Enrichment of food crops with selenium: controlled production of Se enriched plants to delay fruit ripening and plant senescence, and to increase nutritive value and health benefits.

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U

NIVERSITY OF

P

ISA

Enrichment of food crops with selenium: controlled

production of Se enriched plants to delay fruit ripening

and plant senescence, and to increase nutritive value and

health benefits.

by

Martina Puccinelli

Ph.D. Thesis

Agriculture, Food and Environment

Department of Agriculture, Food and Environment

University of Pisa

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U

N IV E R S IT Y OF

P

IS A

Enrichment of food crops with selenium: controlled

production of Se enriched plants to delay fruit

ripening and plant senescence, and to increase

nutritive value and health benefits.

by

Martina Puccinelli

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy in

Agriculture, Food and Environment

Candidate: Martina Puccinelli

Supervisors

Dr. Beatrice Pezzarossa

Prof. Fernando Malorgio

Accepted by the Ph.D. School

The Coordinator

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Declaration

This thesis is a presentation of my original research work. Wherever contributions of others are involved, every effort is made to indicate this clearly with due reference to the literature and acknowledgement of collaborative research and discussions.

This thesis contains no material that has been submitted previously, in whole or in part, for the award of any other academic degree or diploma.

Signature Date

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Preface

This dissertation is submitted for the degree of Doctor of Philosophy at the University of Pisa. The research described herein was conducted at the Department of Agriculture, Food and Environment, University of Pisa, and at Institute for Studies of Ecosystem of National Research Council, Pisa, between November 2014 and October 2017. The research was conducted under the supervision of Prof. Fernando Malorgio (Department of Agriculture, Food and Environment, University of Pisa) and Dr. Beatrice Pezzarossa (Institute for Studies of Ecosystem of National Research Council, Pisa).

Part of this work has been presented in papers published or under review in peer-reviewed journals as follows:

CHAPTER 1

Puccinelli M, Malorgio F, Pezzarossa B (2017) Selenium Enrichment of Horticultural Crops. Molecules 22:933. doi: 10.3390/molecules22060933

CHAPTER 2

Puccinelli M., Malorgio F., Maggini R., Rosellini I., Pezzarossa B. Biofortification of

Ocimum basilicum L. plants with selenium. III International Symposium on Horticulture in Europe - SHE2016. Chania, Crete (Greece), October 17-21, 2016. under review

CHAPTER 3

Puccinelli M, Malorgio F, Rosellini I, Pezzarossa B (2017). Uptake and partitioning of selenium in basil (Ocimum basilicum L.) plants grown in hydroponics. Scientia Horticulturae 225:271–276. doi: 10.1016/j.scienta.2017.07.014.

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Abstract

The ability of some crops to accumulate selenium (Se) is crucial for human nutrition and health. Selenium has been identified as a cofactor of the enzyme glutathione peroxidase, which is involved in the reduction of peroxides that can damage cells and tissues, and can act as an antioxidant. Plants are the first link in the food chain, which ends with humans. Increasing the Se amount in plant products, without exceeding the toxic threshold, is thus a good way to increase animal and human Se intake, with positive effects on long-term health. In many Se-enriched plants, most Se is in its organic form. Given that this form is more available to humans and more efficient in increasing the selenium status than inorganic forms, the consumption of Se-enriched plants appears to be beneficial. An antioxidant effect of Se has been detected in Se-enriched vegetables and fruit crops. In addition, Se appears to be effective in delay fruit ripening and plant senescence. This thus highlights the possible positive effect of Se in preserving a longer shelf-life and longer-lasting quality.

The main goal of the present thesis was to investigate the effects of selenium on plant metabolism in order to use the potential beneficial effects of Se to improve the quality and the shelf-life of leafy and fruity vegetables.

The mechanism of Se uptake, distribution, accumulation, and the effects of Se concentration on plant growth, quality and post-harvest shelf-life in sweet basil (Ocimum basilicum L.) were investigated.

The first two experiments aimed to study the ability of sweet basil to accumulate selenium, and to evaluate the possibility to obtain Se-enriched basil as a Se supplement resource for humans. Different Se concentrations (0.5, 1, 2 and 4 mg Se L-1) were tested in order to determine the optimal Se concentration in leaves that may induce benefits to human health without phytotoxic effects. Floating system was tested as a possible cultivation system for the production of Se biofortified basil plants. The results showed that the addition of selenium as sodium selenate in the nutrient solution significantly increased the Se content in basil, and could be an efficient system for providing enriched basil plants.

On the basis of the results of the first two experiments, a third trial was conducted in order to understand the processes of Se uptake and accumulation in basil plants. Since the amount of Se accumulated in leaves in the previous experiments was far from the Se toxic threshold for humans, higher Se concentrations (4, 8 and 12 mg Se L-1) were tested. The Se

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concentration in all plant organs was measured during the growth cycle, and the relationship among Se uptake rate, Se concentration in the nutrient solution and plant growth was evaluated. Selenium was absorbed by the roots, translocated to the above-ground organs and accumulated particularly in the leaves, without affecting the plant biomass. Se concentration increased during seedling growth, was highest in the younger leaves and then declined before or upon flowering. The growing trend detected in the total Se was more dependent on the biomass, which increased throughout the experiment, than on the Se concentration, which reached the maximum values during the first part of the experiment and then decreased. The fourth experiment aimed at studying the allocation of selenium absorbed by roots in the different plants organs. The remobilization of Se accumulated in the seeds to the seedlings, and the effects of Se on the quality of sprouts were investigated. Basil plants accumulated Se mostly in leaves and roots. High amounts of Se were also accumulated in seeds and then remobilized to sprouts. The sprouts, produced by the seeds of Se-enriched plants, showed higher germination index and antioxidant capacity compared to seeds of control plants. Given the potential action of Se in ameliorating the oxidative stress and delaying senescence, the effects of Se on basil leaf quality and shelf-life were studied in the fifth experiment. The different climate conditions during the two experiments reported in the fifth chapter may have affected the amount of Se taken up by plants. Lower values of cumulative solar radiation during the second experiment may have determined a lower plants growth and transpiration rate, inducing a lower Se accumulation in basil leaves, compared to the first experiment reported in the same chapter. Se-treated plants showed increased phenolic content and antioxidant activity, thus contrasting the reduction of biomass due to the high Se concentrations added to the nutrient solution. This resulted in an improved quality of basil leaves.

As final issue, the effects of Se in tomato fruit were investigated. Tomato plants were grown in solution enriched with selenium to study the effects of Se treatments on fruit ripening and shelf-life. The results obtained in this experiment confirmed the effect of Se in delaying fruit ripening, and the possibility to use Se-treatments to improve the postharvest shelf-life of fruits.

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Acknowledge

I would like to thank my advisors Dr. Beatrice Pezzarossa and Prof. Fernando Malorgio for their support throughout my PhD work.

I also wish to thank Dr. Rita Maggini, Dr. Giulia Carmassi, Dr. Alice Trivellini, Riccardo Pulizzi and Irene Rossellini for helping me in the research activities.

A thank goes to Prof. Lucia Guidi and Dr. Marco Landi for their feedback and valuable discussion which definitely improved my present work.

I am grateful to Prof. Leon Terry who gave me the opportunity to work in his laboratory at the University of Cranfield – United Kingdom. I would especially like to thank Dr. Roberta Tosetti for teaching and helping me during my stay there. A special thank goes to all the beautiful people that I have met there: Dr Antonio Bermejo Bernal, Dr. Natalia Falagán Sama, Dr. Simone Rossi...

I am also thankeful to Dr. Cecilia Stanghellini who gave me the chance to work at the Greenhouse Horticulture Unit of Wageningen University and Research – The Netherlands. I would especially like to thank Ing. Frank Kempkes, Dr. Esteban Baeza Romero and Ilias Tsafaras teaching and helping me during my stay there.

A special thank goes to Prof. Alberto Pardossi.

Finally, I owe many thanks to Prof. Anna Mensuali and Prof. Antonio Ferrante who kindly accepted to act as referee of this thesis.

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List of tables

Chapter 1: General introduction and thesis outline

Table 1.1 Selenium accumulation in the edible parts of leafy vegetables in relation to the concentration and chemical form of Se supplemented to plants, and to the method of Se supplementation.

Table 1.2 Main effects of Se treatments in leafy vegetables.

Table 1.3 Selenium (Se) accumulation in fruit in relation to the concentration and chemical form of Se supplemented to plants, and to the method of Se supplementation.

Table 1.4 Main effects of selenium (Se) treatments in fruit crops.

Chapter 2: Biofortification of sweet basil plants with selenium

Table 2.1 Chronological events of the cultivation cycle and main climatic conditions in the two experiments.

Table 2.2 Biomass production (g m-2) of basil plants subjected to different Se treatments in the two experiments. Values followed by different letters differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant. Table 2.3 Se concentration measured in roots, stems and leaves of basil plants treated with different Se concentrations in the first and in the second experiment. Values followed by different letters differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 2.4 Carotenoids and total phenols content measured in leaves of basil plants treated with different Se concentrations in the first second experiment. Values followed by different letters differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

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Chapter 3: Selenium uptake and partitioning in basil (Ocimum basilicum

L.) plants grown in hydroponics.

Table 3.1 Climate conditions recorded during all the experiment.

Table 3.2 Biomass production (g DW m-2) in basil plants subject to different Se treatments. Value followed by different letters differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 3.3 Se content (g kg-1 DW) in basil plants subject to different Se treatments. Value followed by different letters differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 3.4 Se content (mg plant-1) in basil plants subjected to different Se treatments during the growth cycle. The statistical analysis was made separately for each sampling during the growth cycle. Values are means with standard errors. Value followed by different letters differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Chapter 4: The use of selenium-enriched basil seeds to produce

biofortified microgreens with high nutritional value.

Table 4.1 Biomass, expressed as g plant-1, of different organs of basil plants subjected to different Se treatments. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 4.2 Se concentration in different organs of basil plants subjected to different Se treatments. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 4.3 Proportion of selenium in different plant parts and Se translocation factor (shoot Se concentration/root Se concentration) in basil plants subjected to different Se treatments. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant. Table 4.4 Biomass production and selenium (Se) concentration of microgreens produced from seeds of basil plants treated with different Se concentrations. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

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Chapter 5: Effects of selenium treatments on quality of basil leaves.

Table 5.1 Climate conditions recorded in the periods before the first, second and third cut of basil plants in the first experiment, and before the first and second cut in the second experiment.

Table 5.2 Leaf biomass production (g DW m-2) in basil plants treated with different Se concentrations. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 5.3 Antioxidant capacity, expressed as µmol Fe(II) mg-1 DW, in leaves of basil plants treated with different Se concentrations measured at harvest in the first experiment, and at harvest and after 5 days of storage in the second experiment, in subsequent cuts. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 5.4 Total phenols content, expressed as mg Gallic acid equivalent (GAE) g-1_{DW, in}

leaves of basil plants treated with different Se concentrations measured at harvest in the first experiment, and at harvest and after 5 days of storage in the second experiment, in subsequent cuts. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 5.5 Rosmarinic acid content, expressed as mg g-1 DW, in leaves of basil plants treated with different Se concentrations measured at harvest in the first experiment, and at harvest and after 5 days of storage in the second experiment, in subsequent cuts. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 5.6 Chlorophylls content, expressed as mg g-1 DW, in leaves of basil plants treated with different Se concentrations measured at harvest in subsequent cuts of the first experiment. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 5.7 Nitrate content, expressed as mg NO3- kg-1 FW, in leaves of basil plants treated

with different Se concentrations measured at harvest in subsequent cuts of the first and second experiments. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 5.8 Transpiration rate (E) (mmol H2O m-2 s-1), stomatal conductance (GS) (mmol H2O

m-2 s-1) and net photosynthetic rate (Pn) (µmol CO2 m-2 s-1) in leaves of basil plants treated

with different Se concentrations, measured at harvest in subsequent cuts of the first experiment. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 5.9 Transpiration rate (E) (mmol H2O m-2 s-1), stomatal conductance (GS) (mmol H2O

m-2 s-1) and net photosynthetic rate (Pn) (µmol CO2 m-2 s-1) in leaves of basil plants treated

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experiment. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant

Table 5.10 Ethylene production (nL g-1 DW h-1) in leaves of basil plants treated with different Se concentration measured at harvest and after 5 days of storage in subsequent cuts in the second experiment. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Chapter 6: Effect of selenium enrichment on metabolism of tomato fruit

during post-harvest ripening.

Table 6.1 Yield and qualitative characteristics of tomato fruit (cv. Red Bunch) of plants subjected to different Se treatments. Data are the means of 3 replicates.

Table 6.2 Se concentration in tomato fruit of plants subjected to different Se treatments. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant. Table 6.3Firmness during post-harvest ripening in tomato fruit of the first), second and third truss in plants treated with different selenium concentrations. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

Table 6.4 Activity of antioxidant enzymes (ascorbato peroxidase (APX), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px)) and lipid peroxidation expressed as malondialdehyde equivalents (MDA eq) content in red fruit of tomato plants subjected to different Se treatments. Values followed by different letters in the same column differ significantly at 5% level by the LSD test. Significance level: *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns = not significant.

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List of figures

Chapter 2: Biofortification of sweet basil plants with selenium.

Figure 2.1 Nitrates (NO3-) concentration measured in leaves of basil plants treated with

different Se concentrations in the first (A) and in the second (B) experiment. The statistical analysis was made separately for each cut. Values are means with standard errors (n=4). Figure 2.2 Rosmarinic acid concentration measured in leaves of basil plants treated with different Se concentrations in the first (A) and in the second (B) experiment. The statistical analysis was made separately for each cut. Values are means with standard errors (n=4).

Chapter 3: Selenium uptake and partitioning in basil (Ocimum basilicum

L.) plants grown in hydroponics.

Figure 3.1 Proportion of selenium in different parts (A) and Se translocation factor (shoot Se concentration / root Se concentration) (B) in basil plants subjected to different Se treatments. Values are means with standard errors (n=4).

Figure 3.2 Se uptake rate (µg Se g root-1 day-1) in basil plants subjected to different Se treatments. Values are means with standard errors (n=4). The statistical analysis was made for each sampling separately.

Chapter 4: The use of selenium-enriched basil seeds to produce

biofortified microgreens with high nutritional value.

Figure 4.1 Germination Index (GI) of basil seeds produced by plants treated with different Se concentrations. The statistical analysis was made separately for each cut. Values are means with standard errors (n=4).

Figure 4.2 Antioxidant capacity, expressed as µmol Fe(II) mg-1_DW,_{of microgreens produced}

from seeds of basil plants treated with different Se concentrations. The statistical analysis was made separately for each cut. Values are means with standard errors (n=4).

Chapter 5: Effects of selenium treatments on quality of basil leaves.

Figure 5.1 Se concentration measured in leaves of basil plants treated with different Se concentrations in subsequent cuts in the first (A) and in the second (B) experiment. The statistical analysis was made separately for each cut. Values are means with standard errors (n=4).

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Chapter 6: Effect of selenium enrichment on metabolism of tomato fruit

during post-harvest ripening.

Figure 6.1 Fruit of tomato plants, treated with different selenium concentrations, during post-harvest ripening at 21 °C.

Figure 6.2 Post-harvest ripening process in fruit of the first (A), second (B) and third (C) truss of plants subjected to different Se treatments, on the basis of visual color appearance. Values are means with standard errors (n=4).

Figure 6.3 Post-harvest weight loss in fruit of the first (A), second (B) and third (C) truss in plants subjected to different Se treatments, expressed as percentage of the initial fresh weight. Values are means with standard errors (n=4).

Figure 6.4 Ethylene production during post-harvest ripening in tomato fruit of the first (A), second (B) and third (C) truss in plants subjected to different Se treatments. Values are means with standard errors (n=4).

Figure 6.3 Respiration rate during post-harvest ripening in tomato fruit of first (A), second (B) and third (C) truss of plants subjected to different Se treatments. Values are means with standard errors (n=4).

Figure 6.6 Non-structural carbohydrates, glucose (A) and fructose (B), concentration in tomato fruit of plants subjected to different Se treatments. Values are means with standard errors (n=4).

Figure 6.7 Pigments, Lycopene (A), b-carotene (B), chlorophyll a (C) and chlorophyll b (D) concentration in tomato fruit of plants subjected to different Se treatments during post-harvest. Values are means with standard errors (n=4).

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List of Appendices

Appendix I: Materials and methods

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Chapter 1: General introduction and thesis outline

The ability of some crops to accumulate selenium (Se) is crucial for human nutrition and health. Selenium has been identified as a cofactor of the enzyme glutathione peroxidase (GSH-Px) (EC 1.11.1.9), which is a catalyzer in the reduction of peroxides that can damage cells and tissues, and can act as an antioxidant. Increasing the Se quantity in plant products, including leafy and fruity vegetables, and fruit crops, without exceeding the toxic threshold, is a good way to increase animal and human Se intake, with positive effects on long-term health. An antioxidant effect of Se has been detected in Se-enriched vegetables and fruit crops due to an improved antioxidative status and to a reduced biosynthesis of ethylene, which is the hormone with a primary role in plant senescence and fruit ripening. This thus highlights the possible positive effect of Se in preserving a longer shelf-life and longer-lasting quality

1.1. Selenium in soil

Selenium (Se) is a metalloid located in Group VI of the Periodic Table, and its biochemistry resembles that of sulfur (S). Se can exist in four oxidation states: -2 (selenide), 0 (elemental Se), +4 (selenite) and +6 (selenate). Almost all the organic Se compounds are isologues of corresponding Sulphur compounds. In alkaline and well-oxidized soils, the predominant form of Se is selenate, whereas selenite is the predominant form of Se in well-drained mineral soils with pH from acid to neutral. Selenite becomes the dominant form under strongly reduced soil conditions (Elrashidi et al. 1987).

1.2. Selenium in animals and humans

Selenium (Se) is an essential component of selenoaminoacids and selenoproteins. It thus has multiple roles in the growth and functioning of living cells and has many crucial biological functions in animals and humans (Birringer et al. 2002; Tapiero et al. 2003). Selenium is also a cofactor of the enzyme glutathione peroxidase, and a catalyzer of the reduction of peroxides, which can damage cells and tissues (Rotruck et al. 1973). It is thus involved in antioxidant defense (Ursini and Bindoli 1987). As a component of iodothyronine deiodonase and thioredoxin reductase (Arthur et al. 1993; Gladyshev et al. 1999) Se is involved in the formation of thyroid hormones. Selenium plays a role in DNA synthesis, fertility, reproduction, and in muscle function by improving endurance and recovery, and slowing the ageing process (Cabaraux et al. 2006; Suttle 2010). Se also helps prevent certain cancers and

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reduces the incidence of viral infections, cardiovascular damage, arthritis, and altered immunological functions (Shamberger 1981; Rayman 2012).

Se is biologically active at low concentrations for normal growth and development, and at moderate concentrations for the homeostatic function. However, at high concentrations, Se can induce toxicity (Hamilton 2004). The margin between the nutritional requirement and toxicity is quite small, and outside this range, deficiency or toxicity can occur.

Sub-optimal Se intake and status are correlated with a wide variety of human diseases, such as heart diseases, cystic fibrosis, cognitive decline, Alzheimer's, cancer, impairment in immune function, oxidative stress-related disorders, reduced fertility and hypothyroidism (Combs 1980; Rayman 2012). A severe Se deficiency may be associated with a cardiomyopathy called Keshan disease, and with an endemic degenerative osteoarthritis known as Kashin-Beck disease (Birringer et al. 2002).

On the other hand, the short-term ingestion of high levels of Se can cause nausea, vomiting and diarrhea. If the excessive consumption is chronic, it may lead to a specific disease called selenosis, and damage the cardiovascular, gastrointestinal, neurological and hematopoietic systems (Yang et al. 1983; Raisbeck 2000; Dhillon and Dhillon 2003).

1.3. Selenium in plants

There is no definitive evidence regarding whether selenium is essential for vascular plants. However, it has been hypothesized that it may have beneficial biological functions in species that are able to accumulate high amounts of Se and that need Se for their normal growth (Hartikainen et al. 2000; Terry et al. 2000; Djanaguiraman et al. 2005). The accumulation of selenium in plants varies in relation to plant species, and is affected by soil Se concentration, soil properties and the chemical form of Se (Bañuelos and Meek 1990).

The leaf Se concentration in most plants, defined as non-accumulators, is usually below 100 mg Se kg-1 dry weight. Only a selected number of plant species grown in areas rich in selenium can accumulate a high amount of selenium in the leaves. These plants are classified as hyperaccumulators, when they accumulate more than 1000 mg kg-1 (Astralagus L. and Stanleya pinnata (Pursh) Britton), and indicators or secondary accumulators when accumulating 100-1000 mg kg-1 (Brassica juncea (L.) Czern., Melilotus (L.) Mill., Atriplex L.) (Ellis and Salt 2003).

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Most agricultural crops have a much lower tolerance (<50 mg Se kg-1), however brassicaceae, onion, garlic and some mushrooms have a high Se concentration due to their high content of sulphur compounds. Selenium has a close similarity in terms of properties to sulphur and it can play the same role as S in biochemical systems. Uptake, translocation and metabolism of Se mimics those of S, thus the substitution of sulphur with selenium results in selenium analogue compounds that increase the selenium content (Brown and Shrift 1982; Terry et al. 2000; White et al. 2004). Legumes, especially lentils, contain a high Se concentration, however nuts rich in proteins, such as pistachios, walnuts and Brazil nuts, have been found to have the richest selenium content. Fruits usually contain a low selenium content, probably due to the low protein and high-water content (Navarro-Alarcon and Cabrera-Vique 2008).

At appropriate concentrations, Se positively affects seed germination and plant growth as well as food processing (Hartikainen et al. 2000; Xue et al. 2001; Hu et al. 2003; Turakainen et al. 2006). Se protects plants from several abiotic stresses, including ultraviolet light, heavy metals and arsenic, and biotic stress, including pathogens and herbivores (Schiavon and Pilon-Smits 2017). Se counteracts oxidative stress by inhibiting lipid peroxidation (Seppänen et al. 2003; Djanaguiraman et al. 2005), and increases GSH-Px activity (Xue et al. 2001; Cartes et al. 2005; Mora et al. 2008). The enhanced antioxidation associated with an increase in glutathione peroxidase activity may delay plant senescence and decrease postharvest losses.

At high concentrations, Se acts as a pro-oxidant, inhibiting the growth and germination of seeds and reducing yields (Hartikainen et al. 2000; Xue et al. 2001; Carvalho et al. 2003). Selenium can affect the quality of vegetables and fruit. An increased cellular content of linoleic acid and sterols and a decreased oleic acid content have been observed in Camelia oleifera plants treated with selenium (Song et al. 2015). Se treatment had a positive effect on maintaining the sensory and the postharvest quality by reducing the respiratory intensity and ethylene production in broccoli (Lv et al. 2017), by decreasing PAL activity and ethylene production in lettuce and chicory (Malorgio et al. 2009), and by diminishing ethylene production in tomato (Pezzarossa et al. 1999, 2014). In green tea, Se increased plant yield, total amino acid, and vitamin C content (Hu et al. 2003). In peach and pear Se spraying of the canopy slowed down the rate of fruit softening, and thus increased the shelf-life (Pezzarossa et al. 2012). The application of selenium may be effective in controlling

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postharvest gray mold disease in tomato fruits caused by Botritis cinereal Pers. (Wu et al. 2016).

Due to its ability to increase the antioxidant defense of plants (Terry et al. 2000; Djanaguiraman et al. 2010; Hasanuzzaman and Fujita 2011), Se has been found to delay plant senescence (Hartikainen et al. 2000; Xue et al. 2001; Malorgio et al. 2009) and fruit ripening (Pezzarossa et al. 2012, 2014, Zhu et al. 2016, 2017b) in several horticultural species, which could lead to decreased postharvest loss. The antioxidant capacity of Se and an improved GSH-Px activity are related (Hartikainen et al. 2000; Xue et al. 2001; Djanaguiraman et al. 2005), which suggests the presence of a Se-dependent GSH-Px (Hartikainen et al. 2000).

1.4. Strategies for increasing selenium uptake by humans

Several strategies can improve a suboptimal selenium status, including a diversified diet, food supplements, fortification of food stuff, and the biofortification of plants (Rayman 2004, 2008, Broadley et al. 2006, 2010; Fairweather-Tait et al. 2011).

A diversified diet can provide a good intake of minerals, proteins and vitamins, however in many socio-economic contexts around the world, access to diverse diets is not possible (Benemariya et al. 1993; Thomson 2004; Williams et al. 2009; Johnson et al. 2010; Fairweather-Tait et al. 2011).

Se supplements include sodium selenate and sodium selenite (inorganic forms), and selenium-enriched yeast, selenomethionine and selenocysteine (organic forms). However, since 2002 in the EU only inorganic forms of selenium are permissible as food supplements. Se-enriched yeast appears to have a high variability with respect to its Se content and speciation, but represents a good way to increase the consumption of Se by humans (Rayman 2004).

The addition of nutrients (minerals, vitamins) to increase the nutritional quality of processed food during manipulation is called “fortification” (Gómez-Galera et al. 2010; Calvo and Whiting 2013).

The ability of some crops to accumulate selenium is crucial for human nutrition and health. Plants are the first link in the food chain which ends with humans. Increasing the Se content in plant products, including leafy and fruity vegetables, fruit crops, cereals, without

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exceeding the toxic threshold, is thus a good way to increase animal and human Se intake and may have positive effects on long-term health.

Increasing the concentration of micronutrient in plants in order to improve the nutritional quality of plant-based food during plant growth rather than during crop processing is known as biofortification (Bañuelos and Lin 2009; Carvalho and Vasconcelos 2013; Hefferon 2015; Díaz-Gómez et al. 2016). The main strategies to increase both mineral levels and their bioavailability in the edible part of staple crops include agronomic intervention and plant breeding. Agronomic biofortification is performed through the application of mineral elements with a good mobility, such as I, Zn, and Se, in the soil and in the plants (White and Broadley 2009; Landini et al. 2011; Carvalho and Vasconcelos 2013). Se biofortification programs have mainly been conducted in north and central Europe. Agronomic Se biofortification at a national scale was successfully adopted in Finland in 1984, where Se was added to all agricultural fertilizers because of the very low consumption of Se by Finnish people. The program was successful in increasing Se concentration in foodstuffs and Se intake in humans (Eurola et al. 1991; Broadley et al. 2006).

Se biofortification of fruits, vegetables and cereals is a good way to increase the supplementation of selenium by humans and has been reviewed elsewhere (Broadley et al. 2006; White and Broadley 2009; Zhu et al. 2010; Malagoli et al. 2015; Schiavon and Pilon-Smits 2017). In many Se-enriched plants most Se is in the major organic form (selenomethionine, selenocysteine and methylselenocysteine). It is more available to humans and more efficient in increasing the selenium content, especially in the blood, than the inorganic forms. The consumption of Se-enriched plants thus appears to be beneficial (Finley 2005; Hartikainen 2005; Broadley et al. 2006; Rayman 2008). For example, in onions, garlic and broccoli, Se is mostly present as Se-methylselenocysteine or γ-glutamyl-Se-methylselenocysteine, with some differences according to the plant species and application doses. In Se-enriched onions the main Se chemical form is γ-glutamyl-Se-methylselenocysteine (about 63% of total Se), followed by selenate (10%), and selenomethionine (5%) (Kotrebai et al. 2000; Hurst et al. 2010). γ-glutamyl-Se-methylselenocysteine is the predominant Se species (73%) which can also be found in enriched garlic. Other chemical species are present at lower concentrations: seleniomethinine (13%), γ-glutamyl-selenomethionine (4%), Se-methylselenocysteine (3%) and selenate (2%) (Ip et al. 2000). In enriched broccoli Se mostly consists of

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Se-17

methylselenocysteine (45%) and small amounts of selenomethionine and selenate (Finley et al. 2001).

The potential of Se fortification of crops by genetic manipulation is still not clear. Some evidence indicates that Se content can be increased in grains by breading, thus providing an alternative to agronomic fortification and minimising the use of Se fertilizers (Eurola et al. 1991; Landini et al. 2011; Hefferon 2015; Malagoli et al. 2015), however further efforts are needed.

The amount and the chemical form of Se found in natural products is well known, and the main foods providing selenium in the diet are nuts, bread, cereals, meat, fish, eggs, and milk/dairy products, as reviewed by Fairweather-Tait et al. (2011).

The Food and Nutrition Board of the Institute of Medicine (USA) has proposed a Recommended Dietary Allowance (RDA) of 55 µg of Se per day-1 for adults and a tolerable upper intake of 400 µg of Se per day-1_{(Krinsky et al. 2000). The accumulation of selenium}

in food crops induced by biofortification must satisfy a rational approach to selenium supplementation according to the recommended RDA, without leading to Se intoxication and without producing phytotoxic effects or reducing the agricultural production.

1.5. Se biofortification of plants

There are four main methods for enriching plants with selenium: i) adding Se to the soil; ii) soaking seeds in a Se solution before sowing; iii) foliar or fruit spraying; iv) hydroponic cultivation with a nutrient solution containing Se.

The addition of Se fertilizers to the soil is an appropriate way to biofortify high amounts of foodstuffs and to increase the Se content in soils that have a low selenium content (Mechora et al. 2011, 2014; Premarathna et al. 2012; Fernandes et al. 2014). However, high amounts of Se need to be applied to the soil to obtain Se plant concentrations equal to other fertilization methods (Broadley et al. 2010; Chilimba et al. 2012). The soil selenium content can be increased by fertilizing soil with salt (selenate or selenite) or by the incorporation of Se-hyperaccumulator plants in the soil. Good results in the biofortification of carrots and broccoli have been obtained by adding Se-enriched Stanleya pinnata plants to soil (Bañuelos et al. 2015). As Se-hyperaccumulator, Stanleya pinnata can be used for the phytoremediation of soil with a high concentration of Se (Freeman and Bañuelos 2011), and the Se-enriched

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plant material can be then incorporated into the soil thus combining phytoremediation and biofortification (Bañuelos et al. 2010).

Selenium spraying has been used to enhance the Se content in potato (Poggi et al. 2000), rice (Chen et al. 2002), soybean (Yang et al. 2003), buckwheat and pumpkin (Stibilj et al. 2004), garlic (Smrkolj et al. 2005) carrot (Kápolna et al. 2009), broccoli (Šindelářová et al. 2015), cabbage (Mechora et al. 2014), radish (Schiavon et al. 2016), basil (Hawrylak-Nowak 2008; Kopsell et al. 2009; Barátová et al. 2015; Mezeyová et al. 2016), tomato (Schiavon et al. 2013; Zhu et al. 2016), peach and pear (Pezzarossa et al. 2012), and grapes (Zhu et al. 2017a). Results of spraying depend on the characteristics of the leaf and fruit surface, such as the presence of hairs, the characteristics of the epicarp, the chemical composition of the epicuticular wax or the deposition of wax platelets (Pezzarossa et al. 2012). Foliar application of Se has proved effective for the biofortification of Chicorium intybus L. plants: the Se concentrations in sprayed plants (Germ et al. 2007) were found to be higher compared with plants grown in a nutrient solution containing Se (Diaz et al. 2007; Malorgio et al. 2009). In addition, foliar spraying is preferable to soil application due to the lower amount of Se generally used, and because no residual effects have been observed. Foliar application involves a minimum consumption of Se salts and is an effective, safe and economically acceptable way of improving Se content in crops (MacLeod et al. 1998).

The results of biofortifying plants by soaking seeds in a solution containing Se are still not well known, however good results have been obtained in grains. Ožbolt et al. (2008)(Ožbolt et al. 2008) found an increased Se content in buckwheat plants without any decrease in production, and an improvement in drought tolerance was found by Nawaz et al. (2013) in wheat. However, Se concentrations detected in these biofortified plants were lower compared to the other methods.

Hydroponic culture enriched with selenium is useful for providing Se-enriched vegetables. Studies have been conducted on lettuce (Xue et al. 2001; Carvalho et al. 2003; Malorgio et al. 2009; Ramos et al. 2010; Smoleń et al. 2014), sweet basil (Hawrylak-Nowak 2008), chicory (Malorgio et al. 2009), spinach (Ferrarese et al. 2012), chard (Hernández-Castro et al. 2015) and tomato (Carvalho et al. 2003; Pezzarossa et al. 2014). The use of a floating system makes it possible to control the concentration of Se in the growth medium and to easily adapt the Se supply to the growth stage of plants, thus avoiding salt loss (Malorgio et al. 2009).

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In general, the supplementation of Se by foliar application or in the growth medium increases Se concentration in plant tissues without a loss of production or qualitative characteristics.

1.6. Selenium enrichment of leafy vegetable: effects on yield, quality and

senescence

Se biofortification of leafy vegetables has been widely studied. Plants have been grown on selenium enriched substrates (Pezzarossa et al. 2007; Businelli et al. 2015), in a nutrient solution with selenium added (Zhu et al. 2004; Diaz et al. 2007; Ríos et al. 2008, 2009, 2013; Malorgio et al. 2009; Blasco et al. 2010; Saffaryazdi et al. 2012; Ferrarese et al. 2012; Hawrylak-Nowak 2013; Hernández-Castro et al. 2015) or treated with Se foliar application (Germ et al. 2007; Hawrylak-Nowak 2008; Kopsell et al. 2009; Oraghi Ardebili et al. 2015; Barátová et al. 2015; Mezeyová et al. 2016). Table 1.1 summarizes the various studies on the Se enrichment of leafy vegetables.

The addition of selenium has been found to significantly increase the Se content in plants, in general without negatively affecting the biomass and the quality of leaves, as in spinach (Zhu et al. 2004; Germ et al. 2007; Saffaryazdi et al. 2012; Ferrarese et al. 2012; Hawrylak-Nowak 2013), lettuce (Pezzarossa et al. 2007; Ramos et al. 2010), basil (Hawrylak-Hawrylak-Nowak 2008; Mezeyová et al. 2016), and chicory (Malorgio et al. 2009). The addition of low doses of Se, approximately 1.5 mg Se L-1 in the nutrient solution (Zhu et al. 2004; Pezzarossa et al. 2007; Malorgio et al. 2009; Ramos et al. 2010; Saffaryazdi et al. 2012; Ferrarese et al. 2012; Hawrylak-Nowak 2013), or 1 mg Se L-1 by foliar fertilization (Germ et al. 2007) were not found to induce toxic effects in chicory plants. Similarly, foliar fertilization with 50 mg Se L-1 (Hawrylak-Nowak 2008) or 25 mg Se m-2 (Mezeyová et al. 2016) did not negatively affect the biomass of sweet basil plants. Se could also have positive effects on plant growth, increasing yield in basil (Hawrylak-Nowak 2008; Oraghi Ardebili et al. 2015), cichory and lettuce (Diaz et al. 2007; Malorgio et al. 2009; Ramos et al. 2010; Hawrylak-Nowak 2013). Ríos et al. (2008) observed a positive effect of Se on plant growth at application rates of 5-20 µmol Se L-1, no effects at 40 µmol Se L-1 and a reduced production of biomass at an application rate of 120 µmol Se L-1 (Ríos et al. 2008, 2009, 2010, 2013).

The chemical form of Se applied influences the toxicity in plants. In general, selenate is less toxic than selenite, and biomass production is higher when selenate is supplied (Ríos et al. 2008, 2009, 2010, 2013; Ramos et al. 2010; Saffaryazdi et al. 2012). In addition, selenite is toxic for plants at lower concentrations compared to selenate (Ramos et al. 2010).

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An antioxidant effect of Se has been detected in Se-enriched leafy vegetables due to the increase in the antioxidant enzyme activity: lypoxigenase (LOX) (Ríos et al. 2008), superoxide dismutase (SOD) (EC 1.15.1.1) (Ríos et al. 2009; Ramos et al. 2010), catalase (CAT) (EC 1.11.1.7) (Ramos et al. 2010), ascorbate peroxidase (APX) (EC 1.11.1.11), and glutathione peroxidase (GSH-Px) (EC 1.11.1.9) (Ríos et al. 2009). An improved oxidative state was also detected by measuring the content of some oxidative markers and compounds, such as MAD, DPPH, H2O2, phenols and flavonoids, and by measuring the antioxidant

activity by the FRAP test. A reduction in DPPH, H2O2 (Hawrylak-Nowak 2013), MDA

content (Hawrylak-Nowak 2008), an increase in antioxidant capacity (measured by the FRAP test), phenols (Hawrylak-Nowak 2008; Ríos et al. 2008; Saffaryazdi et al. 2012; Oraghi Ardebili et al. 2015),and flavonoid content were detected in plants treated with low concentrations of selenate (Ríos et al. 2008).

The reduced glutathione content is also an indicator of the oxidative status of plant tissues, and Se increases the content of this compound (Oraghi Ardebili et al. 2015). However, sometimes Se does not induce changes in phenolic compounds content (Barátová et al. 2015; Mezeyová et al. 2016), but it may have a positive effect on the anthocyanin content (Hawrylak-Nowak 2008). Due to its antioxidant action, Se may have a positive effect at low concentrations thus increasing plant growth. In contrast, at high concentrations Se, especially as selenite, can act as a pro-oxidant inducing oxidative stress in leafy vegetables, by increasing H2O2 (Ríos et al. 2009), MDA (Ríos et al. 2008; Hawrylak-Nowak 2013) content and lipid peroxidation (Ríos et al. 2010).

The effect of Se on photosyntethic pigments is not clear. In general, low concentrations of Se have not been found to affect the photosynthetic pigment content in basil (treated with 50 mg Se L-1 or lower) (Hawrylak-Nowak 2008) , lettuce (treated with 0.316 mg Se L-1 selenite, 2.37 mg Se L-1 selenate or lower) (Ríos et al. 2010; Hawrylak-Nowak 2013), and chicory (1 mg Se L-1 or lower) (Malorgio et al. 2009). On the other hand, higher concentrations of Se were found to decrease the content of photosynthetic pigments both in lettuce (treated with 0.474 mg Se L-1_{selenite, 3.16 mg Se L}-1_{selenate or higher) (Hawrylak-Nowak 2013) and}

basil (30 mg Se L-1_{or higher) (Oraghi Ardebili et al. 2015).}

An increase in carotenoids in basil (treated with concentrations from 30 to 120 mg Se L-1) (Oraghi Ardebili et al. 2015), and of chlorophylls in spinach (treated with 1 mg Se L-1₎

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The effect of Se on nitrogen metabolism is not clear. Ríos et al. (2010) found a reduced nitrate content in Se-treated lettuce and basil plants, respectively, whereas no effects were detected in chicory (Malorgio et al. 2009), spinach (Ferrarese et al. 2012), and chard (Hernández-Castro et al. 2015).

A longer shelf life and preserved quality, in association with lower rates of ethylene biosynthesis have been observed in lettuce and chicory (Diaz et al. 2007; Malorgio et al. 2009). In addition, in senescing lettuce plants, Se has been found to increase stress tolerance by preventing the decrease in tocopherol concentration and by increasing the activity of superoxide dismutase (SOD) (EC 1.15.1.1) (Xue et al. 2001).

Table 1.2 summarizes the main physiological effects induced by selenium in leafy vegetables.

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22 Re fe re n ce D iaz et a l. 2 0 0 7 Pezzaro ss a et al . 2 0 0 7 Pezzaro ss a et al . 2 0 0 7 Mal o rg io et al . 2 0 0 9 Rí o s et al . 2 0 0 8 , 2 0 0 9 , 2 0 1 0 , 2 0 1 3 ; Ma lo rg io et al . 2 0 0 9 ; Ramo s et a l. 2 0 1 0 ; H aw ry la k -N o w ak 2 0 1 3 H aw ry la k -N o w ak 2 0 1 3 Bu si n el li e t al . 2 0 1 5 D iaz et a l. 2 0 0 7 G erm et al . 2 0 0 7 Mal o rg io et al . 2 0 0 9 H aw ry la k -N o w ak 2 0 0 8 ; K o p sel l et al . 2 0 0 9 ; Barát o v á et a l. 2 0 1 5 Mezey o v á et al . 2 0 1 6 K o p se ll et a l. 2 0 0 9 Z h u et al . 2 0 0 4 Ferra res e et al . 2 0 1 2 Saffary azd i et a l. 2 0 1 2 H ern án d ez -Ca st ro et al . 2 0 1 5 [S e] in e d ib le p ar t 26 mg kg -1 DW 20 mg kg -1 DW 170 mg kg -1 DW 26 mg kg -1 DW 10 to 43 .3 mg kg -1 DW 30. 6 mg kg -1 DW 219 mg kg -1 DW 30 mg kg -1 DW 45 mg kg -1 DW 29. 1 mg kg -1 DW 7. 86 to 150 mg kg -1 DW 7. 86 to 150 mg kg -1 D W 41. 5 mg kg -1 DW 12 mg kg -1 FW 15. 5 mg kg -1 DW 3. 89 mg g -1 DW 1393 µ g o f S e pe r shoot S u p p lem en ta tion m et h od s E nr ichme nt o f nut rient solut ion S oil f er ti li za ti on S oil f er ti li za ti on E nr ichme nt o f nut rient solut ion E nr ichme nt o f nut rient solut ion E nr ichme nt o f nut rient solut ion P ea t fe rti li za ti on E nr ichme nt o f nut rient solut ion F oli ar f er ti li za ti on E nr ichme nt o f nut rient solut ion F oli ar f er ti li za ti on F oli ar f er ti li za ti on F oli ar a ppli ca ti on E nr ichme nt o f nut rient solut ion E nr ichme nt o f nut rient solut ion E nr ic hment o f nut rient solut ion E nr ichme nt o f nut rient solut ion S e com p ou n d se lena te se lenite se lena te se lena te se lena te se lenite se lena te se lena te se lena te se lena te se lena te se lenite se lena te se lena te se lena te se lena te [S e] s u p p le m en te d 0. 5 and 1 mg S e L -1 1. 5 and 5 mg S e kg -1 5 mg S e kg -1 0. 5 and 1 mg S e L -1 0. 16 to 5. 12 mg S e L -1 fr om 0 .16 to 2. 4 mg S e L -1 1 to 1000 mg S e kg -1 0. 5 and 1 mg S e L -1 1 mg S e L -1 0. 5 and 1 mg S e L -1 1 to 50 mg S e L -1 25 and 50 mg m -2 2 to 32 mg S e L -1 0. 8 and 1 .6 mg S e L -1 0. 2 to 0. 4 mg S e L -1 1 to 10 mg S e L -1 10 and 20 mg S e L -1 P lan t sp ec ies L ac tuca sati va L . va r. Ac epha la L ac tuca sati va L . va r. Ac epha la L ac tuga sati va L. L ac tuca sati va L . va r. Ac epha la L ac tuca sati va L. L ac tuca sati va L . cv. J us tyna L ac tuca sati va L. C hic or ium int ybus L. C hicor ium int ybus L. C hicor ium int ybus L. Oc imum bas il icum L. Oc imum bas il icum L. Oc imum bas il icum L. Spinacia oler ac ea L. Spinacia oler ac ea L. Spinacia oler ac ea L. B eta vulgar is s ubs p. V ulgar is L. T a b le 1 .1 S el en iu m a cc u m u la ti o n i n t h e ed ib le p a rt s o f le a fy v eg et a b le s in r el a ti o n t o t h e co n ce n tr a ti o n a n d c h em ic a l fo rm o f S e su p p le m en te d t o p la n ts , a n d t o t h e m et h o d o f S e su p p le m en ta ti o n .

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23 Re fe re n ce Barát o v á et al . 2 0 1 5 Bu si n el li e t al . 2 0 1 5 D iaz et a l. 2 0 0 7 D iaz et a l. 2 0 0 7 Ferra res e et al . 2 0 1 2 H aw ry la k -N o w ak 2 0 0 8 H aw ry la k -N o w ak 2 0 1 3 H ern án d ez -Ca st ro et al . 2 0 1 5 Mal o rg io et al . 2 0 0 9 Mal o rg io et al . 2 0 0 9 Mezey o v á et al . 2 0 1 6 O rag h i A rd eb il i et al . 2 0 1 5 Pezzaro ss a et al . 2 0 0 7 Ramo s et a l. 2 0 1 0 Rí o s et al . 2 0 0 8 Rí o s et al . 2 0 0 9 Rí o s et al . 2 0 1 0 Saffary azd i et al . 2 0 1 2 Z h u et al . 2 0 0 4 E th ylene p rod u ct ion dec re a se d ec re a se d ec re a se d ec re a se Nit rat e con te n t

none none none de

cr ea se P h ot os yn th et ic p igm en ts none d ec re a se a t h ig h [ S e] none dec re a se a t h ig h [ S e] in cr ea se Ant ioxi d an t com p ou n d s none in cr ea se none in cr ea se im cr e as e in cr ea se Ant ioxi d an t en zym es in cr ea se in cr ea se in cr ea se Oxi d at ive m ar k er s im p ro v e im p ro v e B iom as s none d ec re a se a t h ig h [ S e]

none none none none none n.d

.

none none none n.d

. none none d ec re a se a t h ig h [ S e] d ec re a se a t h ig h [ S e] d ec re a se a t h ig h [ S e] none none P lan t sp ec ies O ci m u m b a si li cu m L. L a ct u ca s a ti va L. L a ct u ca s a ti va L . v ar . A ce p h al a C h ic o ri u m i n ty b u s L. S p in a ci a o le ra ce a L. O ci m u m b a si li cu m L. L a ct u ca s a ti va L . cv . Ju st y n a B et a v u lg a ri s su b sp . V u lg a ri s L. C h ic o ri u m i n ty b us L. L a ct u ca s a ti va L . v ar . A ce p h al a O ci m u m b a si li cu m L. O ci m u m b a si li cu m L. L a ct u ca s a ti va L . v ar . A ce p h al a L a ct u ca s a ti va L . cv . V er a L a ct u ca s a ti va L . cv . P h il ip u s L a ct u ca s a ti va L . cv . P h il ip u s L a ct u ca s a ti va L . cv . P h il ip u s S p in a ci a o le ra ce a L. S p in a ci a o le ra ce a L. T a b le 1 .2 M a in e ff ec ts o f S e tr ea tm en ts i n l ea fy v eg et a b le s.

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1.7. Selenium enrichment of fruit crops: effects on yield, quality and

senescence

Due to the antioxidant capacity of Se and its influence on ethylene biosynthesis, several experiments have been conducted to study the effects of Se biofortification on fruit quality and post harvest. In tomato (Solanum lycopersicon L.) selenium, as sodium selenate, has been added to nutrient solutions (Lee et al. 2007; Schiavon et al. 2013; Pezzarossa et al. 2013, 2014; Businelli et al. 2015; Wu et al. 2016; Zhu et al. 2017b) or given to plants by foliar application. Only a few studies have been conducted on the Se-enrichment of fruit trees. Peach (Prunus persica Batch) and pear (Pyrus communis L.) (Pezzarossa et al. 2012) were sprayed with selenate solution, table grape (Vitis vinifera L.) (Zhu et al. 2017a) was sprayed with organic selenium, and pear (Pyrus bretschneideri cv. Huangguan), grape (Vitis vinifera L., cv. Kyoho), and peach (Prunus persica Batch, cv. Jinliuzaohong) were sprayed with amino acid-chelated selenium solution.

The addition of Se has been found to increase the Se concentration in the fruits and leaves of the treated plants of both tomato (Lee et al. 2007; Schiavon et al. 2013; Pezzarossa et al. 2013, 2014; Businelli et al. 2015; Wu et al. 2016; Zhu et al. 2017b) and fruit trees (Pezzarossa et al. 2012; Zhu et al. 2017a), without affecting the yield. Table 1.3 summarizes the various studies on the Se enrichment of fruit crops.

Evidence of the positive effects of Se on the quality parameters of tomato fruit has been reported, such as soluble solid content (Lee et al. 2007; Zhu et al. 2016, 2017b), titrable acidity (Lee et al. 2007; Zhu et al. 2016), glucose, fructose, and total sugar content (Lee et al. 2007), and firmness (Zhu et al. 2016). Se also positively affects the soluble solid content in peach and pear (Pezzarossa et al. 2012), glucose, fructose, organic acid and protein contents in grape (Zhu et al. 2017a). In addition, during storage, Se delayed the decline of firmness (Pezzarossa et al. 2012; Zhu et al. 2016) and titrable acidity, as well as weight loss in tomato fruits (Zhu et al. 2016). In tomato fruits, Se may also induce an increased content of pigments and of antioxidant compounds (Schiavon et al. 2013; Pezzarossa et al. 2013, 2014; Zhu et al. 2017b).

An increased net photosynthetic rate and a decreased stomatal conductance and transpiration rate were detected in pear, grape, and peach foliar sprayed with amino acid-chelated selenium (Feng et al. 2015).

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Treatments with Se showed a positive effect in improving the oxidative status of fruit and in delaying tomato fruit ripening, thus positively affecting the post-harvest shelf-life of fruit without negatively affecting the quality (Pezzarossa et al. 2013; Zhu et al. 2016, 2017b). Se may increase the antioxidant enzyme activity (Businelli et al. 2015; Zhu et al. 2017b) and decrease various reactive oxygen species during storage (Zhu et al. 2017b). Experiments conducted by Pezzarossa et al (2014) found a delay in the onset of fruit ripening in Se-enriched tomato, which also showed a reduced rate in colour change, and an earlier harvesting of control plants compared to Se-treated plants. The delay in fruit ripening may depend on a reduced ethylene production. This reduction could be due to the higher cellular concentration of Se-Met than Met, which is a precursor of ethylene in the ethylene biosynthesis pathway (Brown and Shrift 1982; Yang et al. 1984). Another hypothesis regards the genes of the enzymes involved in the ethylene biosynthesis pathway. In this pathway, the conversion of S-adenosyl methionine to 1-Aminocyclopropane-1-carboxylic acid (ACC) is catalysed by the enzyme ACC synthase (ACS). The ACC is converted into ethylene by ACC oxidase (ACO) (Yang et al. 1984). Four genes belong to the ACO gene family: ACO1, predominantly expressed in tomato fruit (Barry et al. 2000), ACS2 and ACS4, which during ripening primarily govern the ethylene production of system 2 (Nakatsuka et al. 1998). Se may have an indirect positive effect on the post-harvest storage of vegetables by reducing germination and the mycelial growth of some harmful fungi such as Botrytis cinerea Pers. (Wu et al. 2016; Zhu et al. 2016) and Pennicillium expansum Link (Wu et al. 2014). Botrytis cinerea Pers. causes gray mold decay, one of the main pre- and post-harvest diseases in fruit and vegetables (Quinn et al. 2010; Youssef and Roberto 2014), leading to economic losses (Soylu et al. 2010; Cabot et al. 2013). Se also counteracts infections by Alternari brassicola (Schwein.) Wiltshire and Fusarium sp. Link and enhances resistance to fungal diseases (Hanson et al. 2003), for example reducing the damage due to Fusarium wilt infection in tomato (Companioni et al. 2012). In addition, high Se concentrations in plant tissues may have a positive effect by reducing invertebrate herbivory damage (Hanson et al. 2003). Table 1.4 summarizes the main physiological effects induced by selenium in fruit crops.

In conclusion, biofortified food with selenium can address Se deficiencies in humans, thus increasing the amount of selenium in the diet and preventing the risks of excess Se intake which mineral supplements can induce. In horticultural crops, selenium has potential benefits in terms of storage and shelf-life. In fruit, Se may modulate the ripening process,

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probably through its antioxidant and anti-senescence properties with beneficial effects in terms of post-harvest commercial life, and greater benefits for human health. Further studies are needed in order to fully understand the molecular and biochemical mechanisms directly or indirectly affected by selenium in fruit tissues at ripening and during the postharvest phase.

Enrichment of food crops with selenium: controlled production of Se enriched plants to delay fruit ripening and plant senescence, and to increase nutritive value and health benefits.

U

P

Enrichment of food crops with selenium: controlled

production of Se enriched plants to delay fruit ripening

and plant senescence, and to increase nutritive value and

health benefits.

by

Martina Puccinelli

Ph.D. Thesis

Agriculture, Food and Environment

Department of Agriculture, Food and Environment

University of Pisa

U

P

Enrichment of food crops with selenium: controlled

production of Se enriched plants to delay fruit

ripening and plant senescence, and to increase

nutritive value and health benefits.

by

Martina Puccinelli

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy in

Agriculture, Food and Environment

Candidate: Martina Puccinelli

Supervisors

Dr. Beatrice Pezzarossa

Prof. Fernando Malorgio

Accepted by the Ph.D. School

The Coordinator

Declaration

Abstract

Acknowledge

Table of Contents

List of tables

Chapter 1: General introduction and thesis outline

Chapter 2: Biofortification of sweet basil plants with selenium

Chapter 3: Selenium uptake and partitioning in basil (Ocimum basilicum

L.) plants grown in hydroponics.

Chapter 4: The use of selenium-enriched basil seeds to produce

biofortified microgreens with high nutritional value.

Chapter 5: Effects of selenium treatments on quality of basil leaves.

Chapter 6: Effect of selenium enrichment on metabolism of tomato fruit

during post-harvest ripening.

List of figures

Chapter 2: Biofortification of sweet basil plants with selenium.

Chapter 3: Selenium uptake and partitioning in basil (Ocimum basilicum

L.) plants grown in hydroponics.

Chapter 4: The use of selenium-enriched basil seeds to produce

biofortified microgreens with high nutritional value.

Chapter 5: Effects of selenium treatments on quality of basil leaves.

Chapter 6: Effect of selenium enrichment on metabolism of tomato fruit

during post-harvest ripening.

List of Appendices

Chapter 1: General introduction and thesis outline

1.1. Selenium in soil

1.2. Selenium in animals and humans

1.3. Selenium in plants

1.4. Strategies for increasing selenium uptake by humans

1.5. Se biofortification of plants

1.6. Selenium enrichment of leafy vegetable: effects on yield, quality and

senescence

1.7. Selenium enrichment of fruit crops: effects on yield, quality and

senescence