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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Report activities and pubblications

Title: 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.

Candidate: PUCCINELLI Martina

Tutor(s): Dr. PEZZAROSSA Beatrice, Prof. MALORGIO Fernando

Institution(s): Department of Agricultural Food and Environment; CNR, Institute for Ecosystem Studies, Pisa.

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COOPERATION

• CNR, Istituto per lo Studio degli Ecosistemi, Pisa. • Scuola Superiore Sant’Anna, Pisa.

• DAFE, Department of Agricultural, Food and Environment, University of Pisa. • Chemistry section of DAFE of Pisa.

SUPPORTING PROJECTS

Biological and productive behaviour of horticultural species (research line DAFE).

ANNUAL REPORT: 1° YEAR [2014-2015] Research activity

During the first months of the year I carried out a bibliographic research focused on the topics of the project. In the second part of the year two experiments on basil plants and one experiment on tomato plants were conducted in a greenhouse at the Department of Agriculture, Food and Environment. The experiments had the objective to investigate the effects of Se on plant growth and metabolism, in particular on oxidative status and fruit ripening and maturation.

1st_experiment

Plants of Ocimum basilicum L., cv Tigullio, were grown in hydroponics using a floating system which allows an appropriate discharge of the waste nutrient solution and a monitoring of the Se plant uptake. 16 plants for each replicate and four replicas for each treatment were used.

The plants were sown on October 20th_{and transplanted on November 17}th_{to cell plug-trays measuring}

0.32×0.54 m. Polystyrene plug-trays were placed in floating tanks of 60 L capacity, containing the nutrient solution. The oxygenation of the nutrient solution was obtained by insufflating air with air pumps in the tanks.

The experiments were conducted in a heated greenhouse (minimum night air temperature at 7–8 °C) and supplementary lighting was provided by high pressure sodium lamps (HPS, SON-T 400 W, Philips) at a constant daylength of 9 hours.

A week after transplanting selenium (Se), as sodium selenate (Na2SeO4), was added to the nutrient

solution at rate of 0 (control), 0.5, 1 and 2 mg Se L-1. The experiment ended on December 30th. Plants were then harvested and the fresh weight (FW) of leaf material was determined. At harvest ethylene production in fresh leaves was measured by gas chromatography. Chlorophyll and carotenoid content were measured by a spectrophotometric method using fresh material.

Then, part of the samples was oven-dried oven dried at 50 ◦C up to constant weight, and the dry weight (DW) was recorded; the remaining part was frozen with liquid nitrogen and stored at -80°C.

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3 The biomass production was calculated and expressed as g DW/plant

The oven-dried ground leaf material was used for the analysis of Se, nitrate and other mineral elements. Se content was measured by hydride generation atomic absorption spectrophotometry after sample digestion with nitric and perchloric acid, and reduction by hydrochloric acid. The contents of calcium (Ca), zinc (Zn), potassium (K), magnesium (Mg), manganese (Mn) and iron (Fe) were measured by absorption spectrophotometry after sample digestion with nitric and perchloric acid.

The frozen leaf material was used for the analysis of rosmarinic acid and total phenols content. The rosmarinic acid content was measured by HPLC and the total phenol content by a spectrophotometric method. The frozen material was analyzed also for ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity, as well as for the content of reduced and total glutathione (GSH), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and malondialdehyde (MAD). I carried out these analyses in the chemistry section of DAFE to evaluate the oxidative status of plants in relation to the leaf Se content.

The results obtained in the experiment were elaborated and statistically analyzed.

- 2° experiment

The second experiment was carried out on plants of Ocimum basilicum L., cv Tigullio grown under the same conditions of the experimental plan described above. Due to the low content of Se detected in the first experiment and on the little effect observed on plant metabolism, in this experiment a higher dose of Se was used. The treatments were: control (no Se added), 1 mg Se L-1, 2 mg Se L-1 and 4 mg Se L-1. The plants were sown on January 12th and transplanted on February 16th. A week after transplants Se was added as sodium selenate to the nutrient solution. The experiment ended on March 19th and the analyses on fresh, oven-dried and frozen samples were the same performed in the first experiment. The methodology is described above.

The ethylene production was measured at harvest, after 2 and after 6 days of storage at 8°C, by gas chromatography.

- 3° experiment

This experiment was performed with the aim to investigate the effect of Se on fruit ripening and plant senescence.

Plants of Microtom, a dwarf cultivar of tomato (Solanum Lycopersicum L.), were grown under greenhouse. Microtom cultivar was chosen because of the shorter growing season and the small plant size that makes easier the management of the plants if compared to other tomato cultivar.

The plants were sown on March 2nd, transplanted on March 31st in pots and grown in hydroponics. The substrate used for cultivation was made of 70% peat and 30% perlite. The plants were irrigated with a nutrient solution.

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4 A week after transplant Se, as sodium selenate, was added to the nutrient solution at rate of 0, 0.5 mg Se L-1, 1 mg Se L-1. Treatment with Se lasted for 20 days; after this period the irrigation with nutrient solution without Se has been restarted.

The harvest began on June 3rd. Two different samplings were performed. In the first sampling, 8 plants for each treatment were harvested when 50% of fruit overall reached the ripe stage. Leaves, root, shoot and fruit were separated, fruits were separated in red, breakers and green, according to the maturation stage. Part of these samples were oven dried at 50 °C up to constant weight and chopped and the dry weight (DW) was recorded; the remaining part was frozen with liquid nitrogen and stored at -80 °C. The dried sample were used for analysis of

For the second sampling we chose 18 plants for each treatment. Fruit were harvested gradually when they reached the ripe stage and we monitored their post-harvest maturation. The fruit harvested at ripe stage were placed on a sheet of paper and photographed approximately every four days. Among these fruit, three were chosen from each group and the ethylene production of these fruit were measured approximately every three days.

In vivo ripening and maturation of fruit of four plants for each treatment was monitored taking pictures of each plant approximately every three days.

Other activities

Courses completed

- Academic English, Adrian Wallwork. Course for Phd students organized by DAFE, University of Pisa.

- Statistic. Course for Phd students organized by DAFE, University of Pisa.

Seminars

- November 7th_{2014. “Elsevier Publishing Connect workshop”, Elaine van Ommen Kloeke – Pisa,}

Italy.

- December 1st_{2014. Workshop "Dal sole alla tavola: la figura di Gian Franco Soldatini dallo studio}

della fotosintesi sino alla qualità del cibo".

- December 5th_{2014. “LABOR: i laureati in WBQ e WBV incontro le aziende: quali i bisogni, le}

opportunità, le richieste del mondo del lavoro?” – Pisa, Italy.

- December 11th_{2014. "L’agrofarmaceutico, una evoluzione del terzo millennio. Una visione}

imprenditoriale", Cav. Lav. Valentino Mercati – Pisa, Italy.

- February 2015 6th_{. Il cibo della salute. Nutraceutica e alimenti funzionali Convegno del ciclo "Pisa}

verso EXPO 2015. Il cibo tra scienza e conoscenza". Nutrafood center – Pisa, Italy. PhD Program On Agriculture, Food and Environment, University of Pisa.

- February 2015 25th_{. Corso su CAB Abstracts e AGRIS su piattaforma OvidSP. Maurice Clementi}

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5 - March 2015 13th_{. Scientific thinking and method. G. Zanni, G. Benelli, M. Landi. PhD Program}

On Agriculture, Food and Environment, University of Pisa.

- May 2015 8th_{. "I mestieri del laureato in Biosicurezza e Qualità degli Alimenti e di quello in}

Biotecnologie Vegetali e Microbiche”.

Short stay abroad

Cutting of tomato samples with cryomicrotome at the Department of Biology, Biotechnical faculty, University of Ljubljana,Jamnikarjeva 101, SI-1000 Ljubljana. From 26th_{July 2015 to 30}th_{July 2015.}

References

• Hawrylak-Nowak B. 2008. Enhanced selenium content in sweet basil (Ocimum basilicum L.) by foliar fertilization. Vegetable crops research bulletin 69: 63-72.

• Pezzarossa B., Rosellini I., Borghesi E., Tonutti P. and Malorgio F. 2014. Effects of Se-enrichment on yield. Fruit composition and ripening of tomato (Solanum lycopersicum) plants grown in idroponics. Scientia Horticuiturae 165: 106-110.

• Rios J J, Blasco B, Rosales M A, Sanchez-Rodriguez E, Leyva R, Crevilla L M, Romero L and Ruiz J M. 2010. Response of nitrogen metabolism in lettuce plants subjected to different doses and forms of selenium J Sci Food Agric 2010; 90: 1914–1919

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ANNUAL REPORT: 2° YEAR [2015-2016]

Research activity

During the first months, I carried out the analyses at TwinMic Beamline of Elettra-Sincrotrone Trieste S.C.p.A. on the plant samples prepared in the first year. After that, five experiments on basil plants and one experiment on tomato plants were conducted with the aim to investigate the effects of Se on plant growth and metabolism, in particular on plant senescence, fruit ripening and maturation.

- Analyses at TwinMic Beamline

The microscope was operated in scanning transmission mode (STXM) with at 1.6keV, in order to acquire Se L edge fluorescence signal. Simultaneously the signal of others mineral elements have been also collected. The X-Ray Fluorescence map (75x75 pixel) and the X-ray absorption and phase contrast images of the same area with 270 nm diameter probe size were performed on 4 different samples from selenium-enriched tomato plants and on 2 samples from the control tomato plants. The very high resolution of the STXM images and the good phase contrast allowed us to clearly reveal the internal structures of the plant tissue and to discriminate the presence of ticker or denser areas. The selenium signal was present and it was possible to estimate the distribution, but the data obtained are not statistically reliable because the aluminum signal interfered with selenium.

- 1st_experiment

In order to study the potential positive effect of Se on basil plants subjected to salt stress, Ocimum basilicum L. cv Tigullio plants were grown in hydroponics with the following treatments: 0 mg Se L-1 and 2 mM NaCl (T1); 0 mg Se L-1 and 25 mM NaCl (T2); 4 mg Se L-1 and 2 mM NaCl (T3); 4 mg Se L

-1_{and 25 mM NaCl (T4). Chlorophylls and carotenoids content, and ethylene production were measured}

in fresh leaves at harvest and after 3 days of storage at 10°C. Total phenolic and rosmarinic acid content, and photosynthetic activity were determined in leaves at harvest. Total Se content was determined in oven–dried ground leaves, stem and roots samples. The same samples were analyzed for K, Ca, Mg, Zn, Mn, and Fe contents.

- 2nd_experiment

In order to evaluate the effect of Se on plant senescence and on the leaves quality of subsequent mowings, plants of Ocimum basilicum L. cv Tigullio were grown in hydroponics, and Se was added to the nutrient solution at rate of 0 (control), 4, 8 and 12 mg Se L-1. Sampling consisted in three mowing, after the first one, plants were let to grow and then cut again. At each mowing ethylene production, chlorophyll, carotenoids, total phenols, rosmarinic acid content and antioxidant capacity were measured on fresh leaves material at harvest and after 5 days of storage at 10°C. The oven-dried ground leaf material was used for the analysis of Se, nitrate and calcium, potassium and magnesium.

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7 - 3rd_experiment

In order to further investigate the role of Se in delaying plant senescence and the effects of Se on the quality of leaves obtained by mowing, plants of Ocimum basilicum L. cv Tigullio were grown in hydroponics following the experimental plan described above. Plants were mowed two times.

The results obtained in the experiment were elaborated and statistically analyzed. - 4th_experiment

This experiment was carried out to evaluate the rate and the dynamic of Se uptake in basil plants. Ocimum basilicum L. cv Tigullio plants were grown in hydroponics and added with 0 (control), 4, 8 and 12 mg Se L-1_._{One plant from each replicate was harvested immediately after the selenium treatment and then}

every 2 weeks for 5 times. DW was collected and data were used to calculate the RGR (Relative Growth Rate, mg DW mg DM-1 d-1). Total selenium content was determined in all plant organs. The rate of Se uptake was calculated according to Pitman (1972) and expressed as µg g root-1 day-1. The translocation

factor (TF) was calculated as the ratio of Se accumulated in the shoot to selenium accumulated in the root (Renkema et al. 2012).

In order to evaluate the Se distribution in roots, stem, leaves, inflorescences, seeds and sprouts, Ocimum basilicum L. cv Tigullio plants were grown in hydroponics and treated with 0 (control), 4 and 8 mg Se L-1_{. The effects of Se on seeds germination and the antioxidant capacity of sprouts were also evaluated.}

Plants were cut and separated in roots, stem, leaves and inflorescences. Leaves were then separated in first, second and third node, and seeds were separated from the inflorescences. The seeds were sown and sprouted, then the sprouts were analyzed for Se content. Seeds produced by plants treated with 4 and 8 mg Se L-1 were germinated in Petri dishes on wet sheets of paper (1st test). The experiment was replicated with seeds produced by plants treated with 4 mg Se L-1 (2nd test). Purchased seeds treated with water containing 4 mg Se L-1 were also sprouted (3rd test). In the 1st and 2nd test the antioxidant capacity of sprouts was measured.

In order to investigate the effect of selenium on fruit composition at harvest and ripening physiology, plants of tomato “Red Bunch” (Solanum Lycopersicum L.) were hydroponically grown under greenhouse. Se, as sodium selenate, was added to the nutrient solution at rate of 0, 1 and 1.5 mg Se L-1. Nine plants for each treatment were selected for the harvesting of ripe tomato, evaluation of fruit quality and measure of antioxidant activity in fruit, the others plants were used to collect breakers tomatoes for monitoring the ripening in laboratory. Sugar content, antioxidant enzyme activity (GSH-Px, APX, SOD

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8 and CAT), solid soluble content, titrable acidity and pH were measured in the harvested fruit. In addition the taste index and maturity index were calculated using the equation proposed by Navez et al. (1999). Fruit let to ripe in laboratory were harvested at breakers stage. Every about two days, for about two weeks, non-destructive analysis (ethylene production, weight loss, hardness and visual evaluation of fruit color) and destructive analysis (chlorophyll, lycopene and sugar content) were carried out. Some analysis will be carried out at Agrifood Center, Cranfield University, Cranfield, Bedforshire, UK.

The experiment is still ongoing. References

- Navez, B., Letard, M., Graselly, D., Jost, M. (1999). Les criteres de qualité de la tomate. Infos-Ctifl, 155, 41-47.

- Pitman, M.G. (1972). Uptake and transport of ions in barley seedling. Aust. J. Biol. Sci. 25: 905-919 - Renkema H, Koopmans A, Kersbergen L, Hale B, Berkelaar E (2012) The effect of transpiration on

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ANNUAL REPORT: 3rd YEAR [2016-2017]

Research activity

From September 2016 to February 2017 I have carried out some biochemical analyses at the Agrifood Center, Cranfield University, Cranfield, Bedforshire. In particular, I have analyzed the content of non-structural carbohydrates, carotenes and chlorophylls in tomato fruits collected at different ripening stages during the 6th experiment of the 2nd year. The activity of some antioxidant enzymes and the antioxidant status of red fruit were also analyzed. The goal of the experiment was to investigate the effects of selenium on tomato fruit composition at harvest and during post-harvest ripening. For this experiment, plants of tomato “Red Bunch” (Solanum Lycopersicum L.) were hydroponically grown under greenhouse. The setup of this experiment is described above. The results obtained in the experiment were elaborated and statistically analyzed.

From March to July 2017, I have spent four months working in the Greenhouse Technology group of Wageningen University and Research, The Netherlands. During my internship, I have conducted an experiment to study the effect of high light availability on cucumber yield.

During the last months of the third year, I have finished to elaborate the data and I have written my PhD thesis.

References

- Brown TA, Shrift A (1982) Selenium: Toxicity and Tolerance in Higher Plants. Biol Rev 57:59– 84. doi: 10.1111/j.1469-185X.1982.tb00364.x

- Fa Yang S, Hoffman NE, Yang SF, Hoffman NE (1984) Ethylene biosynthesis and its regulation in higher plants. Ann Rev Plant Physio 35:155–189.

- Zhu Z, Chen Y, Shi G, Zhang X (2017) Selenium delays tomato fruit ripening by inhibiting ethylene biosynthesis and enhancing the antioxidant defense system. Food Chem 219:179–184. doi: 10.1016/j.foodchem.2016.09.138

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International conferences

- III International Symposium on Horticulture in Europe - SHE2016. Chania, Crete (Greece), October 17-21, 2016

National conferences

- Convegno Nazionale Postraccolta2017, Pisa (Italy). September 28-29, 2017.

Pubblications

Journal articles

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

- Puccinelli M, Malorgio F, Rosellini I, Pezzarossa B (2017) Uptake and partitioning of selenium in basil (Ocimum basilicum L.) plants grown in hydroponics. Sci Hortic 225:271–276. doi:

10.1016/j.scienta.2017.07.014 National Conference

- Puccinelli M., Malorgio F., Rosellini I., Maggini R., Pezzarossa B. Increase of the nutritional value of sweet basil (Ocimum basilicum L.) by selenium fertilization. XI Giornate Scientifiche SOI Bolzano, 14-16 settembre 2016. Acta Italus Hortus 20, pp. 60.

- Puccinelli M., Pezzarossa B., Malorgio F. Effect of selenium enrichment on metabolism of tomato fruits during ripening. Convegno Nazionale Postraccolta2017, Pisa. September 28-29, 2017.

International conference

- Puccinelli M., Malorgio F., Maggini R., Rosellini I., Pezzarossa B. Biofortification of Ocimum basilicum L. plants with selenium. Acta III International Symposium on Horticulture in Europe - SHE2016. Chania, Crete (Greece), October 17-21, 2016.

Papers not related to the PhD thesis

- Pardossi A. Romani M., Carmassi G., Guidi L., Landi M., Incrocci L., Maggini R., Puccinelli M., Vacca W., Ziliani M. (2015). Boron accumulation and tolerance in sweet basil (Ocimum basilicum L.) with green or purple leaves. Plant Soil, 395: 375–389.