 Figure 2. Sulfur-containing defense compounds. GR: Glutathione Reductase, APX: Ascorbate Peroxidase, GPX: Glutathione Peroxidase, MDHAR: Monodehydroascorbate Reductase, DHAR: Dehydroascorbate Reductase, APS: 5’-adenylylsulfate, PAP: 3’-phosphoadenylylsulfate, APSK: APS kinase, APSR: APS reductase, OAS-TL: O-acetylserine thiol lyase (modified from Rausch and Wachter 2005; Mendoza-Cózatl et al. 2005). 39 Cys is synthesized in the last stage of photosynthetic assimilation of sulfate in plant cells and is the first organic compound containing reduced S. Cys has essential roles in the function, structure and regulation of proteins, being the precursor of many important S-containing compounds involved in plant defense signaling and plant development (Gotor et al. 2014). Macroarray analysis revealed an integrated signaling pathway in plant defense gene expression in Arabidopsis, with upregulation by MeJA of several genes related to S metabolism, GSH, Cys and Met biosynthesis and S-rich defense proteins involved in GS metabolism (Jost et al. 2005; Guo et al. 2013). Methionine biosynthesis Met synthesis links Cys biosynthesis to the aspartate-derived amino acid biosynthetic pathway (for review, see Hawkesford and Kok 2006; see also other papers published by the group of M. Hawkesford; for reviews on the aspartate pathway, see Azevedo et al. 1997; 2006). Biosynthesis of Met from Cys involves three enzymatic steps. O-phosphohomoserine (OPHS, EC 2.7.1.39) derived from the aspartate pathway is a common substrate for both threonine and Met synthesis, catalyzed by threonine synthase (TS, EC 4.2.3.1) and methionine synthase (MS, EC 2.1.1.13), respectively (Azevedo et al. 1997). Cystathionine γ-synthase (CgS, EC 2.5.1.48) catalyzes the synthesis of cystathionine from Cys and OPHS by trans-sulfuration (Hawkesford 2005). Cystathionine is then converted to homocysteine (a β-cleavage reaction) by cystathionine β-lyase (CbL, EC 4.4.1.8). Homocysteine is exported from chloroplasts and converted (by methylation) into Met through MS activity. The activity of CgS and TS will influence biosynthesis of Met and threonine, respectively. CgS activity almost certainly has a large effect on flux and is most likely feedback-regulated by Met or a derivative (Azevedo et al. 1997; 2006). Similarly, TS activity is regulated by S-adenosylmethionine (SAM, also known as S-AdoMet), which is a derivative of Met (Azevedo et al. 1997; Wang and Frey, 2007). These controls effectively maintain the Met pool within close constraints. Rather small gene families encode the proteins of this pathway (CgS: 2 genes, CbL: 1 gene, MS: 3 genes). Furthermore, Met is a gateway to 40 many other important S-containing metabolites, including S-methylmethionine (SMM), SAM and dimethylsulfonio-propionate (DMSP). SMM is a transportable derivative of Met. It can revert to Met by donating a methyl group to homocysteine in a reaction catalyzed by homocysteine S-methyltransferase (HMT, EC 2.1.1.10). Under some circumstances, SMM may be the major S constituent of the phloem sap, and it has a role in delivering S to sink tissues such as seeds (Hawkesford 2005). SAM is one of the most important S-compounds in plant metabolism (Azevedo et al. 2006); it is involved in many processes and is the main methyl donor involved in transmethylation of proteins, nucleic acids, polysaccharides and fatty acids (Ma et al. 2003). SAM is also a precursor of the polyamine (PA) synthetic pathway (spermidine/spermine biosynthesis pathway) and of nicotinamide biosynthesis (important for Fe nutrition in plants). SAM is known as the ‘activated Met form’ (Bürstenbinder and Sauter 2012), and up to 80% of the Met pool may be converted to SAM at the expense of adenosine triphosphate (ATP) utilization (Ravanel et al. 1998; Iqbal et al. 2013) by SAM synthetase (SAMS, EC 2.6.1.6, five genes in the family). Spermidine and spermine have multiple proposed roles, including stress response, pH regulation, DNA replication and senescence processes. Consumption of SAM may increase S demands to meet these needs, although ultimately Met is recycled. SAM is also the precursor for ET, a potent modulator of plant growth and development that is involved in stress signaling (Wang et al. 2002). The synthesis of ET from SAM is catalyzed by 1-aminocyclopropane-1-carboxylic acid synthase (ACCS, EC 4.4.1.14) and ACC oxidase (ACCO, EC 1.14.17.4). Met is not consumed in this reaction but is recycled, resulting in no net S demand. A side product of the final biosynthetic step for ET is cyanide, which is detoxified to β-cyanoalanine by β-cyanoalanine synthase (CAS, EC 4.4.1.9), an isoform of OAS-TL (Hatzfeld et al. 2000). DMSP is produced in high concentrations in many marine algae and in some higher plants, such as marsh grasses in the genus Spartina, sugar cane and Wollastonia biflora. It is synthesized in higher plants via SMM, but it is generally present in low concentrations in other plant species. Several roles have been proposed, including salt tolerance and herbivore deterrence. 41 SAM, in decarboxylated form and catalyzed by S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50), provides 5´-desoxy-(5´-),3-aminopropyl- (1), a methylsulfonic salt required for PA biosynthesis (Roy and Wu 2002; for a review in PA: Alcázar et al. 2010; Hussain et al. 2011; Bitrián et al. 2012). PA l metabolites are essential to plant survival and have been correlated with biotic and abiotic stress resistance in many plant species; studies have employed exogenous PA application and genetic manipulations of different plant species (Bitrián et al. 2013). Increased biosynthesis of putrescine and spermidine in transgenic tobacco plants that had human SAMDC inserted into their genomes resulted in greater resistance to salt, drought and biotic stress (Waie and Rajam 2003). Microarray, transcriptomic and proteomic studies have demonstrated the role of PA in signaling cascades that increase plant tolerance or resistance to biotic and abiotic stress (Hussain et al. 2011). Sulfur compounds related to plant defense Environmental stress usually affects plant cell homeostasis and development, increasing ROS production and leading to oxidative stress (Arruda and Azevedo 2009; Azevedo et al. 2011; Cia et al. 2012; Boaretto et al. 2014). Abiotic stress is a consequence of the effects of a wide variety of distinct external agents on plants, such as temperature (heat or chilling), water (drought or flooding), salinity, proton toxicity, heavy metals, overexposure to ultraviolet rays, ozone and others (Azevedo et al. 1998; Gratão et al. 2005; Monteiro et al. 2011; Bulbovas et al. 2014; Nogueirol et al. 2015). All living organisms have a series of pathways to combat environmental stress. In plant species, changes in photorespiration, enzymatic and non-enzymatic antioxidant pathways, regulation and responsive gene expression, and morphological and anatomical adaptations have been identified and investigated (Foyer and Shigeoka 2011). Excessive ROS generation has been considered a negative process for many years, but it is an essential component of signaling processes that prompt adjustments in gene expression and cellular structure in response to environmental changes (Shao et al. 2008; Foyer and Shigeoka 2011; Monteiro et al. 2011). 42 Plants can respond to abiotic stresses in a number of ways. These include, as a primary step, the induction of a network of signaling pathways and, at later stages, the response by specific proteins, metabolites and other compounds triggered by the signal transduction of the first step (Shulaev et al. 2008). Molecular analysis revealed that both short-term and long-term responses are important for understanding the progression of signaling events when the external and then the internal nutrient supply become depleted (Schachtman and Shin 2007). Similarly, it is critical to understand how experiments are designed because chronic and acute treatments with a stressor can produce completely different responses (Gratão et al. 2008). Furthermore, these distinct responses may improve the understanding or identification of the mechanisms involved in stress tolerance or of plants that are tolerant to the induced stressful condition. Signal transduction and detection networks that control plant responses to nutrient deprivation are not characterized for N and S to the extend it should considering how important these elements are. As already emphasized in this review, the S assimilation pathway is related to plant responses to abiotic stress and to defense mechanisms. It is a source of reduced S for many cellular processes and for synthesis of Cys, which is used in Met synthesis and/or incorporated into proteins or GSH (Siddiqui et al. 2012). It is important to bear in mind that ROS is naturally produced by the cell metabolism. Oxidative stress occurs when the redox balance is disturbed and excess ROS induces a range of stress defense mechanisms (Gratão et al. 2005). ROS is an upstream mediator of nutrient signaling and increases rapidly after mineral nutrient deprivation, as indicated by Schachtman and Shin (2007). This is a major problem because the literature concerning the effect of an element/nutrient on a plant is extensive, but studies on the secondary responses and effects on the uptake and translocation of other essential elements are more limited. The resulting knowledge gap causes uncertainties regarding the full effects of the stressful conditions to which the plants were subjected. More integrated studies must be conducted. Research on oxidative stress induced by heavy metals is increasing dramatically. The number of studies being published is astonishing, although the majority confirms known information. Nonetheless, it appears that the stress 43 induced by metals increases demand for reduced S, activating the expression of SO4 2- transporters and enzymes of the assimilatory pathway (Hawkesford 2005; Hawkesford and Kok 2006; see also other papers published by the group of M. Hawkesford). Recent studies indicate that SO4 2- transport in the plant vascular system, its assimilation in leaves and the recycling of S-containing compounds are related to drought stress signaling and response (Takahashi et al. 2011; Hawkesford 2012). Plants are known to synthesize and release H2S in a process catalyzed by L-cysteine desulfhydrase (LCD, E.C. 4.4.1.1) and involving conversion of L- Cys to H2S, pyruvate and ammonia (García-Mata and Lamattina 2010). H2S was formerly considered toxic to plant development, inducing excessive production of ROS. However, as understanding of its role in metabolic stress responses has increased, several studies have shown that its main function in plants is as a signaling molecule that controls physiological and biochemical processes (Jin et al. 2011; Li et al. 2012). For example, a study of spinach plants fumigated with H2S gas demonstrated that approximately 40% of the H2S was converted into GSH in plant leaves (Lisjak et al. 2011). H2S had an essential role in alleviating the stress damage caused by aluminum chloride (AlCl3) in germinating wheat seedlings, increasing esterase and amylase activity and maintaining low malondialdehyde (MDA) and H2O2 levels (Zhang et al. 2010). Pre-treatment with sodium hydrosulfide (NaHS, a H2S donor) resulted in increased activity of guaiacol peroxidase (GPX, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11) and catalase (CAT, EC 1.11.1.6) and decreased aluminum (Al) uptake in Al pre- treated seeds of wheat; this confirmed H2S as a signaling molecule in response to abiotic stress (Zhang et al. 2010). In Vicia faba, A. thaliana and Impatiens walleriana, H2S also induced stomatal closure and ABA-dependent signaling, possibly through the regulation of ABC transporters in guard cells under drought, and enhanced tolerance of water stress (García-Mata and Lamattina 2010). Tobacco cells pretreated with NaHS also exhibited heat tolerance and regrowth after stress exposure (Li et al. 2012). A. thaliana exposed to the same pretreatment produced more H2S, exhibited drought tolerance by limiting stomatal aperture and increased the production and expression of drought marker genes (Jin et al. 2011). 44 In plants, incorporation of O-acetylserine sulfhydrylase (OASS, EC 2.5.1.65) and serine acetyltransferase (SAT, EC 2.3.1.30) into the cysteine synthase (CysK, EC 2.5.1.47) complex plays a regulatory role in S assimilation and Cys biosynthesis (Francois et al. 2006). The molecular mechanisms for the coordination of S, nitrogen and carbon assimilation are not yet known in detail. O-acetylserine, a precursor of Cys, was proposed as the signal regulating SO4 2- assimilation, but it is not likely to be the outgoing signal for N and C metabolism. In S-deprived plants, for example, the level of glucose, fructose and phosphoenolpyruvate decreased, while starch concentration increased (Lunde et al. 2008). The reduction in photosynthetic rate, increase in oxidation of the ferredoxin:thioredoxin system, changes in starch synthesis and degradation, and the lower use of carbohydrates as an energy source can explain the changes in carbohydrates content (Lunde et al. 2008). Complementary deoxyribonucleic acid (cDNA) array analysis revealed that expression of genes involved in auxin synthesis is induced upon S- starvation, suggesting a possible role of phytohormones (Kopriva and Rennenberg 2004). Clearly, and despite significant progress in understanding the regulation of SO4 2- assimilation and GSH synthesis, their coordination with N and C metabolism is not yet fully understood, and the several potential signal molecules identified are still far from being sufficiently explanatory (Kopriva and Rennenberg 2004). Stressed plants usually exhibit decreased rates of cellular division and elongation and, consequently, reduced or inhibited growth. This response may not only be a means of preserving energy for the defense process but may also function as protection against hereditary damage (Li et al. 2014). The chloroplasts play a major role in modulating the plant response, being both sensitive to abiotic stress factors and a major site for S assimilation (Biswal et al. 2008). These organelles are also important for ROS production because of the interaction between electrons escape from the photosynthetic electron transport chain and molecular oxygen (Foyer and Noctor 2012). Chloroplasts can therefore coordinate the C, N and S metabolic pathways, providing essential precursors for the synthesis of S-containing compounds (Jamal et al. 2006; Biswal et al. 2008). Another interesting aspect is that Fe-deficiency can result in severe disruption to the thylakoid lamellae, with loss of grana, but such http://apps.isiknowledge.com/DaisyOneClickSearch.do?product=WOS&search_mode=DaisyOneClickSearch&doc=4&db_id=&SID=U1imC78Fbha46n9IAEJ&name=Francois%20JA&ut=000244177000023&pos=1&cacheurlFromRightClick=no 45 damage to the photosynthetic apparatus can be diminished by S nutrition. Photosynthetic activity and sucrose synthase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activity are also closely related to S status (Muneer et al. 2014). Glutathione (GSH), metallothioneins (MTs) and phytochelatins (PCs) The stress response involves enzymatic antioxidant and other defense systems, including sulfur-containing compounds such as the essential macronutrient sulfur (S); glutathione (GSH: a S-containing thiol tripeptide, γ-L- glutamyl-L-cysteinyl-glycine); a class of phytochelatins [PCS: (g-Glu-Cys)n-Gly, n = 2 to 5 usually]; S-rich proteins; S-amino acids; hydrogen sulfide (H2S); and a range of secondary metabolites (Gratão et al. 2012). The GSH pool determines the degree of expression of genes linked to defense. It is controlled by many signaling pathways before and during stress, establishing a direct link between stress defense gene expression and GSH biosynthesis. Microarray, reverse transcription polymerase chain reaction (RT- PCR) and high performance liquid chromatography (HPLC) analyses of Arabidopsis plants exposed to cadmium (Cd) revealed that plants activate the S assimilation pathway by increasing transcription of specific genes that enhance the supply of GSH for PCS synthesis (Kawashima et al. 2011; Jobe et al. 2012). Moreover, roots and leaves have also been shown to exhibit distinct responses to Cd stress (Herbette et al. 2006; Gallego et al. 2012). Metallothioneins are proteins with two structural domains (Cys-rich and metal-binding) involved in metal homeostasis and detoxification (Majic et al. 2008; Choppala et al. 2014; Gu et al. 2014). But in the case of metal stress, the action of PCS is essential. PCS are Cys-rich peptides related to GSH and most likely are synthesized in the same pathway (Qureshi et al. 2007; Zagorchev et al. 2013). Plants can inactivate the toxic effects of excessive ROS by intracellular chelation of the metallic ion by GSH and/or PCS in the cytosol. Depending on the plant species, these complexes can be transported into the vacuole by a specific metal-requiring enzyme (Pál et al. 2006; Yadav 2010; Hossain and Komatsu 2013). Increased GS can be considered a sulfate reserve for PCS synthesis, however the identity of the S-compounds that are reduced 46 when GS increases and the impact of metals on their metabolism are unknown, despite the hypothesis that GS provides an additional S source under metal stress condition (Ernst et al. 2008; Bell and Wagstaff 2014). Additionally, a common response by metal-stressed plants may be the activation of the ascorbate–glutathione cycle, either for the removal of H2O2 or to ensure the availability of GSH for the synthesis of these metal-binding proteins (Vitória et al. 2001; Jozefczak et al. 2012). Glucosinolates Glucosinolates are amino acid-derived secondary metabolites consisting of a thioglucose moiety, a sulfonated aldoxime, and a side chain derived from either aliphatic or aromatic amino acids (Halkier and Gershenzon 2006). When plant tissues are disrupted the glucosinolates are hydrolyzed by the highly active plant enzyme myrosinase, a thiolglucosidase. The cleavage of the glucose thioester linkage produces an unstable intermediate that rearranges into biologically active thiocyanates, isothiocyanates, nitriles and oxalidine-2- thiones, depending on reaction conditions and presence of additional proteins (Hell and Kruse 2006). These products are chemically very reactive and may interfere with proteins and free amino acids. They are generally caustic and potentially toxic, hence their antimicrobial activities in plant defence. Although the glucosinolates also occur in a number of other plant families, the economic importance of oilseed rape, mustard, and the cabbage subspecies raised further interest in the biochemistry and molecular biology of glucosinolates biosynthesis and degradation (Hell and Kruse 2006). In the reductive S assimilation pathway, the activities of enzymes in the pathway are influenced by S supply, with some (in vitro) evidence that ATPS and APR can form a complex to by-pass a branch point in the S assimilation pathway catalyzed by adenosine-5´-phosphosulfate kinase (APSK). APSK forms 3´-phosphoadenosine 5´-phosphosulfate (PAPS), an important substrate for the formation of the secondary S-containing metabolites including glucosinolates, and is therefore a significant enzyme in members of Brassicaceae (Leung et al. 2006). Four APSK genes have been cloned from Arabidopsis, for example, APSK1, APSK2, and APSK4, all localized in the 47 plastid, while APSK3 is a cytoplasmic isoform (Mugford et al. 2009). The use of mutants has shown that APSK1 is able to produce sufficient PAPS to maintain normal plant growth (Mugford et al. 2010), while disruption of APSK1 and APSK2 expression reduces the biosynthesis of glucosinolates (Mugford et al. 2009) demonstrating that the expression of APSK genes are strongly linked to the biosynthesis of glucosinolates. However, in non-glucosinolate accumulating species, including for example onion, the secondary (APSK-mediated) pathway must also operate to generate important pools of sulfate esters. In order to elucidate the synthesis and degradation of glucosinolates in plants, (Mugford et al. 2009) observed highly significant alterations in the levels of glucosinolates and their desulfo-precursors in A. thaliana apk1, apk2, apk3, apk4 and wild-type mutants. The levels of each individual glucosinolates were reduced in the leaves of the mutant so that total glucosinolate levels reached only 15% of that in wild-type mutant. The reduction was accompanied by a massive increase in desulfo-precursors, which reached a ten-fold higher concentration than the mature glucosinolates in wild-type leaves. A similar reduction in glucosinolate levels was detected in the seeds of the mutant plants, however, the sulfo-precursors did not accumulate in the seeds. These results confirm that glucosinolates are not synthetized in seeds but are transported in the mature sulfate form (Magrath and Mithen 1993). Final Considerations S is an essential chemical nutrient for plant growth and survival. S- containing defense compounds play significant roles in plant metabolism, stress response, cellular acclimation and adaptation (Fig. 3). They have gained much attention worldwide as biochemical genetics, plant physiology and breeding have been integrated to produce stress-tolerant plants and to identify preferential tolerance mechanisms. The development of transgenic plants with multi-stress tolerance should be the focus of research in the near future because stress factors rarely occur singly. Yet, the use of natural occurring or induced mutants cannot be left. Our knowledge of the effects of transgenic S-containing compounds is still limited, partially because of the complex regulatory mechanisms described 48 previously. Significant advances in metabolic analyses with the “omics” techniques can improve identification of non-target gene products, enabling simultaneous consideration of gene expression, enzyme activity and metabolites. In conclusion, this literature review is expected to improve our understanding of the essential mechanisms involved in oxidative stress in plants and the induction of S-containing compounds. The increased understanding may aid researchers in overcoming problems that occur in contaminated environments. Figure 3. Sulfur assimilation is linked to multiple metabolic pathways responsible for a diverse range of physiological functions. Three major areas include primary metabolite biosynthesis, stress responses and pathogen defense. GSH: glutathione; PCS: phytochelatins; SAM: S- adenosylmethionine (S-AdoMet); SMM: S-methylmethionine; DMSP: dimethylsulfonioproprionate (Modified from Hawkesford 2005). 49 Acknowledgments We would like to thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP – Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – Brazil) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES - Brazil) for continuous financial support over the years for our projects on this and related subjects. References Alcázar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C, Carrasco P, Tiburcio AF (2010) Polyamines: molecules with regulatory functions in plant abiotic stress tolerance. Planta 231:1237-1249 Al-Whaibi MH, Siddiqui MH, Basalah MO (2012) Salicylic acid and calcium induced protection of wheat against salinity. 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