Physiology and Biochemistry of 'Rocha' Pear During

PHYSIOLOGY AND BIOCHEMISTRY OF ‘ROCHA’ PEAR DURING RIPENING

AND LONG-TERM CONTROLLED ATMOSPHERE STORAGE

ADRIANO ARRIEL SAQUET

ORIENTADOR: Professor Doutor Domingos Almeida COORIENTADOR: Doutor Josef Streif

TESE ELABORADA PARA OBTENÇÃO DO GRAU DE DOUTOR EM ENGENHARIA AGRONÓMICA

2017

PHYSIOLOGY AND BIOCHEMISTRY OF ‘ROCHA’ PEAR DURING RIPENING

AND LONG-TERM CONTROLLED ATMOSPHERE STORAGE

TESE APRESENTADA PARA A OBTENÇÃO DO GRAU DE DOUTOR EM ENGENHARIA AGRONÓMICA

ADRIANO ARRIEL SAQUET

ORIENTADOR: Doutor Domingos Almeida, Professor, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal. COORIENTADOR: Doutor Josef Streif, Investigador, Universidade de Hohenheim, Stuttgart, Alemanha.

JÚRI Presidente: Doutora Maria Helena Mendes da Costa Ferreira Correia de Oliveira (Professora Associada, Instituto Superior de Agronomia, Universidade de Lisboa);

Vogais: Doutora Inmaculada Recasens Guinjuan (Professora Catedrática, Escola Tècnica Superior d’Enginyeria Agrària, Universitat de Lleida, Espanha); Doutor Ricardo Manuel de Seixas Boavida Ferreira (Professor Catedrático, Instituto Superior de Agronomia, Universidade de Lisboa); Doutor Josef Streif (Senior Scientist, University of Hohenheim, Alemanha); Doutor Fernando José Cebola Lidon (Professor Associado com Agregação, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa); Doutora Maria Dulce Carlos Antunes (Professora Auxiliar, Faculdade de Ciências e Tecnologia, Universidade do Algarve).

2017

ACKNOWLEDGEMENTS

I extend my sincere gratitude to Prof. Dr. Domingos Almeida for his supervising of this research. Thanks for all the advices and considerable patience on planning and reading this thesis. My grateful thanks to Dr. Josef Streif, Competence Center for Fruit Science of Bavendorf, University of Hohenheim, Germany, for helpful suggestions in this research project and reading this thesis. I thank Dr. Daniel Neuwald for the partnership and support in carrying out additional storage trials with ‘Alexander Lucar’ pear at Competence Center for Fruit Science Bavendorf, Ravensburg, Germany. Thanks to the Federal Institute of Science and Education, Brazil, for granting this training period in Portugal. My sincerely thanks to my colleagues Rita Galvão Gonçalves, Carla Alegria, Tiago Daniel Vieira, Pedro Figueiredo and Cristina Couto for friendship and valuable help in laboratory. My thanks to Daniel Duarte and Diana Faria from the laboratory of Food Chemistry for friendship and kind laboratory help. Special thanks to Paula Gonçalves from de Laboratory of Analytical Chemistry for helpful support in various laboratory procedures. I thank very much Prof. Dr. Luisa Carvalho, from the Laboratory of Plant Physiology for her kindly assistance in showing the operation of the multi-reader before and during my measurements of adenylates. Thanks to Dr. Mariana Mota from the Laboratory of Plant Physiology for kindly laboratory support. Finally, I thank my mother Antonieta, a great mother and a great person, my father Edi in memoriam, my brother Marcos and his family, and especially my family, wife Solange, son Leonardo and daughter Manuela, for their unconditional friendship, support and nice time spended togheter in Lisbon.

“The Earth is the great school of souls, where it educates students of all ages” (Francisco Cândido Xavier)

TABLE OF CONTENTS Acknowledgements ...... ii Table of contents ...... iii Abbreviations ...... vi Abstract ...... viii Resumo ...... ix Resumo alargado ...... x Scientific contributions directly related to the doctoral thesis ...... xiii

Chapter 1. General introduction ...... 01 1.1. Pear fruit quality ...... 01 1.2. The ‘Rocha’ pear ...... 02 1.3. The main physiological and biochemical changes during ripening of pear ...... 03 1.4. Storage systems for pear ...... 03 1.4.1. Cold storage in air ...... 03 1.4.2. Controlled atmosphere storage ...... 04 1.5. Postharvest treatment with 1-methylcyclopropene ...... 05 1.5.1. General information on 1-methylcyclopropene ...... 05 1.5.2. Effect of 1-methylcyclopropene on pear ripening and storability ...... 06 1.6. Physiological disorders during storage of ‘Rocha’ pear ...... 07 1.6.1. Internal storage disorders...... 07 1.6.2. Superficial scald ...... 08 1.7. Macro- and micronutrients related to fruit quality and storage disorders ...... 09 1.8. Mechanisms of internal storage disorders ...... 10

Aim and outline of the thesis ...... 15

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Chapter 2. Mapping the gradients of adenylate nucleotides and energy charge within ‘Rocha’ pear fruit ...... 17 Abstract ...... 17 Introduction ...... 18 Material and methods ...... 19 Results and discussion ...... 20 Conclusion ...... 22

Chapter 3. Ripening physiology and biochemistry of ‘Rocha’ pear as affected by ethylene inhibition ...... 23 Abstract ...... 23 Introduction ...... 24 Material and methods ...... 25 Results ...... 28 Discussion ...... 33 Conclusions ...... 37

Chapter 4. Sensory and instrumental assessments of ripening changes in ‘Rocha’ pear: Effect of temperature and ethylene inhibition ...... 38 Abstract ...... 38 Introduction ...... 39 Material and methods ...... 40 Results ...... 42 Discussion ...... 48 Conclusions ...... 51

Chapter 5. Mineral composition of ‘Rocha’ pear fruit related to storage disorders ...... 53 Abstract ...... 53 Introduction ...... 54 Material and methods ...... 55 Results and discussion ...... 58 Conclusions ...... 69

Chapter 6. Internal disorders of ‘Rocha’ pear affected by oxygen partial pressure and inhibition of ethylene action ...... 70 Abstract ...... 70 Introduction ...... 71 Material and methods ...... 72 Results and discussion ...... 75 Conclusions ...... 87

Chapter 7. Responses of ‘Rocha’ pear to delayed controlled atmosphere storage depend on oxygen partial pressure ...... 88 Abstract ...... 88 Introduction ...... 89 Material and methods ...... 90 Results and discussion ...... 92 Conclusions ...... 98

Chapter 8. General discussion ...... 99 General discussion ...... 99 Conclusions ...... 102 Future research directions ...... 103

References...... 104

ABBREVIATIONS

ADP Adenosine 5’-diphosphate AEC Adenylate energy charge AK Adenylate kinase AMP Adenosine 5’-monophosphate AMR ATP monitoring reagent ANOVA Analysis of variance ATP Adenosine 5’-triphosphate CA Controlled atmosphere DCA Dynamic controlled atmosphere DM Dry mass DPA Diphenylamine ECi Initial electrical conductivity ECf Final electrical conductivity ECt Total electrical conductivity EDTA Ethylene diamine tetra acetic acid FAO Food and Agriculture Organization FID Flame ionization detector FM Fresh mass GC Gas chromatograph INE Instituto Nacional de Estatística INIAV Instituto Nacional de Investigação Agrária e Veterinária kPa Partial pressure LSD Least significant difference 1-MCP 1-Methylcyclopropene MK Myokinase PEP Phosphoenol pyruvate PK Pyruvate kinase pO2 Oxygen partial pressure pCO2 Carbon dioxide partial pressure RH Relative humidity SD Standard deviation TA Titratable acidity

TCA Trichlor acetic acid TCA cycle Tricarboxylic acid cycle TSS Total soluble solids

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Abstract

Long-term storage of pears is a challenge in the absence of treatment with diphenylamine, due to the development of physiological disorders. Aspects of the ripening physiology and biochemistry of pears, particularly those treated with the ethylene action inhibitor 1-methylcyclopropene, also remain unknown. The aims of this thesis were to map the gradients of adenylate nucleotides and energy charge in the fruit and their changes during fruit ripening and storage period, to compare instrumental and sensory assessments of ripening, to relate the fruit mineral composition to the development of internal storage disorders and determine the optimal storage conditions for long-term storage of ‘Rocha’ pear under controlled atmosphere. Significant radial gradient in energy charge from the skin tissues to the fruit center may be related to internal storage disorders. Significant radial gradients in Ca and B decreasing from the skin tissues toward the fruit center were also consistent with the location of internal storage disorders. However, ‘Rocha’ pear were able to adjust the energy charge during ripening and long-term storage even under low respiration rates induced by 1- methylcyclopropene treatment or low oxygen partial pressure. ‘Rocha’ pear was able to ripen immediately after harvest without chilling or exogenous ethylene application. ‘Rocha’ pear tolerated extremely low 0.5 kPa O2 during 257 d storage without developing storage disorders and kept acceptable firmness and skin color after 7 d shelf life. The 46 d delay in the pull down of O2 partial pressure was detrimental to quality maintenance of ‘Rocha’ pear during long-term controlled atmosphere storage.

Keywords: Adenylate nucleotides, mineral nutrition, physiological disorders, postharvest quality, Pyrus communis.

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Resumo

Fisiologia e bioquímica da pera ‘Rocha’ durante o amadurecimento e armazenamento prolongado em atmosfera controlada

O armazenamento prolongado de pera é um desafio quando considerada a ausência da difenilamina, devido ao desenvolvimento de acidentes fisiológicos. Aspectos relacionados à fisiologia e à bioquímica do amadurecimento de pera, em particular quando tratadas com 1- metilciclopropeno, um inibidor da ação do etileno, também permanecem mal compreendidos. Os objetivos desta tese foram de mapear os gradientes dos nucleótidos de adenosina e da carga energética no fruto e suas alterações durante o amadurecimento e armazenamento, comparar avaliações sensoriais e instrumentais durante o amadurecimento da pera, relacionar a composição mineral do fruto com o desenvolvimento de acidentes fisiológicos internos e determinar condições ótimas para o armazenamento prolongado da pera ‘Rocha’ em atmosfera controlada. O significativo gradiente decrescente na carga energética a partir dos tecidos da casca em direção ao centro do fruto pode estar relacionado ao surgimento dos acidentes fisiológicos. Os gradientes decrescentes observados nos nutrientes Ca e B a partir dos tecidos da casca ao centro do fruto coincidiram com a localização dos acidentes fisiológicos no fruto. No entanto, a pera ‘Rocha’ ajustou a carga energética durante o amadurecimento e, também, durante o armazenamento prolongado, mesmo com taxas respiratórias mais baixas, induzidas pelo 1-metilciclopropeno ou pela baixa pressão parcial de O2. A pera ‘Rocha’ amadureceu imediatamente após a colheita sem exposição prévia à baixa temperatura ou à aplicação exógena de etileno. A pera ‘Rocha’ tolerou a pressão parcial de O2 de 0,5 kPa durante 257 dias de armazenamento sem desenvolver acidentes fisiológicos e amadureceu com firmeza e cor da epiderme adequadas após 7 dias a 20 ºC. O retardamento na redução da pressão parcial de O2 em 46 dias foi prejudicial para a preservação da qualidade da pera ‘Rocha’ durante o armazenamento prolongado em atmosfera controlada.

Palavras-chave: Acidentes fisiológicos, nucleótidos de adenosina, nutrição mineral, Pyrus communis, qualidade pós-colheita.

Resumo alargado

Fisiologia e bioquímica da pera ‘Rocha’ durante o amadurecimento e armazenamento prolongado em atmosfera controlada

As recentes alterações regulatórias e tecnológicas alteraram profundamente a conservação da pera ‘Rocha’. A proibição do tratamento pós-colheita com difenilamina, a introdução do 1-metilciclopropeno e as tecnologias para controle da pressão parcial de O2 com elevada precisão vieram criar um novo contexto ao qual o setor se está a ajustar. Para acelerar este ajustamento tecnológico, os objetivos desta tese foram mapear os gradientes dos nucleótidos de adenosina e da carga energética no fruto, bem como os gradientes dos elementos minerais, analisar o metabolismo energético durante o amadurecimento e armazenamento da pera ‘Rocha’, caracterizar sensorialmente os frutos durante o amadurecimento e determinar as condições ótimas para o armazenamento prolongado da pera ‘Rocha’ em atmosfera controlada. Os gradientes dos nucleótidos de adenosina e da carga energética foram analisados radial e longitudinalmente no fruto. Foi constatado um gradiente decrescente na carga energética a partir dos tecidos da casca em direção ao centro do fruto, com valores de 0,80, 0,72 e 0,69 na casca, na camada externa da polpa e na camada interna da polpa, respectivamente, mas não se observou nenhum gradiente longitudinal no fruto. Apesar da menor carga energética no centro do fruto, consistente com a localização dos acidentes fisiológicos internos, o valor parece ser adequado a uma manutenção da homeostasia do fruto durante o armazenamento em atmosfera controlada. O estudo da fisiologia e da bioquímica do amadurecimento da pera ‘Rocha’, tratada ou não com o inibidor de ação de etileno 1-metilciclopropeno (1-MCP) revelou que a pera ‘Rocha’ pode amadurecer normalmente sem exposição prévia ao frio ou aplicação de etileno exógeno, seguindo o padrão típico de frutos climatéricos com relação à respiração e produção de etileno. O tratamento com 1-MCP induziu uma redução transiente nas concentrações de ATP e na carga energética. A carga energética máxima de 0,77 foi aferida em frutos não tratados com 1-MCP durante a primeira semana de amadurecimento, decrescendo posteriormente até valores de 0,70. O tratamento com 1-MCP retardou o amolecimento dos frutos, o amarelecimento da casca e o aumento do efluxo de electrólitos, mas não bloqueou o amadurecimento. O metabolismo conseguiu ajustar a carga energética em valor estável na faixa de 0,70 durante o amadurecimento, mesmo com taxa respiratória mais baixa, induzida pela aplicação de 1-MCP.

A avaliação sensorial e instrumental foi realizada para investigar a qualidade da pera ‘Rocha’ durante o amadurecimento a diferentes temperaturas e com inibição parcial da ação do etileno. Após 30 dias em ar a -0,5 °C dois lotes de frutos foram tratados com 1-MCP nas doses de 150 e 300 nL L-1 e expostos para amadurecerem a 20 °C. A outra metade, foi subdividida em dois grupos e, os frutos mantidos em ar à 10 e 20 °C, sem tratamento com 1- MCP. A 10 °C o amadurecimento foi mais lento, com taxas mais baixas na produção de etileno e da respiração dos frutos e taxas de amolecimento e de amarelecimento inferiores aos frutos tratados com 1-MCP e amadurecidos a 20 ºC. A avaliação sensorial revelou que os provadores caracterizaram os frutos amadurecidos a 10 °C como mais verdes e firmes, menos suculentos e menos doces. Além disso, os provadores perceberam os frutos tratados com 300 nL L-1 de 1- MCP e amadurecidos a 20 °C como mais suculentos, mais doces e flavor mais intenso. A temperatura de amadurecimento teve efeito maior sobre o perfil sensorial do que o tratamento com 1-MCP, que pode ser usado para modular o perfil sensorial da pera. Para determinar a possivel relação entre a composição mineral da pera e o desenvolvimento dos acidentes fisiológicos, foi realizado, inicialmente, um mapeamento detalhado das concentrações dos macronutrientes (N, P, K, Ca, Mg, P e S) e dos micronutrientes (Fe, Mn, Zn, Cu e B) no fruto. Os macronutrientes N, Ca, Mg e S, bem como os micronutrientes Fe, Mn, Zn, Cu e B apresentaram concentrações mais elevadas nos tecidos da casca do que na polpa do fruto. Os elementos P e K apresentaram, no entanto, gradientes crescentes de concentração a partir dos tecidos da casca ao centro do fruto. Longitudinalmente, o Ca esteve menos concentrado nos tecidos da casca e na polpa do terço distal do fruto. A concentração de B foi mais baixa nos tecidos da polpa da região distal. Ao final de cinco meses de armazenamento, a concentração de minerais foi avaliada em frutos sadios e em frutos afetados por acidentes fisiológicos provenientes de quatro pomares. As concentrações de Ca, em frutos oriundos de três dos pomares apresentaram-se mais baixas nos frutos afetados. As relações K/Ca e (K+Mg)/Ca foram mais elevadas nos frutos afetados. Dentre os micronutrientes, o elemento B apresentou concentrações significativamente mais baixas em frutos afetados, procedentes de dois dos pomares. As baixas concentrações de Ca e B na região central do fruto, estão possivelmente, relacionadas com o surgimento dos acidentes fisiológicos internos durante o armazenamento. O armazenamento da pera ‘Rocha’ por 136 dias a -0,5 °C foi avaliado em ar ou em atmosfera controlada com 3.0 e 0.5 kPa de O2 combinados com 0.6 kPa de CO2. Frutos tratados -1 com 150 nL L de 1-MCP também foram armazenados em 3.0 e 0.5 kPa de O2 após 32 dias do retardamento na redução da pressão parcial de O2. Acidentes fisiológicos internos não foram

xi verificados em frutos armazenados em ar ou em 0,5 kPa de O2, mas 10,2 % dos frutos armazenados em 3,0 kPa de O2 foram afetados por acidentes fisiológicos. O tratamento com

1-MCP aumentou a ocorrência de acidentes nos frutos armazenados em 0,5 e 3,0 kPa O2. As baixas pressões parciais de O2 reduziram a produção de etileno e a intensidade respiratória, as quais sofreram uma redução adicional pela aplicação do 1-MCP. As concentrações de ATP e a carga energética mantiveram-se mais elevadas em frutos armazenados em ar do que em 3,0 kPa de O2 e mais baixas em frutos armazenados em 0,5 kPa de O2. Não foi possível estabelecer um limiar nas concentrações de ATP ou na carga energética abaixo das quais os acidentes fisiológicos se desenvolvam. Conclui-se que a qualidade pós-colheita da pera foi melhor preservada em 0,5 kPa de O2 e sugere-se a existência de uma zona de risco nas pressões parciais de O2 em relação à ocorrência dos acidentes fisiológicos internos em ≤ 3.0 kPa de O2 e > 0.5 kPa de O2.

A eficácia do retardamento em 46 dias na redução da pressão parcial de O2 sobre a qualidade da pera ‘Rocha’ foi avaliada. Os frutos foram armazenados de imediato em 0,5 ou

3,0 kPa de O2 ou a redução da pressão parcial de O2 foi retardada em 46 dias antes da colocação em regime da atmosfera controlada com 0,5 ou 3,0 kPa de O2. Após 257 dias de armazenamento, a pera ‘Rocha’ tolerou o armazenamento imediato em 0,5 kPa de O2 sem desenvolver nenhum tipo de acidentes fisiológicos. No entanto, 63,3 % dos frutos armazenados imediatamente em 3,0 kPa de O2 foram afetados. O retardamento, em 46 dias, na redução das pressões parciais de O2 diminuiu a ocorrência de acidentes fisiológicos em frutos mantidos em

3,0 kPa de O2 para 35,5 %, entretanto, aumentou a ocorrência dos danos em frutos armazenados em 0,5 kPa de O2 para 27,3 %. Conclui-se que, em contraste com as referências da literatura em relação à pera ‘Conference’ no Norte da Europa, o retardamento na colocação em regime da atmosfera controlada não previne e ocorrência de acidentes fisiológicos internos em pera ‘Rocha’ produzida na região Oeste de Portugal. O efeito do retardamento na redução da pressão parcial de O2 sobre a ocorrência dos acidentes fisiológicos foi dependente das pressões parciais de O2. Portanto, o retardamento em 46 dias, na redução da pressão parcial de O2 não foi benéfico para preservação da qualidade da pera ‘Rocha’.

Palavras-chave: Acidentes fisiológicos, nucleótidos de adenosina, nutrição mineral, Pyrus communis, qualidade pós-colheita.

xii

Scientific contributions directly related to the doctoral thesis

The following peer-reviewed articles resulting from chapters of this thesis have been published:

1. Almeida, D.P.F.; Saquet, A.A. (2017). Mapping the gradients of adenylate nucleotides within ‘Rocha’ pear fruit. Acta Horticulturae. (in press). 2. Saquet, A.A.; Almeida, D.P.F. (2017). Ripening physiology and biochemistry of ‘Rocha’ pear as affected by ethylene inhibition. Postharvest Biology and Technology, 125, 161-167. 3. Saquet, A.A.; Almeida, D.P.F. (2017). Internal disorders of ‘Rocha’ pear affected by oxygen partial pressure and inhibition of ethylene action. Postharvest Biology and Technology, 128, 54-62. 4. Saquet, A.A.; Streif, J.; Almeida, D.P.F. (2017). Responses of ‘Rocha’ pear during delayed controlled atmosphere storage depend on oxygen partial pressure. Scientia Horticulturae, 222, 17-21.

The following manuscripts are in preparation:

1. Saquet, A.A.; Almeida, D.P.F. (2017). Sensory and instrumental assessments of ripening changes in ‘Rocha’ pear: Effect of temperature and ethylene action inhibition. 2. Saquet, A.A.; Streif, J.; Almeida, D.P.F. (2017). Mineral composition of ‘Rocha’ pear fruit related to storage disorders.

The following methodological articles related to the doctoral research but not included in the thesis have been presented and published in a conference proceeding:

1. Saquet, A.; Streif, J.; Cristóvão, L.; Carreira, P.; Almeida, D. (2016). Experimental device to study temperature effects on food quality. Advances in Refrigeration Sciences and Technologies, Volume VIII, 5 pp. 2. Saquet, A.; Barbosa, A.; Almeida, D. (2016). Cooling rates of fruits and vegetables. Advances in Refrigeration Sciences and Technologies, Volume VIII, 5 pp.

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Chapter 1

1. General Introduction

1.1. Pear fruit quality Quality is a broad umbrella word with several meanings. Modern fruit quality management considers quality as the “degree to which a set of inherent characteristics fulfils requirement” (ISO 9001, 2015). One of the main concepts of product quality relates to ability of the marketers to meet the customer needs (Shewfelt, 1999). The fruit supply chain includes various customers with different needs that have to be articulated throughout the supply chain that delivers fruit to the consumer. Pear fruit quality is judged by consumers primarily from their perception of the acceptability of fruit based on characteristics including the visual appearance (green, yellow or red skin), texture, and flavor (Kappel et al., 1995). Most pear consumers prefer characteristic flavor associated with an equilibrated content of sugars, acids and volatile compounds (Ma et al., 2000), slight yellowed skin cultivars (Kappel et al., 1995), and a buttery-juicy texture (Chauvin et al., 2010; Escribano et al., 2016). Pears undergo physiological and biochemical changes between harvest and consumption, which determine their final quality characteristics. Some of these changes render the fruit desirable for consumption while others lead to economic losses (Wills et al., 2007). The beneficial changes include the conversion of starch to sugars, the reduction in organic acids, the biosynthesis of aroma volatiles, and adequate softening (Sugar and Basile, 2006; Villalobos-Acuña and Mitcham, 2008). The detrimental changes include water loss due to excessive transpiration, excessive softening, postharvest physiological disorders and pathological decay (Raese et al., 1999; Spotts et al., 2007; Calvo et al., 2015; Almeida et al., 2016). Several pear quality changes are related to fruit ripening. Pears are climacteric fruit (Lelièvre et al., 1997), which are characterized by specific physiological and biochemical events and responses. Some of them are the ethylene and respiration rise, color changes and softening (Brady, 1987; Barry and Giovannoni, 2007). The plant hormone ethylene influences many of these ripening dependent events including its own production, and such effects on fruit ripening culminate with the anticipation of senescence (Lelièvre et al., 1997). Therefore, the main challenge of postharvest technologies is to reduce the fruit metabolism and

1 senescence with innovative, adequate strategies and modern storage technologies for keeping quality as long as possible without physiological disorders. However, senescence and deterioration of harvested pear fruit cannot be fully stopped. As a rule, fruit quality offered in the markets is determined by the level of quality achieved up to the harvest time and is only maintained as high as possible during postharvest period, never improving by postharvest handling (Streif, 2002; Hewett, 2006). The challenge of storage managers is, therefore, to minimize quantitative and qualitative quality losses. Responses of pear fruit to storage systems and their conditions depend on cultivar, season, growing conditions and physiological maturity at harvest (Sugar et al., 1998; Streif, 2002). Throughout storage period, fruit quality is generally preserved at high level whereas conditions at several points during the distribution chain are not adequate for fresh commodities (Johnston et al., 2002). ‘Rocha’ pear shows very good storability during cold storage in air or CA storage reaching around 10 months under appropriated CA conditions (Almeida et al., 2016). Meanwhile, as happen with other fruits, this pear cultivar has storage-related problems, which may limit their fruit quality and marketability during and after long-term storage even under CA. Such limitations are mainly related to the occurrence of superficial scald and internal storage disorders.

1.2. The ‘Rocha’ pear The pear cultivar ‘Rocha’ originated in Sintra, Portugal, in the middle of the 19th century. In recent decades, this pear cultivar has become dominant in the Portuguese pear industry, currently accounting for more than 95 % of the crop. ‘Rocha’ is an export pear, with the main markets in the United Kingdom, France, Brazil and Russia (Silva et al., 2005; FAO, 2014). This pear cultivar has become one of the top 10 major pears in the World market (WAPA, 2016). ‘Rocha’ is a summer pear cultivar characterized by medium size, yellow color when ripe, total soluble solids content between 11 and 14 % (Silva et al., 2005), good potential for long-term storage and poststorage handling and market ability (Almeida et al., 2016). Currently, the ‘Rocha’ pear is grown in Portugal in 12,115 ha (INE, 2016), with an average production of 203,000 t in 2014 but reduced to 135,000 t in 2016 (WAPA, 2016). The pear crop is mainly located in the Oeste region in the center of Portugal, with orchards in Alentejo (INE, 2016).

1.3. The main physiological and biochemical changes during ripening of pear Fruit ripening is the process by which fruits attain their desirable flavor, quality, color, palatable nature and adequate textural properties (Barry and Giovannoni, 2007). On the basis on ripening behavior, fruits are classified as climacteric and non-climacteric fruits (Brady, 1987; Barry and Giovannoni, 2007). Many anabolic and catabolic processes happen during pear ripening. The main changes are: a) changes in cell walls with softening of fruit (Ben-Arie et al., 1979; Ahmed and Labavitch, 1980); b) changes in the skin color with degradation of chlorophylls and appearance of other pigments such as carotenoids and anthocyanins (Knee and Tsantili, 1988); c) an increase in ethylene production (Biale, 1960; de Wild et al., 2003); d) changes in flavor and aroma volatiles (Shiota, 1990; Song and Bangerth, 1996); e) degradation of starch to simple sugars (Knee and Tsantili, 1988); f) the increase in the membrane permeability (Blackman and Parija, 1928; Sacher, 1973); g) changes in the respiration rate with an increase in the glycolytic rate and ATP concentrations (Solomos, 1983; Watkins and Frenkel, 1987).

1.4. Storage systems for pear 1.4.1. Cold storage in air Quality preservation of pears is possible in air at low temperature and high relative humidity. The decrease of temperature has a significant effect in reducing the fruit metabolism (Paul, 1999) and the development of pathogens (Spotts et al., 2007), and water loss (Streif, 2002). Lower temperature slows drastically the respiration rate, therefore diminishing the degradation of nutritional and health compounds prolonging poststorage life of fruits (Yahia, 2011). It has long been known that temperature management is one of the most important factors affecting the quality of fruits. Porritt (1964) showed early that storage life of ‘Anjou’ and ‘Bartlett’ pears was respectively 35 and 40 % larger at -1 than at 0 °C. Pear fruit is not chilling sensitive (Knee, 1987; Drake et al., 2004); therefore, the theoretical optimal temperature for its storage is just above the freezing points. However, practical limitations related to temperature fluctuations, the thermostat is set at a temperature above the critical temperature, the thermostatic oscillation in temperature does not result in storage temperature falling below the critical temperature (Mitchel, 1987). An appropriated RH management in the storage rooms is indispensable for keeping fruit quality (Lidster, 1990). High RH prevents excessive weight loss, however if RH is too high (>95%), it stimulates the development of molds and some physiological disorders (Lidster, 3

1990). Although it is difficult to maintain high RH under regular air storage, it is necessary to do a very rational management of this factor during storage period. RH is dependent upon the surface area of the refrigeration evaporator coil in the storage room and temperature difference between the coil and the air, along with air exchange rates, temperature distribution in the room, commodity and packing material used (Paul, 1999). The main European pear cultivars in the world market are suitable for long-term storage. At temperature of -1 to 0 ºC and relative humidity higher than 90 % pears can be stored in air for 3 to 6 months (Porrit, 1964; Agar et al., 2000; Drake et al., 2004; Wang and Sugar, 2013), after which time postharvest life is limited by advanced ripening (Raese et al., 1999), internal breakdown (Drake et al., 2004), poor pedicel condition (Drake et al., 2004), decay (Spotts et al., 2007), and superficial scald (Blaszczyk, 2010; Calvo et al., 2015). All these negative changes during long-term cold storage in air of pears culminate with a significant reduced external appearance as well as internal poor quality.

1.4.2. Controlled atmosphere storage

The traditional CA storage is characterized by the reduction of the pO2 and, when possible, depending on the tolerance of the pear cultivar, the increasing of pCO2 in the storage room (Smock, 1979; Kader, 1997). The control of the concentration of ethylene is also desirable, and low-ethylene CA storage can be devised depending on fruit in store. Pears, in general, are more difficult to store than apples. They tend to be less tolerant to low pO2 and high pCO2 (Kupferman, 2001; Streif et al., 2003; Thompson, 2010). These conditions, when not adequately controlled, lead to the development of physiological disorders such as browning disorders, frequently with formation of cavities in the pear fruit flesh. These disorders are externally not visible and can lead to significant losses under unfavorable storage conditions (Streif, 2002; Franck et al., 2007). ‘Rocha’ pear shows good storability during CA storage. Conditions for CA storage depend not only on the cultivar, but also on the growing regions (Kupferman, 2001). In Portugal

‘Rocha’ pear is normally stored under 2.5 kPa O2 plus 0.7 kPa CO2 (Isidoro and Almeida, 2006;

Almeida et al., 2016) or under 3.0 kPa O2 plus 0.9 kPa CO2 (Gago et al., 2013). In Brazil, however, the CA conditions with 1 kPa O2 plus 1 kPa CO2 or 1 kPa O2 plus 2 kPa CO2 showed promising results in keeping quality of ‘Rocha’ pear without development of internal disorders (de Martin et al., 2015).

1.5. Postharvest treatment with 1-methylcyclopropene 1.5.1. General information on 1-methylcyclopropene The effects of ethylene on plant growth and development was first discovered by Neljubov around 1900 (Neljubov, 1901). After this pioneer finding and with the rapid technological development, the synthesis and action of this simple plant hormone has since been elucidated. One of the most studied examples of ethylene regulation is the ripening of climacteric fruit, in which, contrary to non-climacteric fruit, the ripening process is accompanied by a burst of ethylene production followed by an increase in respiration rate (Biale, 1960; Lelièvre et al. 1997). During postharvest life and storage of climacteric fruits, and in the case of pear, a typical climacteric fruit, the effects of ethylene are partially desired. Fruit is harvested commercially at the mature-green preclimacteric stage and stored for several months at low temperature. This induces in fruit, after rewarming, uniform ripening and the development of aroma and texture characteristics through the induction of ethylene biosynthesis (Agar et al., 2000; Fonseca et al., 2005). Drouet and Hartmann (1982) suggest that low temperature exposure activates a system, which produces small amounts of ethylene; on warming this ethylene induces a second system, which produces ethylene more rapidly and initiates other ripening processes, which are dependent upon transcription and translation of new mRNA. The findings of Agar et al. (2000) with ‘Bartlett’ pear suggest that 4 weeks of cold storage at -1 °C or treatment with 10 Pa C2H4 at harvest stimulates ACC synthase and ACC oxidase activities upon transfer of the fruit to 20 °C and results in satisfactory ripening. On the other side, ethylene triggers the process of ripening anticipating the senescence of fruits and reducing drastically the storage period. In ‘Barttlet’ pear, some effects of ethylene on fruit quality and storage include the yellowing and softening of fruits and the induction of some internal disorders and superficial scald (Bower et al., 2003). ‘Conference’ (De Wild et al., 1999) and ‘Rocha’ (Fonseca et al., 2005) pears are not different in this regard and also show quality losses when ethylene is high in storage rooms. The inhibition of ethylene action by 1-MCP was discovered by Sisler and his collaborators in the 1990s (Sisler and Serek, 1997). This new tool has been added to the list of options for extending shelf life and quality of a range of plant organs. At physiological temperature and pressure, 1-MCP (C4H6) is a gas with a relative molecular mass of 54. 1-MCP occupies the ethylene receptors such that ethylene cannot bind and elicit action (Sisler and Blankenship, 1996). Sisler and Serek (1997) proposed a model of 1-MCP action at the ethylene receptor. The affinity of 1-MCP for the receptor is approximately 10 times greater than that of

5 ethylene. Compared with ethylene, 1-MCP is active at much lower concentrations. 1-MCP also influences ethylene biosynthesis in some species through feedback inhibition (Blankenship and Dole, 2003).

1.5.2. Effect of 1-methylcyclopropene on pear ripening and storability 1-MCP has been experimentally used in pear (Baritelle et al., 2001; Chiriboga et al., 2011; Rizzolo et al., 2014; Almeida et al., 2016) and is in commercial use in the Portuguese pear industry since 2011. Pears have been shown a different behavior when treated with 1-MCP at shelf life or before storage in air or under CA. Depending on 1-MCP concentration, fruit maturity stage and storage conditions, some pear cultivars show ripening impairment and fail to achive a satisfactory skin color, juiciness, and texture (Watkins, 2006). Streif and Saquet (2006) working with ‘Conference’ and ‘Alexander Lucas’ pears concluded that, first: 1-MCP concentrations in these pear varieties have to be much lower than those for apples, second: 1-MCP keeps good quality of pear in both tested storage systems, i.e., RA and CA storage, and third: 1-MCP keeps good quality either in pears late harvested. Pear cultivars such as ‘d‘Anjou’ showed lower ethylene production and respiration rates (Argenta et al. 2003), ‘La France’ had better firmness and lower respiration rates (Kubo et al., 2003) when treated with 1-MCP. The duration and concentration of 1-MCP-induced responses was dependent on 1-MCP treatment concentration (Ekman et al., 2004; Argenta et al., 2003; Argenta et al., 2016). 1-MCP at 420 nL L-1 can prevent ‘d’Anjou’ pear ripening at 1 ºC for 4 months, while the threshold concentration of 1-MCP to inhibit fruit ripening for longer storage periods is higher (Argenta et al., 2003). Reapplication of 420 nL L-1 of 1-MCP can extend storage life of ‘Barttlet’ pear while allowing proper ripening during cold storage intervals (Argenta et al., 2016). Exposure to 200 to 400 nL L-1 1-MCP reduced physiological disorders and skin browning, and slowed the rate of ripening at room temperature (Ekman et al., 2004). Effects of 1-MCP in maintaining green skin color in pears are well documented (Mattheis and Rudell, 2011; Gago et al., 2015; Rizzolo et al., 2015; Vanoli et al., 2016). However, in some situations 1-MCP application can impair the ripening process in pears (Chiriboga et al., 2011). Moreover, the effect of 1-MCP on pear internal disorders is not clear: It has been shown to increase the occurrence of internal disorders during CA storage in ‘Alexander Lucas’ (Hendges et al., 2015), but to alleviate it in ‘Abate Fetel’ (Vanoli et al., 2016), and in ‘Rocha’ pear (Almeida et al., 2016). The Portuguese ‘Rocha’ pear has two main storage problems that can be mitigated by 1-MCP treatment. Superficial scald affects fruit when stored under oxygen partial pressure in

6 the range of 3 kPa (Isidoro and Almeida, 2006), and the internal disorders in fruits stored under low oxygen combined with carbon dioxide higher than 0.7 kPa (Silva et al., 2010; Almeida et al., 2016). The challenge is to optimize long-term CA storage for ‘Rocha’ pear preventing superficial scald and internal disorders, and at the same time keeping acceptable high fruit quality during further shelf life conditions.

1.6. Physiological disorders during storage of ‘Rocha’ pear During fruit growth and development on the plant, after harvest, during storage and marketing fruit damages occur that are not caused by diseases or pests, but by alterations in fruit metabolism, which are called physiological disorders. Some of physiological disorders affect drastically the external appearance and the flavor of fruits. However, in all situations, these damages affect drastically the quality and marketability of fruits causing relevant economic losses around the world.

1.6.1. Internal storage disorders

Low pO2 or high pCO2 injuries result from holding fruit in atmospheres below pO2 or above pCO2 tolerance. The symptoms of internal disorders can include external skin or flesh browning, and in many cases with formation of cavities in the fruit flesh (Höhn et al., 1996; Saquet et al., 2000; Franck et al., 2007; Lum et al., 2016). The major problem of the internal storage disorders is that neither the packer nor the consumer can detect what is inside the fruit until it is cut for consumption (Streif et al., 2003; Franck et al., 2007). The confusing description, classification, and terminology regarding pear internal disorders is recognized (Franck et al., 2007) and there is still no consensus regarding their nature and etiology. Core browning in pear has been described by the damage in the core region and is considered a senescence related disorder (Larrigaudière et al., 2004). Other authors use a generic term to classify all internal disorders observed in fruit stored under CA storage, such as brown heart (Streif et al., 2001; Saquet et al., 2003a) or core breakdown (Lammertyn et al., 2003), who used the same term to brown tissue and the cavities combined. Pear cavities are considered a late stage of development of the brown tissue (Lammertyn et al., 2000; Lammertyn et al., 2003; Franck et al., 2007). CA storage conditions that induce browning increase the membrane permeability intensifying the water loss of the cells resulting in dry and browned tissues, which may further dehydrate originating the cavities in fruit flesh (Xuan et al., 2001; Lammertyn et al., 2003). Flesh browning is generally spread in the flesh of pear fruit, but without cavity formation (Franck et al., 2007). This symptom may result from the

7 development of core browning or coexist together but in both situations the core region of fruit damaged by flesh browning is also brownish (Franck et al., 2007). Several pre- and postharvest factors affect the occurrence of internal disorders in pear fruit. As preharvest factors, pear trees with low crop load are more susceptible to the development of cavities and internal browning than those with balanced load (Kupferman, 2002); early harvested fruit are less susceptible than fruit with advanced maturation (Elgar et al., 1997); late harvested fruits are more susceptible to CA-related disorders (Sugar, 2002; Streif et al., 2003); boron field sprays can alleviate internal disorders in ‘Conference’ pear (Xuan et al., 2003; 2005); calcium treatment is conflicting, but in some situations showing protective effects (Curtis et al., 1990; Raese and Drake, 2006). As postharvest factors, the delay in the establishment of CA conditions alleviates internal disorders in ‘Conference’ pear (Höhn et al., 1996; Saquet et al., 2001), and ‘Braeburn’ apple (Saquet et al., 2003), but not necessarily is effective for ‘Rocha’ pear (Morais et al., 2001;

Almeida et al., 2016); high pCO2 immediately after harvest, especially at low pO2 induce internal disorders (Kader, 1995); low pCO2 rising to higher partial pressures over time did not promote internal disorders as fruit become acclimated (Kupferman, 2002); rapid establishment of CA conditions is benefic for some apples such as ‘Golden Delicious (Lau et al., 1983) and ‘Gala’ apples (Brackmann and Saquet, 1999); the new technology of dynamic controlled atmosphere storage has been shown to be effective for storage of apples (Gasser et al, 2008; Brackmann et al., 2014; Wright et al., 2015), and is promising for pear (Prange et al., 2011; Deuchande et al., 2016).

1.6.2. Superficial scald Superficial scald is a common physiological disorder of apples and pears that affect the fruit skin and can seriously compromise fruit quality after long-term storage (Whitaker et al., 2009; Lurie and Watkins, 2012). Symptoms are brown or black patches on the skin and typically worsen after removal from storage, rendering the fruit unmarketable. Superficial scald in apples and pears is mainly caused by the damage due the oxidation of the sesquiterpene α-farnesene with accumulation of conjugated trienols (Ingle, 2001; Whitaker et al., 2009; Lurie and Watkins, 2012). Pre-storage of pears with the antioxidant diphenylamine inhibits the oxidation of α- farnesene and largely controls superficial scald development (Isidoro and Almeida, 2006). However, this treatment has been banned from the European Union and can no longer be used. In addition, the exposure of pear fruit to the blocker of ethylene action 1-MCP greatly curtails

α-farnesene production and markedly reduces superficial scald incidence and severity (Chen and Spotts, 2005; Almeida et al., 2016). Results from the studies with 1-MCP have shown that ethylene production and perception, and tissue responsiveness to ethylene, are involved in regulation of α-farnesene synthesis and induction of superficial scald in apples and pears (Whitaker et al., 2009). Other factors that influence the occurrence and severity of superficial scald in pears include fruit maturity stage at harvest, O2 and CO2 partial pressures during CA storage, the plant mineral nutrition, climate conditions and orchard location (Ingle, 2001; Whitaker et al., 2009; Lurie and Watkins, 2012).

1.7. Macro- and micronutrients related to fruit quality and storage disorders The concentrations and balance of nutrients play a very important role in the cell structure and metabolism, consequently influencing the susceptibility of pears to internal disorders during postharvest period (Raese and Drake, 2006; Xuan et al., 2003 and 2005; Brunetto et al., 2015). Detailed studies on concentrations of macro- and micronutrients in pears related to fruit quality and storability are scarce. Plants require macronutrients such as calcium, magnesium, nitrogen, phosphorus, sulfur and potassium in relatively large amounts, normally more than 0.1 % of dry mass, and each of these so-called macronutrients are considered essential for plants to complete their life cycle (Marschner, 2011; Maathuis, 2009). The micronutrients, boron, chloride, copper, iron, manganese and zinc are involved in the regulation of metabolic functions as cofactors of enzymes (Hänsch and Mendel, 2009). Several of these elements are redox-active that make them essential as catalytically cofactors in enzymes, others have enzyme-activating functions, and others fulfill a structural role in stabilizing membrane proteins (Kirkby and Römheld, 2007). In pears, appropriate concentrations of macro- and micronutrients are essential for postharvest quality and storage ability. Optimum N concentration in pear fruit allows a proper development of skin color, fruit size and flavor, however, excess of this macronutrient induces large fruit size, lower firmness and increased susceptible to storage disorders such as core breakdown (Watkins, 2009; Brunetto et al., 2015). Excessive N in soil may delay fruit maturation turning fruit more susceptibility to pests and decay (Sugar et al., 1992; Watkins, 2009). Due to little plant requirement in P, deficiency of this macronutrient is unlikely to occur, however it has been observed that P concentration decreases toward the fruit center (Faust et al., 1969) and pear fruit with low P may have high incidence of senescent breakdown and low

9 temperature damage (Watkins, 2009). Higher Mg and K concentrations are normally associated the development of storage disorders in ‘Anjou’ pear (Curtis et al., 1990) and may also increase the occurrence of decay (Watkins, 2009). The role of Ca and others minerals in apples are well known (Bangerth, 1979; Ferguson et al. 1999; De Freitas and Mitcham, 2012), however, in pear the information regarding this nutrient is less abundant, mainly regarding to internal storage disorders. Ca is one of the most studied nutrient in pome fruits, mainly in apples, in relation to the incidence of various pre- and postharvest physiological disorders. Calcium is involved in many reactions and cell structure, but it plays important roles in metabolism like pH regulation, signaling molecule in the cytosol, and cell wall and middle lamella structure of cells (Bangerth, 1979; De Freitas and Mitcham, 2012; Brunetto et al., 2015). Preharvest treatment with Ca sprays was shown to diminish the occurrence of senescent breakdown and flesh browning in pears (Curtis et al., 1990; Watkins, 2009). Reuscher et al. (2014) show that B and Ca play an important role in cell wall formation, and in the case of ‘La France’ pear lower concentrations of these elements were positively associated to the cork spot occurrence. Xuan et al. (2003 and 2005) investigating preharvest B sprays found very interesting results regarding internal browning in ‘Conference’ pear. The beneficial effect of B was shown in the reduction of the membrane permeability, respiration rate, preventing the occurrence of internal browning during long-term of CA storage of ‘Conference’ pear (Xuan et al., 2003 and 2005).

1.8. Mechanisms of internal storage disorders Despite the continuous and relatively rapid advances in postharvest technologies and the development of many biochemical, physiological and transcriptomic laboratory protocols in the last years, the exact mechanism of internal disorders in apples and pears during CA storage is not clear. Low pO2 or high pCO2 under CA storage affect differently the metabolism of stored fruit (Kader, 1995). Investigations involving gas diffusion properties in ‘Cox Orange Pippin’ (Rajapakse et al., 1990) and ‘Braeburn’ apples (Dražeta et al., 2004) as well as in ‘Hosui’, ‘Kosui’ and ‘Conference’ pears (Ho et al., 2010); the antioxidant potential and cell defense system (Franck et al., 2003; Silva et al., 2010) as well as the role of macro- and micronutrients (Bangerth, 1979; Curtis et al., 1990; Xuan et al., 2005) and the fermentative metabolism (Smagula and Bramlage, 1977; Saquet and Streif, 2006; Saquet and Streif, 2008) have been carried out to address the mechanism of internal disorders development. However, they cannot explain the exact point of origin of the cascade effects leading to internal disorders

10 in apples and pears during CA storage. The delay in the establishment of CA conditions (Höhn et al., 1996) showed positive results in alleviating internal disorders in ‘Conference’ pear. The effectiveness of this procedure was further confirmed and used at biochemical level in ‘Conference’ pear (Saquet et al., 2001), ‘Braeburn’ (Saquet et al., 2003a) and ‘Elstar’ apples (Streif and Saquet, 2003) to investigate factors, which could be related to the origin of internal disorders. Studies on the activity of the TCA cycle enzyme succinate dehydrogenase and the respective accumulation of succinate in pome fruit under CA storage more than 40 years ago showed the effect of high pCO2 in inhibiting the enzyme activity (Knee, 1973; Shipway and Bramlage, 1973). Many other key enzymes of the glycolysis (Embden-Meyerhof-Parnas Pathway), TCA cycle and of the respiratory electron transport chain have been shown to be affected at different manner by changing pO2 and pCO2 during CA storage (Solomos, 1982; Kerbel et al., 1988; Hess et al., 1993; Stanley, 1991; Solomos, 1997; Lange and Kader, 1997). Mellidou et al. (2014) conducted a very detailed transcriptomic study about events associated with internal disorders in apples during postharvest storage and concluded that there are several alterations in metabolic pathways including a repression of the TCA cycle as well as the up-regulation of the electron transport chain and fatty acids oxidation. However, taken all these results in account the mechanism of internal storage disorders remains to be fully explained. More than confirming the beneficial effects of the delay in the establishment of CA conditions in apples and pears (Höhn et al., 1996; Saquet et al., 2001; Saquet et al., 2003a; Streif and Saquet, 2003, Verlinden et al., 2002), consistent results were found in three biochemical research fields during CA storage of ‘Conference’ pear, ‘Jonagold’, ‘Braeburn’ and ‘Elstar’ apples: a) the energy metabolism, monitored mainly by changes in the adenylate energy charge (Saquet et al., 2000; Veltman et al., 2003); b) the membrane lipid composition in fruit flesh and respective alterations during storage (Saquet et al., 2003a and 2003b); and c) the fermentative metabolism monitored by the ethanol and acetaldehyde accumulation (Saquet and Streif, 2006, 2008). The lactate fermentative pathway was measured during CA storage of ‘Conference’ pear (Saquet and Streif, 2006) and ‘Jonagold’ apple (Saquet and Streif, 2008), however it was shown no relevant role in this process. When carefully analyzed, the behavior of ‘Jonagold’ apple and ‘Conference’ pear, stored at 1 kPa O2 plus 3 kPa CO2 and mainly under 0.5 kPa O2 plus 6.0 kPa CO2, it can be observed that in ‘Conference’ pear the energy charge, expressed by the ATP:ADP ratios was significant lower than in ‘Jonagold’ apple (Saquet et al., 2000). Furthermore, AEC in tissue of

‘Jonagold’ apple increased continuously during six months CA storage (Saquet et al., 2000). In both investigated pome fruits, this trend was confirmed by an increase in the glycolytic rate measured by higher activities in the enzymes alcohol dehydrogenase and pyruvate decarboxylase with concomitant accumulation of ethanol and acetaldehyde contents, respectively (Saquet and Streif, 2006; 2008). However, the contents of ethanol and acetaldehyde in ‘Jonagold’ apple corresponded to only 13 and 42%, respectively, when compared with the same compounds measured in ‘Conference’ pear fruits under the same CA conditions (Saquet and Streif, 2006; 2008). These results indicate that ‘Jonagold’ apple can maintain the glycolytic flux, but with moderate accumulation of ethanol and acetaldehyde at non-toxic levels and at low levels, which could not change significantly the membrane fluidity and permeability expressed in this case by measurements in electrolyte leakage (Saquet, 2001).

The electrolyte leakage in ‘Jonagold’ apple increased only 14 % even under 0.5 kPa O2 plus

6.0 kPa CO2 from the harvest time to the end of storage period and no significant difference between all CA-conditions were found, while in ‘Conference’ pear it increased 60 % starting to increase already from the second month of storage period (Saquet, 2001). Observing the lipids and their respective fatty acids of cell membranes in the fruit flesh of ‘Conference’ pear, it was found a continuous increase in myristic, palmitic and stearic free fatty acids during storage time indicating the lipid hydrolysis from cell membranes (Saquet, 2001). The free myristic fatty acid increased about 50 % and the stearic acid 100 % after six months CA storage of ‘Conference’ pear. On the other hand, the contents of myristic, palmitic, linoleic and linolenic acids of the polar lipids in tissues of pear fruits decreased continuously, while the same fatty acids in fruit flesh of ‘Jonagold’ apple were either not affected or even increased during storage period (Saquet, 2001). Investigations studying the effectiveness of the delay in the establishment of CA- conditions in ‘Braeburn’ (Saquet et al., 2003a) and ‘Elstar’ (Streif and Saquet, 2003) apples, and in ‘Conference’ (Saquet et al., 2003b) pear reinforced the involvement of the AEC and lipids of cell membranes in this mechanism. In this situation, delayed-stored apple and pear fruits had higher AEC and higher contents of lipids concomitantly with lower occurrence of internal disorders during CA storage. This overview of energy metabolism in pome fruits together with data from other plant cells, tissues or organs under severe hypoxia or total anoxia conditions confirm, at least partially, that the biochemical mechanism for internal disorders in pome fruits during long-term CA storage is associated to energy and membrane lipid metabolism as well as the fermentative metabolism.

Maize (Zea mays) (Saglio et al., 1988; Xia and Saglio, 1992; Bouny and Saglio, 1996) and Trifolium subterraneum roots (Aschi-Smiti et al., 2003) showed stable or even increasing

AEC during hypoxic conditions of 1.5 kPa O2. Pfister-Sieber and Brändle (1995) showed that the total adenine nucleotide pool of normoxic potato tubers remained constant over a 3-d period, while under hypoxia at 1 kPa O2, a continuous decrease in the adenylates took place at the beginning of the treatment. Intermediate adenylate energy charge at a value of 0.6 was reached, whereas in air control tissues were measured energy charge of about 0.85 or higher.

Low pO2 triggers responses to decrease the need for oxygen consumption and increase the efficiency of oxygen use (Geigenberger, 2003). Under hypoxic conditions, the TCA cycle pathway gives more flexibility to the overall metabolism (Toro and Pinto, 2015). In Lotus japonicas, alanine aminotransferase generates a link between glycolysis and the TCA-cycle through the conversion of 2-oxoglutarate to succinate. This generates NADH that is used in the transformation of oxaloacetic acid into malate; together with succinate CoA ligase, both contribute to the generation of ATP under oxygen deficit conditions (Geigenberger, 2003).

Under some conditions, when the availability of molecular O2 is reduced, adaptation to stress may occur through two strategies: the first is a decrease in ATP consumption, which leads to a metabolic crisis at cellular level (Igamberdiev et al., 2010), and the second is characterized by an increase in the glycolytic flux (Mancuso and Marras, 2006). The latter consists of a progressive acceleration in carbohydrate metabolism, that allows the plants to maintain their energy level, especially during the early phase of acclimation to oxygen deficiency (Greenway and Gibbs, 2003; Camacho-Pereira et al., 2009). Another mechanism to compensate the severe ATP deficiency in plants under hypoxia is the induction of alternative pathways, which can use inorganic pyrophosphate (PPi) instead of ATP for phosphorylation reactions (Mustroph et al., 2014). Weiner et al. (1987) were the first to suggest, that pyrophosphate could operate as a secondary energy donor in the cytosol of plant cells. Transcriptomic and proteomic studies using anoxia tolerant rice and anoxia intolerant Arabidopsis, have provided evidence for the selective adoption of PPi over ATP as high energy donor molecules, which may contribute to anoxia tolerance in these kind of plants (Huang et al., 2008). According to Mustroph et al. (2014), this mechanism is considered one potential strategy to cope with this energy crisis to the induction of enzymes that use PPi as the energy source as an alternative to ATP using enzymes. However, according to the same authors, this field remains not fully understood and further investigation is need. In potato cells under severe hypoxic or anoxic conditions it was possible to establish a threshold for ATP concentrations below where the hydrolysis of membrane lipids occurred

(Rawyler et al., 1999; Rawyler et al., 2002). Crawford and Brändle (1996) reported, that polar lipids in hypoxia tolerant Acorus calamus remained stable and the free fatty acids increased only slightly during pO2 deprivation stress, while the polar lipids fraction in Iris germaniaca, a susceptible specie to low pO2, decreased strongly, and at the same time, the contents of free fatty acids increased significantly. The need of molecular oxygen for fatty acids biosynthesis is well known as was reported early by Brown and Beevers (1987) in rice coleoptiles, where under aerobic conditions the amounts of total fatty acid, phospholipid, and total lipids per coleoptile increased by 2.5- to 3-fold between days three and seven, whereas under anoxia, the increases were all less than 60 %. The total content of phospholipids in coleoptiles exposed to 7 days of anoxia represented only 25.8 % of the total phospholipids of coleoptiles exposed the same time in air. The fatty acids of the phospholipids of rice coleoptiles most affected by 7 days of anoxia were the stearic, oleic and linoleic acids.

Aim and outline of the thesis

The aim of this doctoral thesis was to advance the understanding of ripening physiology and biochemistry of ‘Rocha’ pear and to determine the conditions for long-term controlled atmosphere storage of this pear cultivar after the ban of diphenylamine.

Specific objetives were: 1. To assess the concentrations and the distribution of andenylate nucleotides and the adenylate energy charge whitin ‘Rocha’ pear fruit; 2. To characterize the physiology and biochemistry of ripening of ‘Rocha’ pear; 3. To assess and compare the sensory and instrumental quality in ripening ‘Rocha’ pear as affected by temperature and ethylene inhibition; 4. To carry out a detailed mapping of macro- and micronutrients in ‘Rocha’ pear, and investigate their possible relationship to internal disorders development during long- term CA storage; 5. To investigate the possible involvement of adenylate nucleotides and energy charge in the development of internal disorders during CA storage of ‘Rocha’ pear; 6. To investigate the effectiveness of a delayed CA storage procedure in alleviating the occurrence of internal disorders during long-term CA storage of ‘Rocha’ pear.

The general introduction (Chapter 1) presents the significance of the research questions and summarizes the current knowledge regarding ripening physiology of pears and storage conditions for their preservation. As proposed by Saquet et al. (2000) and Veltman et al. (2003b) the cellular energy level is an indicator of cellular homeostasis and may be involved in the development of storage disorders in pears. An examination of the gradients of adenylate nucletides within the pear fruit is presented in Chapter 2. After this preliminary analysis of adenylate nucleotides within the fruit, changes in each nucleotide and in energy charge during ripening in relation to respiration rate and ethtlene biosynthesis was investigated (Chapter 3). Sensory and instrumental analyses were carried out following to investigate the sensory profile in ripening ‘Rocha’ pear as affected by partial ethylene inhibition (Chapter 4). Applied storage trials begun with the study of the macro- and micronutrients within the pear fruit and after 5 months CA storage period to investigate the possible involvement of mineral nutrients during internal storage disorders development (Chapter 5). The storability of ‘Rocha’ pear

15 under extreme low 0.5 kPa O2 and 1-MCP treatment was investigated and the results presented and discussed in Chapter 6. Another storage trial addressed the effectiveness of delayed CA storage procedure in alleviating the development of internal storage disorders (Chapter 7). The workflow underlining the current thesis and its structure is represented in Fig. 1.

Fig. 1. Schematic representation of the thesis workflow and structure.

Chapter 2

Mapping the gradients of adenylate nucleotides and energy charge within ‘Rocha’ pear fruit

Abstract: Cellular energy status is central for metabolic regulation. Pear fruit is stored for several months and its fruit anatomy, surface-to-volume ratio, and gas exchange properties may result in uncharacterized gradients in adenylate nucleotides. The aim of this work was to characterize the axial and radial gradients of adenylate nucleotides and AEC in pear fruit. ‘Rocha’ pear (Pyrus communis L.) fruit with 55-60 mm were sampled after 3 months under controlled atmosphere storage, in a mature but unripe stage. Fruit were sectioned transversely in skin (1.5 mm thick), outer flesh (10 mm thick under skin), and inner flesh (10 mm thick under outer flesh) and longitudinally in three-thirds, proximal, medial, and distal. The sections were freeze-dried and ATP, ADP and AMP measured by bioluminescence. AEC was calculated as: AEC = [[ATP] + 0.5 [ADP]] / [[ATP] + [ADP] + [AMP]]. A radial AEC gradient was measured in the fruit but no significant gradient was observed from the proximal to the distal fruit sections. Total pool of nucleotides, on a fresh mass basis, was 865.7 nmol g-1 in the skin, 438.1 nmol g-1 in the outer flesh, and 585.6 nmol g-1 in the inner flesh. AEC was 0.80, 0.72, and 0.69 in the skin, outer, and inner flesh, respectively. AMP concentrations were less than 11 % of the total nucleotide pool. ATP accounted for 70, 53, and 46 % of the total adenylate nucleotides, in the skin, outer, and inner flesh, respectively. In conclusion, there was variation in AEC in the radial direction, with lower values at the fruit center. Whether this transversal gradient is related to susceptibility of internal browning disorders remains to be clarified.

Keywords: ATP, ADP, AMP, fruit quality, Pyrus communis L.

Introduction Cell metabolism comprise changes in chemical energy among metabolites. Energy released by catabolic reactions is stored in the phosphate bonds of adenylate nucleotides, which can then supply energy to enable thermodynamically unfavorable reactions (Geigenberger et al., 2009). Three adenylate nucleotides are interconverted to store and release chemical energy: ATP, ADP and AMP. The nucleotides are composed of an adenine base attached to a ribose sugar. These adenylate nucleotides are linked to 3, 2 or 1 phosphate groups in ATP, ADP, and AMP, respectively. High-energy phosphoanhydride bonds in the adenylate nucleotides pool store and release energy and AMP, ADP, and ATP are interconverted in biochemical reactions (Haferkamp et al., 2011). Cells require energy to maintain homeostasis and to drive anabolic processes involved in growth, development, and defense from biotic and abiotic stresses (Geigenberger et al., 2009). In harvested fruits, ATP is generated in mitochondria via oxidative phosphorylation, in the tricarboxylic acid cycle, and by the glycolytic pathway in cytoplasm (Fernie et al., 2004; Sweetlove et al., 2010). The energy status of a cell can be quantified by its AEC. The AEC is defined as ([ATP] + 0.5 [ADP]) / ([ATP] + [ADP] + [AMP]) and its value range from 0 to 1 (Atkinson and Walton, 1967). These authors argued that all three nucleotides should be considered to account for the energy status in cell metabolism, rather than ATP and ADP alone. Several enzymes involved in energy metabolism are regulated in opposite ways by ATP and ADP or by ATP and AMP, and ratios between nucleotides are important for metabolic regulation. The AEC of most cells under normal conditions ranges from 0.8 to 0.95 (Atkinson and Walton, 1967). However, AEC can decrease to lower values under increased demand for ATP or under conditions that impair ATP-generating processes like starvation or low oxygen partial pressures (Saquet et al., 2003; Geigenberger et al., 2009). Since ATP-generating pathways are inhibited by a high AEC (Atkinson, 1968), anabolic processes are favored in cells with a high energy charge. Pear fruit is a pome composed of a core, flesh, and skin. Different fruit structures are likely to have metabolic differences that affect the adenylate nucleotide pool. Moreover, resistance to oxygen diffusion in the fruit may limit respiratory activity (Ho et al., 2006; 2009; 2010) and, therefore, the energy charge of tissues. In support of this assumption, ATP and ADP concentrations are higher in the skin than in the flesh of ‘Golden Delicious’ apple (Tan, 1999). Energy charge is likely to affect internal disorders of pear (Saquet et al., 2000; Saquet et al., 2003; Veltman et al., 2003) and the fruit’s ability to maintain homeostasis during long-term

18 storage. Therefore, adequate sampling for studies of the adenylate nucleotide pool require an understanding of their gradients within the fruit. However, the distribution of adenylate nucleotides and AEC within a pear fruit is unknown. The aim of this study was to characterize the axial and radial gradients of adenylate nucleotides and AEC within a pear fruit to support sampling decisions for further studies on the energy status of pear during ripening or storage.

Material and methods

Plant material and sampling ‘Rocha’ pear (Pyrus communis L.) fruit were harvested at commercial maturity from an orchard in Bombarral, Portugal, in August 2014. Fruit with 55-60 mm diameter were stored for

3 months at -0.5 °C and 1.0 kPa O2 + 0.7 kPa CO2 and subsequently sampled for analyses. Pear fruit were sectioned transversely in skin with 1.5 mm thick, outer flesh with 10 mm thick under the skin, and inner flesh with 10 mm thick under outer flesh. Fruit were also sectioned longitudinally in three-thirds: Proximal, medial, and distal. The fruit sections were immediately frozen in liquid nitrogen, freeze-dried at -50 °C and -100 kPa, and stored at -30 °C until nucleotides analyzes.

Extraction and measurement of adenylate nucleotides Extraction and assessments of ATP, ADP and AMP were performed as described by (Saquet et al., 2003a). Lyophilized and powdered tissue samples (1 g) were placed in 10 mL of TCA at 5 % and EDTA at 2 mM solution and extracted during 30 min on ice. The samples were then centrifuged under refrigeration (4 °C) at 21,000 g for 30 min. A 0.1-mL aliquot of supernatant was diluted 30 times with Tris (hydroxymethyl aminomethane)-EDTA buffer at pH 7.75. The reaction mixture to determine ATP was composed by 10 µL extract, 40 µL ATP monitoring reagent (AMR) (BioThema AB, Handen, Sweden) and 150 µL of Tris-EDTA buffer (2 mM, pH 7.75). The bioluminescence was measured with a multi-mode reader (Model Synergy II, BioTek, Winooski, USA). An internal standard of 10 µL of ATP at 2 µM was fed, and the luminescence recorded again in each sample. Before measurement, ADP was converted to ATP by incubating the sample with PK. Samples were incubated with PK at 120 U mL-1 in PEP buffer for 30 min at 25 °C. Total ATP concentration was determined and ADP concentration calculated by difference. AMP was converted to ADP by incubation of the

19 samples with a mixture of myokinase (MK, 180 U mL-1 in PEP buffer), which in turn was converted to ATP by PK. Nucleotide concentrations are expressed on a fresh mass basis. AEC was calculated from the concentration of ATP, ADP, and AMP as AEC = ([ATP] + 0.5 [ADP]) / ([ATP] + [ADP] + [AMP]) (Atkinson, 1968).

Results and discussion Adenylate nucleotide gradients within a pear fruit Gradients in adenylate nucleotides were observed in the radial direction (Table 1), but not in the longitudinal direction (Table 2).

Table 1. Adenylate nucleotides and AEC in the radial section of the fruit. Values are means (SD), n=4.

Adenylate nucleotides (nmol g-1 FM) in the radial section of the fruit Nucleotide or AEC Skin Outer flesh Inner flesh ATP 604.4 (61.5) 231.4 (71.6) 267.1 (38.7) ADP 167.8 (37.4) 169.6 (45.6) 272.1 (42.3 AMP 93.5 (11.6) 37.0 (6.1) 46.4 (10.3) Total pool 865.7 438.1 585.6 AEC 0.80 (0.01) 0.72 (0.05) 0.69 (0.05)

The total pool of nucleotides was 865.7 nmol g-1 in the skin, 438.1 nmol g-1 in the outer flesh, and 585.6 nmol g-1 in the inner flesh (Table 1). ATP accounted for 70.53, and 46.0 % of the total adenylate nucleotides, in the skin, outer, and inner flesh, respectively. AMP concentration was less than 11 % of the total adenylates pool. AEC decreased along the transversal section of the fruit, from 0.80 in the skin to 0.69 in the inner flesh.

Table 2. Adenylate nucleotides and AEC in the longitudinal section of the pear fruit. Values are means (SD), n=4.

Adenylate nucleotides (nmol g-1 FM) in the axial section of the fruit Nucleotide and AEC Proximal Medial Distal ATP 410.2 (67.1) 368.2 (64.2) 498.5 (69.3) ADP 463.7 (72.3) 253.6 (60.9) 151.4 (59.8) AMP 29.9 (6.8) 80.5 (18.3) 84.4 (8.3) Total pool 903.9 702.4 734.4 AEC 0.71(0.07) 0.71 (0.06) 0.78 (0.04)

The sections along the proximal to the distal axis of the fruit combined skin and flesh. Assuming that the skin has higher ATP concentration and AEC than the flesh (Table 1), the results obtained in composite sample of skin plus flesh is likely to underestimate the actual gradient along the longitudinal axis. However, given the relatively small proportion of skin to flesh (ca. 10 %) the comparative results are likely to reflect the actual concentrations in the flesh.

Adenylate nucleotide gradients in harvested fruit There is little information on the distribution of adenylate nucleotides and AEC within fruits. ‘Golden Delicious’ apple has higher concentration of ATP and higher ratios of ATP to ADP in the skin than in the flesh (Tan, 1999). Although the author did not map the gradients in adenylate nucleotides in the fruit, the report is consistent with the results presented herein for ‘Rocha’ pear. In melon under hypoxia conditions, AEC was 0.97 in the outer mesocarp and 0.83 in inner tissues of the mesocarp (Biais et al., 2010). In melon, the total pool of adenylate nucleotides was higher in the pericarp, lower in the outer flesh and increased in the inner of fruit flesh. Similarly, in potato tubers, a bulky plant organ, ATP concentration and AEC was lower at the center than in the outer cortex. The center of the tuber had AEC between 0.45 and

0.60 with pO2 ca. 5 kPa whereas AEC in aerobic tissues at tuber periphery ranged from 0.75 to 0.85 (Geigenberger et al., 2000). Lower ATP concentration and AEC have been reported in the inner flesh of ‘Hass’ avocado (Lange and Kader, 1997), ‘Jonagold’ apple and ‘Conference’ pear (Saquet et al., 2000), ‘Golden Delicious’ apple (Tan and Bangerth, 2001) and ‘Regina’ sweet cherry (Harb et al., 2006). Taken together, these results suggest that AEC decreases from the outer to the inner cell layers in fleshy fruit and other bulky organs, such as potato tuber.

Radial pO2 gradient in these organs are associated with the AEC gradient. Resistance to gas diffusion and respiratory consumption of molecular oxygen create steep oxygen gradients in fruits and in potato tuber (Dadzie et al., 1996; Geigenberger et al.,

2000). These gradients in pO2 from the skin to the center of fruits have been shown in apple cultivars (Rajapakse et al., 1990; Drazeta et al., 2004a), in Asian pears (Rajapakse et al., 1990) and in European pears (Ho et al., 2006; 2010). The low pO2 at the core of pear fruit (Ho et al., 2006; 2010) partially explains the lower AEC in the inner flesh measured in ‘Rocha’ pear (Table 1).

Implications of energy gradients to storage disorders Internal physiological disorders, such as core and flesh browning, and cavities occur in pear during long-term CA storage (Streif et al., 2003). Internal browning disorders in ‘Rocha’ pear under CA storage start at the fruit core or inner flesh at the widest fruit section, the region with lower AEC (Table 1). The lower concentrations of ATP and lower AEC at the fruit core (Table 1) and the decrease in AEC during storage (Saquet et al., 2000; 2001; 2003; Veltman et al., 2003) are consistent with the location of the disorders in the fruit and their occurrence during the storage period. Although the mechanism(s) of internal browning disorders are not fully understood, the AEC of the fruit tissues is likely to play a role in their development. Low AEC shifts metabolic regulation toward catabolism and limited availability of chemical energy can hinder membrane repair leading the irreversible development of browning symptoms. Interestingly, low ATP concentrations are associated with increased membrane permeability in plant cells (Trippi et al., 1996) and a minimum ATP concentration is required to maintain membrane integrity (Rawyler et al., 1999).

Conclusion A radial gradient in ATP concentration and AEC exists in the pear fruit, decreasing from the skin toward the inner flesh, but no significant gradient exists along the longitudinal axis. Tissue sampling should take into account the gradient in adenylate nucleotides and AEC.

Chapter 3

Ripening physiology and biochemistry of ‘Rocha’ pear as affected by ethylene inhibition

Abstract: Pear (Pyrus communis L.) is a climacteric fruit whose ripening behavior is highly cultivar-dependent. This study investigates the postharvest ripening physiology and biochemistry of ‘Rocha’ pear treated with 1-MCP. Fruit from a single orchard harvested at the mature-green stage were treated with 0 (control), 150, and 300 nL L-1 1-MCP, and allowed to ripen in air at 20 °C. ‘Rocha’ pear without exposure to chilling ripened with a typical pattern of ethylene production and respiration rates. Inhibition of ethylene action by 1-MCP delayed the ethylene production peak and reduced its intensity by 60 %. ATP concentration in control fruit was maximum after 7 d and declined toward the end of the ripening period. 1-MCP induced a transient reduction of ATP concentrations and AEC during ripening. AEC of control fruit increased slightly during the first week and decreased to a stable value of 0.7 toward the end of ripening. Skin color and firmness of control fruit changed faster during the first week of ripening. 1-MCP induced a delaying in softening, in yellowing and in electrolyte efflux changes, but not impaired the ripening progress. In conclusion, ‘Rocha’ pear ripened immediately after harvest without chilling treatment or exogenous ethylene application and adjusted the AEC during ripening even with lower respiration rates induced by 1-MCP.

Keywords: ATP, ADP, AMP, adenylate energy charge, respiration, Pyrus communis.

Introduction European pear (Pyrus communis L.) cultivars exhibit a climacteric pattern of respiration and ethylene production (Villalobos-Acuña and Mitcham, 2008). Therefore, several ripening- related processes in pear are regulated by ethylene, including changes in skin color, flesh texture and aroma volatiles emission (de Wild et al., 1999; Hiwasa et al., 2004). Despite this common trait, pear cultivars differ widely in their ripening behavior. Pear cultivars are often grouped into two classes (Villalobos-Acuña and Mitcham, 2008): (i) Winter pears, which require extended exposure to chilling temperatures to induce autocatalytic system II ethylene biosynthesis, are often slow-ripening and suitable to long-term storage, and (ii) Summer pears, which require little chilling exposure for ethylene production and are generally fast-ripening with a shorter storage life. Ripening has been characterized in the main pear cultivars. Ripening-related changes in pear include a burst in ethylene production and the climacteric increase in respiration rate (Biale, 1960) followed by an increase in aroma volatiles production (Shiota, 1990). Increased protein synthesis (Frenkel et al., 1968), namely cell wall enzymes (Ben-Arie et al., 1979) lead to solubilization and depolymerization of cell wall polysaccharides (Ahmed and Labavitch, 1980) and loss of neutral sugars (Gross and Sams, 1984). Chlorophyll degradation (Kräutler, 2008) lead to color changes. All these metabolic processes require chemical energy produced by respiration and stored in the adenosine nucleotide pool (ATP, ADP, AMP). The changes in equilibrium among the three adenylate nucleotides has not been fully investigated in ripening pear, although data on ATP changes have been published (Watkins and Frenkel, 1987). Unlike most climacteric fruit, mature-green pears do not ripe immediately after harvest. Exposure to chilling temperatures are required to induce autocatalytic ethylene biosynthesis and the climacteric rise in respiration rate associated with ripening. This chilling requirement can be replaced by exogenous ethylene. Although the availability of ACC, the substrate for ACC-oxidase, seems to be the limiting factor of pear ripening ability (Lara and Vendrell, 1998), chilling or exogenous ethylene regulate the pool of ACC via the expression of ACC-synthase and ACC-oxidase in pear (Lelièvre et al., 1997). The chilling temperature and period required to induce the climacteric increase in ethylene biosynthesis and normal ripening varies widely among cultivars. ‘Passe Crassane’ requires a 3-month period at 0 °C (Lelièvre et al., 1997), ‘d’Anjou’ (Sugar and Einhorn, 2011) needs 60 d, ‘Comice’ (Sugar and Basile, 2006) about 30 d, and ‘Bosc’ will ripen with less than 10 d of chilling exposure (Chen and Mellenthin, 1982). ‘Bartlett’ will ripen normally after 14

24 to 21 d at -1 to 0 °C (Mitcham et al., 2006) but can also ripen without chilling treatment (Villalobos-Acuña et al., 2011a). The postharvest ripening behavior of ‘Rocha’ pear can be described as intermediate between a typical summer and a typical winter pear. ‘Rocha’ is an early maturing pear that will ripen after harvest with little exposure to chilling temperatures. However, under adequate conditions it has a long storage potential, up to ten months under controlled atmosphere storage (Almeida et al., 2016). The aim of this research was to determine whether ‘Rocha’ pear can ripen immediately after harvest without exposure to chilling or exogenous ethylene application, and investigate changes in individual adenylate nucleotides (ATP, ADP, AMP) and energy charge during ripening. The ethylene action inhibitor 1-methylcyclopropene (1-MCP) was used to investigate the effects of ethylene blockage on ripening initiation and progression.

Material and Methods

Plant material Mature-green fruit of pear (Pyrus communis L. ‘Rocha’) from a commercial orchard located in Cadaval, West Region of Portugal, were used in this study. The maturity stage at harvest was characterized in 3 replicates of 15 fruit each. Pears had an average mass of 200 g ± 24 g, starch index of 8.2 (1 to 10 scale), flesh firmness of 51.8 N, total soluble solids of 10.5 %, titratable acidity of 0.17 % expressed on malic acid equivalents, and a skin hue angle of 105.6 º.

1-MCP treatments and environmental conditions Fruit were placed inside plastic containers (250 L) equipped with a small fan to homogenize the internal atmosphere during the treatment period. Fruit storage density inside the containers was 250 kg m-3. One batch of fruit was treated with 1-MCP (SmartFreshTM, 0.14 % a.i., AgroFresh, Inc., Springhouse, PA, USA) at a dose of 150 nL L-1 and the other batch at a dose of 300 nL L-1 for 15 h at 20 ºC. Untreated control fruit were placed under the same conditions without the 1-MCP treatment. After the treatments, fruit were transferred to plastic crates lined with polyethylene film and maintained at 20 °C (± 0.5). Temperature was monitored in the storage room and inside representative fruit throughout the trial.

Starch pattern index Starch hydrolysis was assessed by the starch-iodine test as described by Almeida et al. (2016). Three samples, in 15 fruit each, were transversely cut and the proximal half surface was dipped in a solution of 1 % (w/v) iodine in 4 % (w/v) potassium iodine for 30 s, allowed dry during 5 min in air and compared with the 10-point scale developed for ‘Rocha’ pear.

Skin color Surface color was measured in CIE L*a*b* color space with a tri-stimulus CR-400 colorimeter (Konica Minolta, Tokyo, Japan). Measurements were made in the widest part of the fruit in three replicate batches of 5 fruit each. Results were expressed as hue angle, a color coordinate suitable to track color changes from the green hue of mature fruit to the yellow hue of ripe fruit (McGuire, 1992).

Flesh firmness Flesh firmness was measured after peel removal with a penetrometer (T.R. Turoni, Forli, Italy) equipped with an 8 mm probe. Firmness was measured twice in each fruit, in opposite sides at the equatorial region, in 3 replicated batches of 5 fruit each. The maximum force to insert the probe into the fruit flesh was recorded.

Total soluble solids and titratable acidity Fruit were cut in the equatorial region and a disc of 10 mm thick was used for juice extraction. TSS was measured in the juice using a digital refractometer (Hanna Instruments, Woonsocket, USA). An aliquot of 10 mL juice was diluted in 90 mL distilled water and the solution titrated with 0.1 M NaOH until pH 8.1. Change in pH were monitored with a potentiometer (JP Selecta, Barcelona, Spain). Titratable acidity was expressed as malic acid equivalents.

Electrolyte efflux Three replicates of five fruit each were used for electrolyte efflux measurements. Cylinders (10 mm in diameter and 30 mm long) were excised from the equatorial region of fruit with the axis parallel to the pear longitudinal axis. After excision, the cylinders were washed with distilled water and blot-dried in cellulose filter paper. A sample of 15 discs, each with 3 mm thick, were washed and dried in cellulose filter paper, immediately placed in 25 mL of 0.45

M mannitol solution and the initial electrical conductivity (ECi) measured with a conductivity

26 electrode (CD 2005, JP Selecta, Barcelona, Spain). The samples were incubated for 2.5 h at 25

°C in a water bath with alternative shacking at 50 rpm. Final conductivity (ECf) was measured at the end of the incubation period and the samples frozen at -30 °C. The samples were thawed and boiled for 15 min under reflux, cooled to 25 ºC, and the conductivity (ECt) measured to determine the total electrolytes. Electrolyte efflux was calculated as (ECf – ECi)/ECt ×100.

Ethylene and respiration measurement Samples of 4 fruit replicated 3 times were placed inside of 2.15 L sealed glass jars and maintained in air at 20 ºC for 2 h. A headspace volume of 0.1 mL was removed from the glass jars with a glass syringe via a rubber septum and injected into a gas chromatograph (Trace 1300, Thermo Fisher Scientific Inc., Marietta, USA) fitted with a capillary column TG bond alumina (Na2SO4) 50 m length and 0.53 mm i.d. (Thermo Fisher Scientific Inc., Marietta, USA). The injector temperature was set at 160 ºC, a FID at 180 °C, the injector temperature at 160 °C, the helium carrier at a flow rate of 15 mL min-1 and the following oven temperature program: hold time of 0.5 min at 100 °C, 20 °C min-1 to reach 180 °C, and a hold time of 0.5 min at 180 °C.

Respiration was measured in the same fruit samples used to measure ethylene. CO2 concentration was measured in the headspace with an infrared gas analyzer (Oxycarb 6, Isolcell, Laives, Italy) with continuous flow at 0.1 L min-1.

Adenylate nucleotides and adenylate energy charge The concentration of adenylate nucleotides were measured at the fruit core region. Cylinders (18 mm thick and 40 mm long) were excised from the core region of the fruit and seeds carefully removed. After excision, the cylinders were cut into 3 mm disks, immediately frozen in liquid nitrogen, and freeze-dried at -50 °C and -100 kPa. The samples were then powdered and used for extraction and measurements of adenylate nucleotides as described by Saquet et al. (2003a) with minor adaptations. Freeze-dried powder (1 g) was suspended in 10 mL of a solution with 5 % TCA, 2 mM of EDTA and homogenized. The suspensions were then extracted during 30 min on ice and centrifuged at 21,000 g for 30 min at 4 ºC. The supernatant was diluted 30 fold with Tris-EDTA buffer (0.1 M Trizma Base and 2 mM EDTA, pH 7.75) before measurements to minimize the inhibitory effects of the anionic TCA on the subsequent reaction. Three replicated extractions and measurements were performed per treatment. The individual adenosine nucleotides, ATP, ADP, and AMP were measured as follows. ATP was determined by the emission of bioluminescence from the reaction mixture composed

27 of 10 µL of extract mixed with 40 µL ATP monitoring reagent (AMR; BioThema AB, Handen, Sweden) and 150 µL of tris-EDTA buffer (0.1 M Trizma Base and 2 mM EDTA, pH 7.75) in a 200 µL wells of a microplate. The bioluminescence of this reaction was measured at room temperature (ca. 25 °C) with a Synergy 2 multi-mode reader (BioTek, Winooski, USA) adjusted with the sensitivity 200 and wavelength of 590 nm. After measurement of each sample, 10 µL of an ATP internal standard of 2 µmol L-1 was fed and the bioluminescence recorded again. ADP was converted into ATP by incubation with PK (EC 2.7.1.40, Sigma-Aldrich, St. Louis, MO, USA) at 120 U mL-1 in a PEP buffer for 30 min at room temperature (ca. 25 °C). Total ATP concentration was assayed as described above and ADP calculated by difference. AMP was converted to ADP and ADP converted to ATP by incubation with a mixture of AK (EC 2.7.4.3, Sigma-Aldrich, St. Louis, MO, USA) at 180 U mL-1 and PK (120 U mL-1) in PEP buffer for 30 min at room temperature (ca. 25 °C). After this incubation time, ATP was measured and AMP calculated by difference. The concentration of adenylate nucleotides are expressed on a fresh mass basis. The AEC was calculated as ([ATP] + 0.5 [ADP]) / ([ATP] + [ADP] + [AMP]) according to (Atkinson and Walton, 1967).

Data analysis Data were subjected to one-way ANOVA with 1-MCP treatment as a fixed factor, according to a completely randomized design using the add-on Action (2014) for Microsoft Excel.

Results

Ethylene production and respiration rates ‘Rocha’ pear showed a typical climacteric pattern of ethylene production and increase in respiration during the ripening period. In control fruit, the maximum level of ethylene (14.7 µg -1 -1 -1 - kg h ) occurred after 19 d at 20 ºC (Fig. 1 A), and the maximum CO2 release (26.8 mg kg h 1 ) was detected at the same time (Fig. 1 B). Treatment with 1-MCP reduced ethylene and CO2 production rates in a dose-dependent manner (Fig. 1 A and B). In addition, 1-MCP delayed the onset of increase in ethylene production, but the dose had no effect on the time frame (Fig. 1 A). 1-MCP reduced, therefore, the respiration rate in relation to untreated fruit by 39.7 % at 150 nL L-1 and by 55.6 % at 300 nL L-1 at day 19 (Fig. 1 B).

Fig. 1. Ethylene production (A) and CO2 release (B) during ripening of ‘Rocha’ pear fruit. Vertical bars are SD, n=3.

Adenylate nucleotides and energy charge of ‘Rocha’ pear ATP concentration at harvest was of 906.4 nmol g-1 (Fig. 2 A). In control fruit, ATP concentration increased slightly during the first 8 d of ripening at 20 ºC, decreasing afterward as ripening progressed. ADP concentration, initially 694.5 nmol g-1, decreased continuously during ripening (Fig. 2 B). AMP accounted for about 7.5 % of the total adenylate pool and its initial concentration (120.2 nmol g-1) remained relatively stable during ripening of control fruit (Fig. 2 C). Partial inhibition of ethylene action by 1-MCP changed the partitioning of the adenylate nucleotides (Fig. 2). In 1-MCP-treated fruit, ATP concentration decreased by 54 % in the first 8 d of ripening, increased thereafter to the initial concentration, to decline again toward the end of ripening (Fig. 2 A). No dose effect of 1-MCP was observed on ATP concentrations (Fig. 2 A). ADP concentrations of 1-MCP treated pear declined in a manner similar to that of control fruit (Fig. 2 B). AMP concentrations increased in 1-MCP-treated fruit, at both doses, reaching the maximum at the day eight and decreased afterward until end of ripening (Fig. 2 C), but remained more than twice (142.6 to 172.2 nmol g-1) the concentration of control fruit (72.8 nmol g-1).

Fig. 2. Concentrations of ATP (A), ADP (B) and AMP (C) during ripening of ‘Rocha’ pear at 20 °C. Vertical bars are SD, n=3.

AEC remained relatively stable at about 0.7 during the full ripening period of ‘Rocha’ pear with a peak of 0.77 at day seven (Fig. 3 A). In 1-MCP treated fruit, a decrease of AEC to 0.55 was observed during the early days followed by a further recovery to 0.71 as ripening progressed (Fig. 3 A). The total pool of adenylate nucleotides (Fig. 3 B) also changed during ripening, decreasing after 8 d in control fruit. In 1-MCP treated fruit, total adenylates decreased at the

30 beginning of ripening, increased after 7 d to decrease again at the end of ripening period. This change pattern on the total pool of adenylates was not affected by 1-MCP dose (Fig. 3 B).

Fig. 3. Adenylate energy charge (A) and total pool of adenylates (B) during ripening of ‘Rocha’ pear. Vertical bars are SD, n=3.

Starch hydrolysis The fruit contained starch at harvest, as indicated by the score 8.2 in the 1 to 10 scale for starch index, but the starch disappeared after 7 d of ripening at 20 ºC (data not shown). At this ripening stage, the starch index was 10 in control and in 1-MCP-treated fruit.

Electrolyte efflux Electrolyte efflux was 3.8 % at harvest and remained stable for 7 d at 20 ºC, after which time it increased sharply to 52.3 % in control fruit at day 19 (Fig. 4). The onset of the increase in electrolyte efflux in 1-MCP-treated fruit also occurred after 7 d, but with a trend different from that of control fruit. The efflux increased slowly initially and at a higher rate after 12 d to reach values similar to those of control fruit by day 19 (Fig. 4). The effect of 1-MCP on electrolyte efflux was similar at 150 nL L-1 or 300 nL L-1.

Fig. 4. Changes in electrolyte efflux during ripening of ‘Rocha’ pear in air at 20 °C. Vertical bars are SD, n=3.

Total soluble solids and titratable acidity TSS in control fruit increased during the ripening period, from 10.5 % at harvest to a maximum of 12.3 % by day 22 (Fig. 5 A). Treatment with 1-MCP at 150 and 300 nL L-1 did not significantly affect the final TSS contents, but slightly delayed their accumulation (Fig. 5A). TA remained in the range of 0.16 to 0.22 % with no consistent effect of 1-MCP treatment at both concentrations (Fig. 5 B).

Skin color Ripening-related color changes in ‘Rocha’ pear can be adequately described by the hue angle, as the surface color changes from green to yellow. Since the color coordinates lightness (L*) and chroma (C*) add little to the description of color changes in ‘Rocha’ pear, and hue angle provides a reliable single measure of color changes in ‘Rocha’ pear (Almeida et al., 2016), hue angle is used herein to describe color changes. Initial hue angle of ‘Rocha’ pear was 105.6 °, corresponding to the greenish hue of the mature fruit, and decreased to 82.4 º in fully ripe yellow control fruit (Fig. 5 C). 1-MCP delayed the yellowing of pear until day 12, an effect that was similar at both doses (Fig. 5 C). At the end of ripening period although the hue angles were not statistically different, 1-MCP treated fruit were visually greener than control fruit. These differences in skin coloration were due to the higher lightness values in control fruits (data not shown).

Flesh firmness Firmness decreased as the pear fruit ripened at 20 °C. The softening rate in control fruit increased from 1.6 N d-1 in the first 6 d to 4.3 N d-1 between day 6 and 12, and decreased again toward the end of ripening period (Fig. 5 D). The treatment with 1-MCP prevented softening during the initial period with no significant dose effect. Subsequent softening of 1-MCP treated fruit occurred at a similar rate to that of control fruit. No mealy texture was observed in ripe fruit.

Fig. 5. Changes in total soluble solids (A), titratable acidity (B), skin color (C) and flesh firmness (D) of ‘Rocha’ pear during ripening in air at 20 °C. Vertical bars are SD, n=3.

Discussion

Ripening of ‘Rocha’ pear in the absence of chilling or exogenous ethylene The results presented herein show that, unlike other European pear cultivars, including ‘Bosc’ (Chen and Mellenthin, 1982), ‘Passe Crassane’ (Lelièvre et al., 1997), ‘Comice’ (Sugar and Basile, 2006) and ‘d’Anjou’ (Sugar and Einhorn, 2011), ‘Rocha’ pear is able to ripen normally immediately after harvest, without exposure to chilling or exogenous ethylene application. Ripening also occurred, albeit at a slower rate, when the fruit was treated with 1- MCP without exposure to low temperature or ethylene. Therefore, ripening induction in ‘Rocha’ pear does not have a strict requirement for chilling or its replacement by exogenous ethylene. The peak of ethylene production in ‘Rocha’ pear was reached after 19 d at 20 ºC (Fig. 1 A), when fruit were very soft (4.6 N) and had high membrane permeability (52.3 % electrolyte efflux). This time to the climacteric peak and firmness end value are similar to the ripening

33 period of ‘La France’ pear with no chilling or exogenous ethylene treatment (Hiwasa et al., 2004). Symptoms of the ripening syndrome were differently affected by the ethylene inhibition of unchilled ‘Rocha’ pear. The delayed onset and reduced peak of ethylene production resulting from 1-MCP (Fig. 1 A) corresponded to a dose-dependent reduction in respiration rate (Fig. 1 B). The reduction of ethylene production by 1-MCP has been documented in several pear cultivars (de Wild et al., 1999; Ekman et al., 2004; Villalobos-Acuña et al., 2011b). Similarly, the reduction in respiration rate by 1-MCP is well documented in pears (Ekman et al., 2004; Neuwald et al., 2015a). In contrast with its effects on ethylene and respiration, 1-MCP delayed fruit softening and skin yellowing but, after the onset, these ripening events progressed similarly in 1-MCP- treated and in control fruit. The increase in electrolyte efflux was both delayed and its rate altered by 1-MCP. Ethylene inhibition reduced the initial rate of TSS accumulation (Fig. 5 A). Other ripening-related changes, namely those in starch hydrolysis and TA (Fig. 5 B) were not affected by the inhibition of ethylene action. Despite the differential change trends, fully ripe pear fruit had the same levels of TSS, TA, electrolyte efflux, skin color, firmness, and energy charge, independently of ethylene action inhibition at the beginning of ripening; however, lower ethylene production and respiration rates persisted until the end of ripening period in 1- MCP-treated fruit. The reported blockage of pear ripening-related softening and color changes by 1-MCP (Chiriboga et al., 2011) was not observed in this study with ‘Rocha’ pear even though the fruit were not cold-stored after harvest.

Adenylate nucleotides and energy charge during ripening The decrease in respiration rate induced by 1-MCP was expected to affect the pool of adenylate nucleotides and the AEC during ripening. Despite the effect of 1-MCP dose on respiration rate (Fig. 1 B), no dose effect was observed on ATP concentrations (Fig. 2 A) and AEC (Fig. 3 A). Moreover, the persistence of 1-MCP effect on respiration throughout ripening contrasts with the transient decrease in ATP concentration and in AEC in the first week of ripening of 1-MCP-treated fruit. Fruit adjusted the AEC after this initial stress period and the equilibrium among total adenylate nucleotides was similar in fruit treated with 150 or 300 nL L-1 1-MCP (Fig. 3 B). Together, these results suggest that the regulation of ATP concentrations do not depend only on respiration rates. Few studies have addressed changes in the adenylate nucleotide pool during fruit ripening, and the reported relationships between ATP concentrations and respiration rates are

34 contradictory. A close relationship between respiration rate and ATP concentrations was reported during ripening of tomato fruit (Chalmers and Rowan, 1971; Xu et al., 2012), and the relationship maintained when respiration rate was reduced by down regulation of alternative oxidase activity (Xu et al., 2012). In ripening ‘Hass’ avocado fruit, ATP concentration closely follows the climacteric pattern of respiration rate (Bennett et al., 1987) but in kiwifruit ATP concentrations remained relatively constant during ripening in contrast with a strong variation on respiration rate (McRae et al., 1992). Contrasting patterns were reported for ‘Bartlett’ and ‘Bosc’ pears (Watkins and Frenkel, 1987). In ‘Bartlett’, ATP accumulated during ripening accompanying a general increase in respiration rate, while in ‘Bosc’ ATP concentration was reported to decrease or remain relatively constant during fruit ripening, with no consistent relationship with respiration rate (Watkins and Frenkel, 1987). The expected correlation between ATP concentration (Fig. 2 A) and respiration rate (Fig. 1 B) was not observed in this study. The initial decrease in ATP concentration in 1-MCP treated fruit paralleled the lower respiration rate of fruit in the first week of ripening. However, after the first 7 d, no direct relationship could be established between changes in respiration and in ATP concentration. The few data available in the literature on ADP concentrations during ripening show a gradual decline in ADP concentrations. This decline has been observed in kiwifruit (MacRae et al., 1992), ‘Braeburn’ and ‘Jonagold’ apples (Saquet et al., 2003b; Xuan and Streif, 2008), ‘Conference’ pear (Saquet et al., 2001), ‘Regina’ sweet cherry (Harb et al., 2006) and ‘Huaizhi’ litchi (Liu et al., 2007). No reports were found on AMP changes during normal ripening of climacteric fruit. AMP concentrations are reported to decrease in ‘Conference’ pear during six months of controlled atmosphere storage (Saquet et al., 2003a) and to increase in ‘Baifeng’ peach during 35 d under chilling conditions at 0 °C (Jin et al., 2012). Similar increases in AMP concentrations were also reported in banana under 10 to 15 kPa O2 (Hill and ap Rees, 1995) and chilling conditions (Wang et al., 2015). The regulation of energy balance during ripening of climacteric fruits remains unclear. The energy needs to sustain normal ripening of ‘Rocha’ pear can be fulfilled with an AEC of 0.7 at the inner flesh. A previous study of the gradients of adenylate nucleotides within ‘Rocha’ pear revealed a clear radial gradient in ATP concentrations and AEC, decreasing from the skin to the inner fruit flesh (Almeida and Saquet, in press; Chapter 2). Pear fruit was able to maintain its AEC relatively constant at 0.7 indicating homeostatic capacity throughout ripening even under respiration rates that differ by 2.3-fold (Fig. 1 B). Plant

35 organs under hypoxia or anoxia leading to strong reductions in respiration rates adapt by decreasing ATP consumption (Geigenberger et al., 2000; Igamberdiev et al., 2010). The mechanism of energy sensing in plant cells and organs remains elusive (Wilson et al., 2006), although a response to the low energy syndrome (Tomé et al., 2014), involving transcriptomic and metabolic adjustments is emerging at the whole plant level and photosynthetic tissues. However, in non-photosynthetic excised plant organs, such as a climacteric fruit in a postharvest environment, the equilibration of energy deficiency cannot rely on photosynthesis or translocation; the mechanism of metabolic adjustment in response to energy deprivation in these situation remains unknown. The ability of the pear fruit to maintain AEC stable under ripening can be attributed to the following strategies: a) a decrease in ATP consumption within cell; b) an acceleration of the glycolytic flux to supply additional ATP; and c) the action of adenylate kinase, as proposed by Hill and ap Rees (1995). Theoretically, AEC can vary from 0 to 1, but actual values reported in plant organs are between 0.3 and 0.9 (Raymond et al., 1985; Geigenberger et al., 2000; Biais et al., 2010; Blanch et al., 2015). Our results suggest that AEC of 0.7 is the basal energy balance required to maintain the normal ripening capacity of ‘Rocha’ pear, whether treated with 1-MCP or not. This AEC is similar to that measured in the inner fruit flesh of ‘Rocha’ pear in an early season (Almeida and Saquet, in press; Chapter 2). However, inconsistencies are evident between the energy-producing respiratory metabolism and AEC. Moreover, the stability of AEC contrasts with ripening-related catabolism of membranes, plastids, and cell walls.

Major disassembly processes during pear ripening Starch hydrolysis with concomitant increase in TSS, changes in membranes leading to higher electrolyte efflux, chloroplast transition to chromoplast with associated chlorophyll breakdown and softening with the associated cell wall disassembly are the major symptoms of the ripening syndrome in ‘Rocha’ pear associated with catabolic pathways. The rapid hydrolysis of starch during the first week of ‘Rocha’ pear ripening was not affected by 1-MCP treatment or dose, indicating complete starch hydrolysis independent of ethylene inhibition (Fan et al., 1999). However, 1-MCP delayed TSS accumulation, which continued during ripening period (Fig. 5 A). Starch hydrolysis is an ATP-consuming process (Tetlow et al., 2004) that occurs at the beginning of the ripening period. Even is this energy- demanding process cannot justify the inconsistency between lower respiration rate and stable AEC in 1-MCP-treated and untreated fruit.

The sudden increase in electrolyte efflux occurred after 7 d of pear ripening at 20 ºC (Fig. 4) indicates a rapid increase in membrane permeability and possible loss of membrane integrity. This increase in electrolyte efflux and membrane permeability is a ripening-related characteristic of fruit (Marangoni et al., 1996). The delay in the electrolyte efflux in 1-MCP treated fruit is consistent with a retention of membrane integrity and retarded senescence during ripening of ‘Rocha’ pear fruit but, after the onset, membrane permeability could not be controlled by 1-MCP treatment (Fig. 4). Maintenance of plant cell membrane integrity is very demanding in energy (Rawyler et al., 1999). However, our results suggest that in ripening pear membrane leakage cannot be prevented by the ATP concentrations and AEC in fruit tissues. This is consistent with the genetically programmed changes in membrane viscosity during fruit ripening (Saltveit, 2002). Color changes related to chlorophyll degradation and chloroplast evolution to chromoplasts (Charoenchongsuk et al., 2015) as well as softening related to cell wall metabolism (Song et al., 2016) are ripening-related features regulated by ethylene and require energy for de novo synthesis of enzymes. In fact, chlorophyllase, the first enzyme in the chlorophyll catabolic pathway is regulated by ethylene (Osorio et al., 2013) as are the various cell wall enzymes, including xyloglucan endotransglucosylase, expansin, and endopolygalacturonase (Song et al., 2016). Yellowing associated with plastid transition and softening related to cell wall metabolism progressed during ripening under a constant AEC of 0.7 indicating that this energy charge was enough to sustain the complex changes involved in these processes.

Conclusions ‘Rocha’ pear ripened normally immediately after harvest without chilling exposure or exogenous ethylene application. Inhibition of ethylene action by 1-MCP delayed the initial part but not impaired the ripening process despite persistent reduction in ethylene production and respiration rate; ATP concentration and AEC were not directly correlated with the respiration rate measured during ripening, and ‘Rocha’ pear was able to adjust AEC after the initial transient reduction induced by 1-MCP treatment. An AEC of 0.7 sustained normal pear fruit ripening.

Chapter 4

Sensory and instrumental assessments of ripening changes in ‘Rocha’ pear: Effect of temperature and ethylene inhibition

Abstract: The sensory profile of pears is changing as new postharvest treatments and storage methods. The inhibition of ethylene action by 1-MCP is used in pear (Pyrus communis L.) to prevent superficial scald and delay ripening. The 1-MCP induced changes in ripening physiology is likely to alter the sensory profile of pears. This study assessed differential effect of 1-MCP and ripening temperature on physiological, physicochemical, and sensory characteristics of ‘Rocha’ pear. Fruit were stored for 30 d at -0.5 °C in air and subsequently treated with 1-MCP at 150 or 300 nL L-1. Untreated fruit were allowed to ripen at 10 ºC or 20 ºC and 1-MCP-treated fruit were ripened at 20 °C. Fruit at 10 ºC ripened at half the rate of fruit at 20 ºC as assessed by ethylene production, respiration rate, softening, and yellowing, even if the fruit at 20 ºC had been treated with 1-MCP. Fruit ripened at 10 ºC were sensory perceived as harder, greener, less juicy and less sweet. After 14 d of ripening, assessors perceived the 300 nL L-1 1-MCP-treated fruit ripened at 20 °C as juicier, sweetest and with higher flavor intensity. Instrumentally measured hue angle and firmness were significantly correlated to the sensory scores of color and hardness. However, measured total soluble solids and titratable acidity did not correlate with the sensory scores of sweetness and acidity. In conclusion, ripening of untreated fruit at 10 °C was slower than that of 1-MCP-treated pears at 20 °C. Ripening temperature had a stronger effect on the sensory profile of pear than 1-MCP treatment, which can be used to modulate the sensory profile.

Keywords: Flavor, 1-methylcyclopropene, Pyrus communis, sensory profile, texture.

Introduction The sensory profile of European pears is changing as new postharvest treatments and storage methods are being introduced in the supply chain. Pears are usually harvested in a mature-green stage and cold-stored before they ripen to a soft and juicy texture (Sugar and Basile, 2006; Villalobos-Acuña and Mitcham, 2008). The chilling requirements to hasten ripening of pears can be replaced by exogenous ethylene application (Sugar and Basile, 2006). These particular fruit ripening requirements are likely to interfere with pear responses to the technological attempts to inhibit the effects of ethylene. The inhibition of ethylene action by the postharvest treatment with 1-MCP is used to prevent superficial scald and delay the ripening process of pears during storage and poststorage shelf life. 1-MCP is very effective in reducing or preventing superficial scald, slowing softening, and delaying skin yellowing in pears (Argenta et al., 2003; Isidoro and Almeida, 2006; Folchi et al., 2014). However, an objectionable ripening blockage is sometimes reported in pears treated with 1-MCP (Chiriboga et al., 2011). In extreme instances, 1-MCP-treated pears do not soften, a condition described as evergreen fruit (Chiriboga et al., 2011). A consistent effect of 1-MCP in pears is the retention of firmness during storage and slower softening during subsequent shelf life (Isidoro and Almeida, 2006; Almeida et al., 2016) and a reduction in respiration rate (De Wild et al., 1999; Kubo et al., 2003; Saquet and Almeida, 2017), not always associated with higher fruit acidity (Moya-León et al., 2006; Saquet and Almeida, 2017). Due to these effects on mechanical properties and on fruit composition, 1- MCP alters the sensory profile of pears. However, conflicting effects of 1-MCP on sensory profiles and consumer acceptance are reported. Cluster analyses of sensory scores clearly discriminate 1-MCP-treated ‘Abbé Fétel’ pear from control fruit whose sensory profile was preferred by assessors (Rizzolo et al., 2014). In contrast, ripe ‘Bartlett’ pear that had been treated with 1-MCP were preferred to ethylene-treated or untreated pear; this preference was attributed to the higher sweetness, juiciness, the typical pear aroma, lower emission of fermentation-related volatiles, gritty texture, and tart taste of 1-MCP-treated pears (Escribano et al., 2016). Addressing the effect of 1-MCP on fruit sensory perception is challenging. Once ripening is blocked or delayed by the inhibition of ethylene action, a comparison at the same sampling date implies differences in ripening stage. Almeida and Gomes (2009) resorted to temperature conditioning to uncouple the effect of 1-MCP from that of the ripening stage per se in kiwifruit. These authors showed that kiwifruit treated with 1-MCP and conditioned in a way that hastened the ripening process were perceived as indistinct from untreated fruit, 39 indicating that consumers respond to differences in the ripening stage, not the 1-MCP treatment per se (Almeida and Gomes, 2009). Instrumental analyses provide a fast, cheap, and objective means to assess pear quality at a commercial scale. Despite their usefulness, the data from instrumental analysis do not always correlate with those from sensory assessments of acceptance or preference (Harker et al., 2002). Therefore, an adequate quality management of pear in the supply chain require a better understanding of the changes in the sensory profiles as affected by postharvest technologies and a better interpretation of the instrumental data from the perspective of consumer acceptance and preference. The purpose of this study was to differentiate the effect of 1-MCP and that of temperature on ripening of ‘Rocha’ pear and to assess whether the sensory profile during ripening was adequately described by the instrumental measurements by methods commonly used in quality control.

Material and methods

Fruit material Pear (Pyrus communis L. ‘Rocha’) fruit with an average weight of 190 ± 25 g were harvested at the physiological maturity from an orchard located in Cadaval, Oeste Region, Portugal. Fruit were sorted by hand for uniform size, absence of disease and external defects and packed into plastic crates containing 14 kg of fruit. Immediately after harvest, fruit were cooled and stored for 30 d in air at –0.5 ºC (±0.3) and relative humidity of 88 to 91 %.

Postharvest treatments and shelf life conditions After 30 d in air at -0.5 °C, fruit were treated with 1-MCP at 150 or 300 nL L-1. During the treatment, fruit were placed inside 250 L plastic containers with a small ventilator and 1- MCP was generated from SmartFreshTM (Agrofresh, Inc., Springhouse, PA, USA). The containers remained sealed for 24 h at -0.5 °C, after which time they were vented to the outside of the facility and fruit were transferred to a room at 20 °C (± 0.3) to ripen. Control fruit were placed under same conditions without the 1-MCP treatment. An additional batch of untreated fruit was ripened in air at 10 °C (± 0.3).

Measurement of ethylene production and respiration rate Ethylene production rate was measured in 3 replicates of 4 fruit each in a closed system at 20 °C (±0.3). Ethylene production was measured at the respective ripening temperatures at 20 or 10 °C. Ethylene in the headspace of 2.15 L glass jars was separated by gas chromatography (GC Trace 1300, Thermo Fisher Scientific, Waltham, MA USA) with a capillary column TG bond alumina (Na2SO4) and detected in a FID detector as described by Saquet and Almeida (2017).

Respiration of pear fruit was expressed by the CO2 release in the headspace of the glass jars. Same fruit samples of ethylene were used for measurements of respiration. Respiration rate was measured at 20 or 10 °C. Immediately after ethylene analysis, the CO2 concentration in the glass jars was measured with an Oxycarb 6 gas analyzer (Isolcell Italia, Laives, Italy) fitted with an infrared sensor and a continuous flow at a rate of 0.1 L min-1.

Instrumental analyses of physicochemical quality characteristics Fruit skin color was measured in the CIE L*a*b* color space with a tri-stimulus colorimeter (CR-400, Konica Minolta, Tokyo, Japan) with the C illuminant. The measurements were made on the widest part of each fruit in three replicated samples of five fruit each. The a* and b* coordinates were converted to hue angle (hº) as described by McGuire (1992). Flesh firmness was measured after skin removal, on pared sides, on the equatorial region of fruit, with a handheld penetrometer T.R. Turoni (FT 327, Forlì, Italy) equipped with an 8-mm diameter tip. The maximum force required to penetrate the probe 6 mm into the fruit flesh was registered in three replications of five fruit each. Juice was extracted from three replicated samples of five fruit each and used for TSS and TA. Juice was obtained from 10 mm diameter discs excised from the widest region of each fruit. TSS was measured directly in the juice with a refractometer (HI 96801, Hanna Instruments, Woonsocket, RI, USA). TA was calculated from volume of NaOH solution at 0.1 M required to increase the pH of 10 mL juice diluted in 90 mL distilled water to a final value of 8.1 and expressed as malic acid equivalents.

Sensory analysis Sensory evaluation was performed after 2, 8 and 14 d of ripening at 10 and 20 °C. Fruit were equilibrated to room temperature and cut into slices (10 mm thick) along the axis. Two slices of pears containing the skin were presented to each panelist inside a lidded transparent polypropylene box coded with a random three-digit number.

A panel of 30 assessors was asked to score the samples for the following attributes: skin color, aroma intensity, hardness, sweetness, acidity, juiciness, flavor intensity, off-flavor and overall appreciation, using a numerical scale ranging from 1 to 9. Skin color was scored from greener (1) to yellower (9), aroma intensity from lower (1) to higher (9); hardness from softer (1) to firmer (9); sweetness, acidity, juiciness, and flavor intensity from lower (1) to higher (9); off-flavor from absent (1) to intense (9); and overall appreciation from dislike (1) to like (9). The panelists were instructed to score the skin color first, then to uncover the box and score for aroma and subsequently to taste the slices and to evaluate the remaining quality attributes.

Data analysis The trial was conducted in a completely randomized design with three replications. Data were subjected to analysis of variance (ANOVA) using the software Action Stat (2014, São Carlos, SP, Brazil). When appropriate, means were separated by Fisher’s protected LSD test at α=0.05. Correlations among the instrumentally measured physicochemical parameters and sensory traits were analyzed using Spearman's rank correlation coefficients and the one-sided null hypothesis tested at α = 0.05.

Results

Ethylene production and respiration rates The ethylene production rate of ‘Rocha’ pear immediately after harvest was below the detection limit. After the transfer from -0.5 to 10 or 20 ºC the ethylene production rate remained very low irrespective of the 1-MCP treatment (Fig. 1 A) and increased during ripening to a maximum of 14.9 µg kg-1 h-1 in control fruit after 15 d in air at 20 ºC (Fig. 1 A). The rate of ethylene production of 1-MCP-treated fruit at 20 ºC was slightly lower than that of control fruit but followed the same trend with no significant dose effect (Fig. 1 A). Untreated fruit maintained at 10 °C produced ethylene at a lower rate than 1-MCP-treated fruit at 20 ºC. Ethylene production by pear fruit at 10 ºC increased at a low rate during the first 10 d, reached a peak of 11.3 µg kg-1 h-1 at day 15 and decreased thereafter (Fig. 1 A). Respiration rate in control fruit at 20 °C increased from 14.2 to 38.6 mg kg-1 h-1 during the first 15 d of ripening (Fig. 1 B). 1-MCP at 150 nL L-1 did not affect the respiration rate in relation to control fruit, but the treatment with 300 nL L-1 reduced the peak of respiration by 27 % (Fig. 1 B). Control fruit at 10 ºC respired at an average rate 55 % lower than that of fruit

42 maintained at 20 ºC (Fig. 1 B). The Q10 of respiration rate between 10 and 20 ºC ranged between 1.6 at the beginning of shelf life to 3.1 by day 10 and decreased to 2.1 by day 15 (Fig. 1 C). On average, the Q10 was 2.4 in control fruit, 2.2 and 2.0 in 1-MCP-treated fruit at 150 and 300 nL L-1, respectively.

Fig. 1. Ethylene production (A) and respiration rate (B) of ‘Rocha’ pear fruit during ripening at shelf life conditions. Bars are SD (n = 3).

Physicochemical changes during fruit ripening Instrumental quality evaluation of ‘Rocha’ pear was based on color, firmness, TSS and TA. At harvest, fruit had a hue angle of 106 º (green hue), flesh firmness of 52 N, TSS of 110 g kg-1 and TA of 2,000 mg kg-1. These fruit characteristics remained unaltered during the 30 d in storage at -0.5 ºC prior to ripening at 10 or 20 ºC. Hue angle is a coordinate suitable to track color changes in ‘Rocha’ pear from the green hue of mature fruit to the yellow hue of ripe fruit (Almeida et al., 2016). At the beginning of shelf life, fruit skin had a hue angle of 104 º (Fig. 2 A). Hue angle decreased faster in control than in 1-MCP-treated fruit during the first 6 d at 20 ºC but the difference in hue was reduced

43 after 15 d with yellow fruit (h = 83 to 90 º). Fruit ripened at 10 °C maintained significant higher hue angle and after 10 d yellowed at a slower rate than those at 20 ºC even if treated with 1- MCP (Fig. 2 A). At 20 ºC, fruit softened at an average rate of 4.7 N d-1 from 54 N to 7 N in 10 d (Fig. 2 B). The softening rates during the initial 5 d were 8.5, 7.4, and 6.9 N d-1 for control fruit and those treated with 1-MCP at 150 and 300 nL L-1, respectively (Fig. 2 B). The final firmness of ripe fruit, however, was similar in 1-MCP treated and control fruit (Fig. 2 B). The softening rate at 10 ºC was of 1.4 N d-1, about 70 % lower than that of control fruit ripened at 20 ºC; at 10 ºC pears required 24 d to reach 8.4 N (Fig. 2 B).

Fig. 2. Changes in skin color (A) and flesh firmness (B) during ripening of ‘Rocha’ pear at shelf life conditions. Bars are SD (n = 3).

TSS ranged from 122 to 134 g kg-1 during ripening and was not significantly affected by 1-MCP treatments or temperature (Fig. 3 A). TA varied from 2,000 to 1,600 mg kg-1 during ripening with no clear effect of temperature or 1-MCP dose (Fig. 3 B).

Fig. 3. Total soluble solids (A) and titratable acidity (B) during ripening of ‘Rocha’ pear at shelf life conditions. Bars are SD (n = 3).

Sensory profiles of ‘Rocha’ pear Sensory profiles of pear fruit sampled 2, 8, and 14 d after the beginning of ripening at 10 and 20 °C are shown in the Fig. 4. After 2 d, the sensory profile of ‘Rocha’ pear was, in general, similar among treatments (Fig. 4 A). Only fruit treated with 150 nL L-1 1-MCP were perceived by panelists as significantly greener, with lower juiciness and lower flavor intensity (Fig. 4 A). After 8 d, fruit ripened at 10 ºC were judged by panelists as harder, more acidic, and greener than those ripened at 20 ºC, although the skin color was similar to fruit treated with 150 nL L-1 1-MCP (Fig. 4 B). This is consistent with the instrumental measurement of firmness (Fig. 2 B), but not with the actual acidity (Fig. 2 D). As ripening progressed further, the sensory profiles became more differentiated. After 14 d, significant differences were perceived in skin color, hardness, sweetness, juiciness, and

45 flavor intensity (Fig. 4 C). Panelists perceived pears ripened at 10 ºC as harder, greener, with lower sweetness, juiciness, and flavor intensity than all other samples (Fig. 4 C). Fruit treated with 300 nL L-1 1-MCP were classified as the sweetest, juiciest and with the better flavor intensity (Fig. 4 C).

Correlation between physicochemical and sensory assessments The closeness of the relationship between the instrumentally measured hue angle, flesh firmness, TSS, and TA and the sensory attributes skin color, hardness, sweetness, and acidity was evaluated using the Spearman's rank correlation coefficients (Table 1). Significant correlations were observed between hue angle and perceived skin color, and between firmness and sensory perceived hardness, but not between TSS and sweetness or between TA and perceived acidity (Table 1).

Skin color* 8 A Overall Aroma intensity appreciation 6 a 4

Off-flavor 2 b Hardness 0 b b a *Flavor intensity Sweetness a

*Juiciness Acidity

Skin color* 8 B Overall a Aroma intensity appreciation 6 4 b 2 a Off-flavor b Hardness* 0 b

Flavor intensity a Sweetness

Juiciness Acidity*

Skin color* 8 C Overall a Aroma intensity appreciation 6 4 b Off-flavor 2 b a Hardness* 0 b b a b a *Flavor intensity Sweetness*

a *Juiciness Acidity

Control 10 °C Control 20 °C 150 1-MCP 20 °C 300 1-MCP 20 °C

Fig. 4. Sensory evaluation of ‘Rocha’ pear at 2 (A), 8 (B) and 14 (C) d of fruit ripening at shelf life conditions. Values are mean scores recorded by the trained panel (n = 25). * Signiﬁcant at P ≤ 0.05.

Table 1. Spearman's rank correlation coefficients between instrumentally measured and sensory assessed characteristics of ‘Rocha’ pear (n = 12).

Instrumental variable Sensory attribute Spearman’s rho1 Hue angle (º) Skin color -0.654* Firmness (N) Hardness 0.661* TSS (%) Sweetness 0.224 n.s. Titratable acidity (%) Acidity 0.510 n.s. 1 *, significant at the 0.05 level; n.s., nonsignificant.

Discussion

Ripening physiology and physicochemical characteristics Pears respond differently to 1-MCP treatment depending on cultivar, maturity stage, dose of 1-MCP, and time of application (Watkins, 2006). ‘Rocha’ pear is able to ripen immediately after harvest without exposure to chilling temperature or exogenous ethylene application (Saquet and Almeida, 2017). Additionally, the inhibition of ethylene action by a treatment with 1-MCP immediately after harvest delays, but does not impair the ripening of ‘Rocha’ pear (Saquet and Almeida, 2017). In the current study, ‘Rocha’ pear fruit were treated with 1-MCP after 30 d in air at -0.5 ºC, a chilling exposure that promotes ethylene biosynthesis in pear (Agar et al., 2000). The effect of 1-MCP on climacteric ethylene production and respiration rate is small or absent when the treatment is applied to pear after initiation of ripening process (Chiriboga et al., 2012). The results reported herein show that 1-MCP at 300 nL L-1 slowed ripening metabolism as indicated by the decrease in ethylene production and respiration rates (Fig. 1). However, ripening temperature had a stronger effect on the metabolic rate of ‘Rocha’ pear; ripening of untreated fruit at 10 °C was slower than that of fruit treated with 150 or 300 nL L-1 1-MCP at 20 °C (Fig. 1 and 2). The change in metabolic rate induced by 1-MCP and by temperature had differential effects on the ripening-related changes of skin color, flesh firmness, total soluble solids and titratable acidity, characteristics that, taken together, are considered as reliable indicators of the eating quality of pears (Kappel et al., 1995). Skin color determines the visual appraisal of pears (Kappel et al., 1995). The treatment with 1-MCP retained the green hue angle during 5 d at 20 ºC and subsequent yellowing occurred

48 at a rate similar to that of untreated fruit at the same temperature. Fruit ripened at 10 °C remained greener that those ripened at 20 ºC for 24 d indicating that lower ripening temperature was more effective in maintaining green skin color than the treatments of cold-exposed fruit with 1-MCP. In contrast to color, the softening rate was not affected by 1-MCP during ripening at 20 ºC. Under these conditions, all pears reached a firmness of 7 N after 10 d. However, ripening temperature had a strong effect on softening rate and the fruit at 10 ºC reached 10 N only after 24 d. Clearly, color and firmness are two physical properties whose change during ripening can be decoupled by a treatment with 1-MCP or by ripening temperature. The differential modulation of color and firmness allowed a range of values at each sampling date to allow the assessment of possible correlations between measurement and sensory perception (see below). The chemical properties TSS and TA are strong determinants of pear flavor and consumer experience. It has been suggested that a minimum of 113 g TSS kg-1 is required for consumer acceptance of pears (Vangdal, 1980). However, TSS alone is not always a reliable indicator of flavor quality. The ratio between TSS and TA provides a better indicator of pear flavor quality and can clarify decision during sensory evaluation (Kappel et al., 1995; Magwasa and Opara, 2015). Neither 1-MCP nor ripening temperature affected TSS, but fruit ripened at 10 ºC had higher TA during the first 5 d than fruit ripened at 20 ºC. Ethylene production, respiration rate and ripening-related yellowing and softening were more effectively slowed down by a temperature of 10 °C than by the 1-MCP treatment in fruit ripening at 20 ºC.

Sensory profiles during pear ripening Skin color, juiciness, flavor intensity, hardness, sweetness and acidity, were the sensory attributes perceived as different during ripening of ‘Rocha’ pear under the various conditions and evaluation times (Fig. 4). Off-flavor was consistently scored as low, and the aroma intensity and the overall appreciation were statistically similar in pears subjected to the different treatments. The sensory profile evolved during the 14 d of ripening and the effect of temperature and ethylene inhibition become more evident in time. 1-MCP applied at 300 nL L-1 after 30 d in air storage at -0.5 °C slightly delayed ripening of ‘Rocha’ pear at 20 ºC without impairment. Pears treated mainly with 300 nL L-1 1-MCP were scored as having significantly higher sweetness, juiciness and flavor intensity. Hardness of these fruit was similar to the other

49 treatments. Flavor maintenance or enhancement by the treatment with 1-MCP have been reported in ‘Packham’s Triumph’ (Moya-León et al., 2006) and ‘Conference’ (Rizzolo et al., 2005) and ‘Spadona’ (Gamrasni et al., 2010) pears stored in air. 1-MCP-treated ‘Packham´s Triumph’ (at harvest with 200 nL L-1 1-MCP) were perceived as better in firmness, color, aroma, sweetness, and a better overall acceptance after 6 months of air storage (Moya-Léon et al., 2006). Similarly, ‘Bartlett’ treated with 600 nL L-1 1-MCP had more desirable sensory traits, as compared with ethylene-treated and untreated pear: higher sweetness, juiciness and typical pear aroma, reduced fermented aroma, gritty texture and tart taste (Escribano et al., 2016). However, the preference for 1-MCP treated pear is not always reported. ‘Abbé Fétel’ pear treated with 300 nL L-1 1-MCP, perceived as firmer and grainy texture, were less preferred by panelists compared to control fruit (Rizzolo et al., 2014). Also, 1-MCP-treated ‘Conference’ pear were described during the sensory evaluation as hard and poor in flavor after six months storage followed by a 10 d shelf life in relation to those ripened with a combination of 1-MCP and ethylene (Neuwald et al., 2015a). Temperature conditioning of 1-MCP-treated pears are likely to play a role in the sensory perception. In kiwifruit, consumers perceived 1-MCP-treated fruit as firmer, less juicy, less sweet and less flavorful than control fruit. However, altering the ripening rate by temperature conditioning to hasten ripening of 1-MCP-treated in relation to untreated fruit made it impossible for panelists to distinguish between treatments (Almeida and Gomes, 2009). Similarly, differences in the sensory evaluation of ‘Rocha’ pear treated with 1-MCP may be associated to different ripening stages induced by treatments, and in this case, mainly due to ripening during shelf life at 20 °C.

Relationship between sensory and instrumental evaluations The Spearman's rank correlation coefficients applies to variables that are not normally distributed and or linearly related necessarily as long as the relationship among variables is monotonic (McDonald, 2014). Hue angle and flesh firmness, the two physical variables that were significantly correlated with their sensory counterparts, skin color and hardness, respectively, were also the two variables that had the widest range of measurements. The hue angle of the pear skin within the interval 104.6 º (green) to 83.3 º (yellow) was significantly correlated with the sensory perception of skin color (Table 1). The negative correlation is due to the definition of the sensory scale from green (low value) to yellow (high value); this direction of color change, related to the progression of ripening, is opposed to the variation of the hue angle from green (higher values) to yellow (lower values). The use of the

50 sensory scale resulted in a coefficient of variation of color scores of 30 %, much higher than the 7 % associated with the hue angles. Flesh firmness of pears between 54.0 and 5.5 N was positively correlated with the sensory perception of hardness (Table 1). The large changes in flesh firmness associated with ripening-related softening (Figure 2 B) had a coefficient of variation of 63 %. The coefficient of variation of sensory hardness scores was 32 %, lower than that of flesh firmness measured with a penetrometer. Texture is a key factor in fruit quality assessment but a reliable relationship between objective measurements and subjective sensory methods is not always feasible (Harker et al., 2002). It has been argued, that no instrumental analyses is currently adequate to replace the human sensory perception (Chauvin et al., 2010). However, practical considerations related to the time, the expertise, and the costs associated to sensory evaluations, instrumental analyses are frequently used in quality control (Chauvin et al., 2010). On the other hand, the correlation between TSS and the perception of sweetness was not significant (Table 1). This is likely due to the narrow interval of TSS in the sampled pears, ranging from 122 and 134 g kg-1. TSS measurements in the pear samples were much less variable that the sensory scores of sweetness, with coefficients of variation of 3 % and 17 %, respectively. The correlation between titratable acidity and the sensory perceived acidity by the sensory taste panel was also non-significant at the 0.05 level although moderately positive (Table 1), likely due to the narrow range found (1.5 to 2.3 mg kg-1). The variation of titratable acidity among pears was similar to that of the sensory acidity scores, with coefficients of variation of 10 % and 14 %, respectively.

Conclusions Ripening of pear at 10 °C progressed at a rate 1.7 fold slower than that of 1-MCP-treated fruit at 20 °C; Ripening temperature is more important to the rate of changes during ripening than 1- MCP; 1-MCP at 150 and 300 nL L-1 after 30 d in air at -0.5 °C reduced the metabolic rate without impairing fruit ripening; Ripening-related changes in skin color and flesh firmness can be modulated by treatment with 1-MCP or by ripening temperature; Fruit ripened at 10 ºC for 14 d were perceived as harder, less sweet, less juicy, and with lower flavor intensity that fruit treated 1-MCP and ripened at 20 °C for the same period;

Instrumentally measured hue angle and flesh firmness were significantly correlated to sensory scores of skin color and hardness, but measurements of total soluble solids and titratable acidity were not related to the sensory scores of sweetness or acidity; A delayed treatment with 1-MCP can be used to modulate the poststorage ripening rate and the sensory profile of ‘Rocha’ pear.

Chapter 5

Mineral composition of ‘Rocha’ pear fruit related to storage disorders

Abstract: Mineral composition is related to pear fruit storability. This study aimed at mapping the concentrations of macronutrients (N, P, K, Ca, Mg, S) and micronutrients (Mn, Zn, Cu, Fe, B) in ‘Rocha’ pear fruit to establish baseline gradients and to investigate the possible relationship of minerals to the occurrence of internal storage disorders. Macronutrients N, Ca, Mg, S, and micronutrients Fe, Mn, Zn, Cu and B had radial gradients with higher concentrations in the skin tissues than in the fruit flesh, while P and K concentrations increased toward the fruit center. Along the longitudinal axis, Ca was less concentrated in the distal region, while Mg increased toward the fruit calyx zone. Concentration of Fe decreased, while Mn increased in the skin tissues from the proximal to the distal region. B was lower in the flesh tissue of the distal region. Ca concentration was lower in fruit with internal disorders than in healthy fruit in three of four orchards. The ratios K/Ca and (K+Mg)/Ca were significantly higher in fruit with internal disorders. Fruit affected by internal disorders had lower B concentration in two of four orchards. Generally, ‘Rocha’ pear showed decreasing radial gradients in Ca and B concentrations from the skin toward the fruit center, but no remarkable gradients longitudinally. The lower concentrations of Ca and B in the fruit center were associated with the occurrence of internal disorders in pears after CA storage.

Keywords: Controlled atmosphere, fruit quality, internal browning, macronutrients, micronutrients, Pyrus communis.

Introduction Mineral nutrition plays a fundamental role in determining the quality and storability of fleshy fruits (Bangerth, 1979; Marcelle, 1995; Brunetto et al., 2015). Pome fruit can develop physiological disorders related to minerals, such as bitter pit in apple (Perring, 1986; De Freitas et al., 2010; Kalcsits, 2016) and cork spot in pear (Curtis et al., 1990; Raese and Drake, 2006). Moreover, storage related disorders in apple (Sharples, 1980; Trentham et al., 2008) and pear (Raese and Drake, 2000; Xuan et al., 2003 and 2005) have been attributed to preharvest plant nutrition and postharvest fruit mineral composition. However, the complex relationships among nutrients and between nutrients and specific quality characteristics hinders the establishment of direct and univocal reasoning regarding preharvest nutrition and storability. Pear is a pome fruit suitable for long-term storage (De Martin et al., 2015; Almeida et al., 2016; Saquet et al., 2017). During long-term storage under CA storage pear can develop internal disorders whose symptoms include browning of flesh and core tissue and formation of cavities (Streif et al., 2003; Almeida et al., 2016; Saquet and Almeida, 2017). Pome fruit derive from an inferior ovary with multiple fused carpels surrounded by a fleshy hypanthium; these fruit are as divided into three tissue zones (Steeves and Sawhney, 2017): i) the core containing seeds and the surrounding vascular tissue separated from the cortex by a papery endocarp; ii) the flesh composed by the hypanthium that is not part of the pericarp but is fused with the exocarp and mesocarp; and iii) the skin. Gradients in mineral composition are expected in fruit with this anatomy due to the spatial distribution of the vascular tissues (Draězeta et al., 2004) and the differential mobility of minerals in the xylem and the phloem (Lewis, 1979; Lang, 1990; Brown and Shelp, 1997; Miqueloto et al., 2014). The characterization of fruit mineral concentrations is relevant not only for recommendations on fertilization but also to modulate fruit quality and storability (Brunetto et al., 2015). Direct effects of mineral nutrients on fruit quality depend upon nutrient content and balance in the fruit (Tagliavini et al., 2000; Kalcsits, 2016). Quality characteristics, such as total soluble solids are positively correlated to fruit K content (Brunetto et al., 2015), while fruit quality maintenance during storage is favored by low N and high Ca fruit concentrations (Tagliavini et al., 2000; Casero et al., 2004) as well as higher B concentrations in pear (Xuan et al., 2001, 2003 and 2005). The objective of this study was to map the gradients of macro and micronutrients within pear fruit and to determine the differences in mineral composition in healthy tissue and tissue with flesh browning.

Material and Methods

Growing conditions and fruit material Fruit were harvested from four orchards of pear (Pyrus communis L.) cultivar ‘Rocha’ grafted onto quince BA 29 (Cydonia oblonga L.), located in the Western region of Portugal. The orchards were installed on soils with a clay-loam texture with a density of 1500 to 2200 trees per hectare and trained as central axis. Trees were 5, 7, 10, and 16 years-old. Fruit ranging in diameter between 55 and 65 mm were harvested at physiological maturity characterized by a flesh firmness of 60 to 70 N and total soluble solids of between 11.8 and 13.0 %.

Storage conditions Fruit were stored for 5 months at -0.5 ºC and 92 to 95 % relative humidity in air or in commercial CA storage room under the following oxygen partial pressures: 3.5; 0.9 and 0.7 kPa O2. The carbon dioxide partial pressure was maintained at 0.5 kPa in all CA storage conditions.

Sample preparation Leaves of pear trees were sampled 100 days after full bloom in 15 trees randomly selected in each orchard. Fully developed leaves (120 per orchard) were collected from the medial third of stem grown during the season. Leaves were oven-dried at 65 ºC until constant weight, ground into a fine powder and analyzed for minerals. Fruit used to determine the gradients of minerals were divided in three radial and three longitudinal sections as follows. On the radial axis (Fig. 1): a) Skin tissue (the outer 1.5 mm), b) outer flesh tissue (the subsequent 10 mm flesh tissue), and c) inner flesh tissue (the subsequent 10 mm flesh tissue). In the longitudinal axis (Fig. 2), fruit were divided in three proportional thirds: a) the proximal region (peduncle part), b) the medial (without core), and c) the distal regions (calyx part), all with and without skin tissue. Samples for these analyzes were replicated three times with 15 fruit each.

Fig. 1. Pear sections analyzed radially (skin tissues, outer flesh and inner flesh tissues).

Fig. 2. Pear sections analyzed longitudinally (1/3 proximal; 1/3 medial; 1/3 distal).

To analyze mineral nutrients in pear fruit after storage period, two cylinders (17 mm thick; 40 mm length) were excised from the healthy and damaged fruit. These fruit samples were taken from the same storage condition. For this procedure, each fruit was cut in three proportional thirds and only the medial portion for sampling was used (Fig. 3). Fruit tissue cylinders were cut in small pieces and dried in a forced air oven at 65 ºC until constant weight.

Dried samples were ground into a fine powder and analyzed for mineral nutrients. Samples for mineral nutrient analyzes were composed by three replicates each containing 15 fruit.

Fig. 3. Tissue sampling for minerals analyzes of healthy and disordered ‘Rocha’ pear fruit after 5 months CA storage.

Mineral analyses Dried and fine powdered samples (5 g) were used for mineral analyzes. For N and S determinations, samples were subjected to catalytic pyrolysis in a Leco CNS 2000 instrument (LECO Corporation, Saint Joseph, MI, USA). N was determined in a thermo conductivity cell and S in an infrared cell. For P, K, Ca, Mg, Fe, Mn, Zn, Cu, and B determinations, samples were solubilized in hydrochloric acid and the minerals determined by inductively coupled plasma optical emission spectrometry (ICP-OES; IRIS Intrepid II, XSP Radial, Thermo Fisher Scientific Inc., Waltham, MA, USA) as described (Wheal et al., 2011). The concentration of nutrients was expressed on a dry mass basis.

Assessment of internal storage disorders Internal storage disorders characterized by the browning of the flesh tissue and the formation of cavities in the fruit flesh were scored after 5 months in storage in three replicates each composed by 45 fruit. To better visualize the damages, individual fruit were cut transversely and longitudinally. The occurrence of internal disorders was expressed in percentage of affected fruit, pooling together browning symptom and cavity formation, independently of the severity of the damages.

Statistical analysis Data on mineral nutrients were subjected to one-way ANOVA according to a completely randomized design using the Statistica TM software version 8.0 (StatSoft Inc., 2007).

Results and discussion

Nutritional status of the orchards The nutritional status of the orchards was assessed by foliar mineral analyses (Table 1). According to the reference mineral nutrients values in leaves of ‘Rocha’ pear trees (INIAV, 2000), N concentrations from all orchards were within the reference values for ‘Rocha’ pear cultivated in the Western Region of Portugal. Foliar P concentration was within the optimal range in orchards 1, 2 and 3 but a low level in orchard 4. The latter is due to the presence of carbonate in this orchard, indicating P deficiency. Foliar K concentration from all orchards were low with an average of the four orchards 27 % lower than the average reference value (12.2 to 14.1 g kg-1). The orchards 1 and 4 had Ca concentrations within the optimal levels indicated for ‘Rocha’ pear, while the orchards 2 and 3 were 29 and 15 % lower, respectively. Mg was 28 and 17 % lower in orchard 1 and 3, respectively. Mg concentrations in plant leaves from orchard 2 was within the optimal values, while the values from orchard 4 was 100 % higher than the reference value. Fe was about double high in leaves from the orchards 1, 2 and 3, while Fe concentration in leaves from the orchard 4 was 47 % lower than the reference indication. Mn reference value for ‘Rocha’ pear cultivated in the Western Region of Portugal is not available. Zn and Cu were much lower in concentrations in all orchards. In an average, Zn and Cu were 33 % lower than the optimal recommended concentrations. Regarding the micronutrient B, the orchards 1 and 2 had satisfactory concentrations, but the orchards 3 and 4 were 33 and 41 % lower than reference value in leaves of ‘Rocha’ pear, respectively.

Table 1. Mineral concentration in leaves of ‘Rocha’ pear trees in four orchards. Concentrations are expressed in a dry matter basis. Values are the mean ± SD (n=3).

Orchard Reference Nutrient 1 2 3 4 values* Macronutrient (g kg-1) N 23.9 ± 0.8 22.3 ± 0.6 22.7 ± 0.0 23.2 ± 0.0 21.1 – 22.1 P 1.6 ± 0.1 1.8 ± 0.1 1.7 ± 0.0 0.3 ± 0.0 1.5 – 1.7 K 8.5 ± 0.2 10.2 ± 0.9 10.7 ± 0.0 8.9 ± 0.0 12.2 – 14.1 Ca 19.8 ± 2.1 12.9 ± 1.2 15.7 ± 0.0 20.1 ± 0.0 17.4 – 19.2 Mg 2.8 ± 0.3 3.8 ± 0.2 3.2 ± 0.0 7.9 ± 0.0 3.6 – 4.1 S - - - - 1.8 – 2.0 Micronutrient (mg kg-1) Fe 86.4 ± 2.1 79.6 ± 15.3 108.2 ± 0.0 34.0 ± 0.0 58.0 – 68.0 Mn 15.6 ± 3.0 75.2 ± 9.1 77.1 ± 0.0 68.0 ± 0.0 - Zn 29.1 ± 3.1 24.9 ± 4.9 30.9 ± 0.0 33.0 ± 0.0 40.0 – 46.0 Cu 8.9 ± 0.4 9.3 ± 0.1 9.1 ± 0.0 6.3 ± 0.0 10.0 – 15.0 B 30.7 ± 5.5 38.8 ± 7.2 20.5 ± 0.0 18.0 ± 0.0 29.0 – 32.0 * INIAV (2000)

Mineral distribution within ‘Rocha’ pear fruit

Dry matter and mineral gradients in pear fruit sections Significant difference in dry matter was measured in the pear fruit (Table 2). Dry matter content was 22.4 % in the skin tissue and 15 % in the flesh tissue. In outer flesh or inner flesh tissues the contents of dry matter were the same. Since the flesh had ca. 67 % of the dry matter content of the skin, higher concentration of some nutrients in the skin tissue are expected.

Table 2. Dry matter in fruit sections of ‘Rocha’ pear fruit.

Fruit section Dry matter (%) Skin tissues 22.4 ± 1.1 Outer flesh tissues 15.0 ± 1.2 Inner flesh tissues 14.9 ± 1.4

Radial mineral concentrations In a pome fruit, gradients along the two axis (longitudinal and radial) are expected considering its anatomy of formation and the spatial distribution of vascular bundles (Kalcsits, 2016). Significant radial gradients within ‘Rocha’ pear fruit were observed for P, K, Ca and Mg but not for N and S (Table 3). The highest N concentration was in the skin (4.8 g kg-1), and the lowest (2.4 g kg-1) in the outer flesh, while the inner flesh had N concentration intermediate (3.6 g kg-1). The concentration of P and K increased from the skin to the center (Table 3). These results contrasts with the observation in apple fruit of Faust et al. (1969), which reported an inverse trend. The concentrations of Ca and Mg decreased from the skin to the inner fruit flesh tissues of ‘Rocha’ pear (Table 3). Ca is often conserved the most important macronutrient for the apple fruit health (Bangerth, 1979; Perring, 1986; Ferguson et al., 1999; De Freitas and Mitcham, 2012), as it relates to pre- and postharvest physiological disorders. Although less investigated in pear, Ca also plays an important role in keeping pear fruit quality (Curtis et al., 1990; Raese and Drake, 2000). Cork spot occurrence in ‘Anjou’ pear was better controlled with higher Ca concentrations in the skin and cortex tissues (Raese and Drake, 2006).

Table 3. Concentration of macro- and micronutrients, expressed on a dry matter basis, in the radial sections of ‘Rocha’ pear. Values are mean ± SD (n=3).

Transversal fruit sections Minerals Skin tissues Outer flesh tissues Inner flesh tissues Macronutrient (g kg-1) N 4.7 ± 0.02 2.4 ± 0.05 3.5 ± 0.12 P 0.4 ± 0.04 0.5 ± 0.04 0.6 ± 0.04 K 4.8 ± 0.04 6.4 ± 0.07 7.1 ± 0.18 Ca 1.1 ± 0.05 0.4 ± 0.04 0.4 ± 0.02 Mg 0.4 ± 0.01 0.3 ± 0.01 0.3 ± 0.03 S 0.3 ± 0.01 0.24± 0.01 0.2 ± 0.01 Micronutrient (mg kg-1) Fe 17.6 ± 1.21 13.3 ± 1.61 8.4 ± 0.82 Mn 3.7 ± 0.11 2.0 ± 0.12 1.5 ± 0.05 Zn 8.1 ± 0.22 5.0 ± 0.44 4.5 ± 0.24 Cu 3.0 ± 0.13 2.7 ± 0.22 0.1 ± 0.00 B 10.3 ± 0.51 8.0 ± 0.14 7.2 ± 0.08

Transpiration is the main driving force for the xylem stream in plants (White and Broadley, 2003), in which Ca seems to move relatively freely, while it is substantially immobile in the phloem (Montanaro et al., 2012; Brunetto et al., 2015). Fruit transpiration is related the surface to volume ratio, which decreases as the fruit develops, negatively affecting the translocation of mineral nutrients via the xylem stream in to fruit (Montanaro et al., 2012; De Freitas and Mitcham, 2012). The fact that fruit are largely phloem fed explains in part, why fruit are generally low in Ca contents and why higher fruit transpiration rates are sometimes associated with increased fruit Ca concentrations (Montanaro et al., 2006 and 2010). Ca and Mg were more concentrated in the skin than in cortex tissue of ‘McIntosh’ apple (Webster, 1981) and in ‘Conference’ pear (Gąstoł and Domagała-Świątkiewicz, 2009). Wilkinson and Perring (1964) stated that accumulation of Ca and Mg in the skin tissue appears to be characteristic of apple fruit. However, the present results with ‘Rocha’ pear are very similar in behavior to that of apple. All micronutrients analyzed showed significant decreasing concentrations from the skin tissue to the inner fruit flesh tissue (Table 3). Fe and B showed the highest concentrations

61 in the skin tissue of ‘Rocha’ pear reaching 17.7 mg kg-1 and 10.3 mg kg-1, respectively (Table 3). The most investigated micronutrient in relation to pear fruit quality during storage is B (Xuan et al., 2003 and 2005; Mielke and Chaplin, 2008), and the present investigation shows results with lower B concentrations in the fruit center, where frequently internal storage disorders develop in pears (Saquet et al., 2000; Almeida et al., 2016; Saquet and Almeida, 2017).

Longitudinal mineral concentrations The longitudinal analyzes of mineral nutrients considered the three thirds of the ‘Rocha’ pear fruit, and each section was analyzed the skin and the flesh tissue separately (Fig. 2). The concentrations of N, P, K, Mg and S did not have longitudinal gradient either in the fruit skin or in the fruit flesh (Table 4). The concentration of Ca was slightly lower in both skin and flesh tissue of the distal fruit region (Table 4).

Table 4. Concentration of macro- and micronutrients, expressed on a dry matter basis, in the longitudinal sections of ‘Rocha’ pear. Values are mean ± SD (n=3).

Skin tissues Flesh tissues Mineral Proximal Medial Distal Proximal Medial Distal Macronutrient (g kg-1) N 5.0 ± 0.02 4.9 ± 0.05 4.5 ± 0.09 3.4 ± 0.02 3.5 ± 0.05 3.5 ± 0.02 P 0.6 ± 0.01 0.4 ± 0.01 0.4 ± 0.01 0.6 ± 0.01 0.7 ± 0.02 0.6 ± 0.01 K 5.7 ± 0.05 5.1 ± 0.09 5.5 ± 0.02 6.4 ± 0.20 6.9 ± 0.09 6.3 ± 0.21 Ca 1.3 ± 0.01 1.3 ± 0.04 1.0 ± 0.05 0.7 ± 0.01 0.6 ± 0.02 0.5 ± 0.01 Mg 0.4 ± 0.01 0.4 ± 0.01 0.5 ± 0.01 0.4 ± 0.01 0.5 ± 0.01 0.4 ± 0.01 S 0.3 ± 0.01 0.3 ± 0.01 0.3 ± 0.01 0.2 ± 0.01 0.3 ± 0.01 0.3 ± 0.01 Micronutrient (mg kg-1) Fe 23.3 ± 1.88 22.3 ± 1.24 19.0 ± 0.81 15.1 ± 0.15 14.1 ± 0.89 12.0 ± 0.05 Mn 3.5 ± 0.05 3.7 ± 0.08 4.3 ± 0.08 2.1 ± 0.05 2.1 ± 0.03 1.9 ± 0.03 Zn 9.3 ± 0.38 9.6 ± 0.17 10.0 ± 0.14 4.7 ± 0.04 5.6 ± 0.05 5.8 ± 0.21 Cu 3.8 ± 0.21 3.3 ± 0.08 3.2 ± 0.17 3.2 ± 0.04 3.6 ± 0.02 3.5 ± 0.29 B 10.3 ± 0.27 9.9 ± 0.05 10.0 ± 0.04 10.3 ± 0.28 10.0 ± 0.03 9.6 ± 0.12

In apple, Ca concentration tends to be higher at the proximal than at the distal region of the fruit, in both skin (Terblanche et al., 1979a) and cortex tissues whereas Mg is frequently

62 higher at the distal than the proximal region (Terblanche et al., 1979b). We observed only a slightly higher Mg concentration in the skin tissue of the distal region. The longitudinal mineral gradient in pome fruit is established between the pedicel and the remnants of the calyx (Lewis and Martin, 1973; Lewis, 1979). The characterization of these longitudinal and radial gradients are relevant for an adequate understanding of the topology of fruit quality, namely the localized development of symptoms of specific physiological disorders associated, directly or indirectly, with tissue mineral concentrations. Among these disorders, bitter pit in apple (De Freitas et al., 2010; Miqueloto et al., 2014, Kalcsits, 2016) and the cork spot in pear are related to Ca concentrations (Curtis et al., 1990; Raese and Drake, 2006; Reuscher et al., 2014). Positive effects of increased B concentrations were reported on reduced internal flesh browning disorders in ‘Conference’ pear (Xuan et al., 2001, 2003 and 2005) and on cork spot in ‘La France’ pear (Reuscher et al., 2014). Ca is transported in the xylem only (Marschner, 2011) and it moves more slowly within the fruit. Because the mobility of calcium is limited, concentrations are thought to decrease with increasing the distance from xylem vessels (Kalcsits, 2016). Vascular bundles in pome fruit such as an apple or a pear are organized into the cortical vascular system surrounding the fruit carpel and branching toward the epidermis, and a carpel vascular system entering the fruit from the peduncle and pass though the carpel to the pistil (Drazeta et al, 2004). Longitudinal and radial mineral gradients have been reported in several fruit, including apple (Faust et al., 1969; Webster, 1981), pear (Curtis et al., 1990; Calouro et al., 2008; Gąstoł and Domagała- Świątkiewicz, 2009), kiwifruit (Ferguson, 1980) and loquat fruit (Gariglio and Agustí, 2005), but a detailed map of the gradients was not found in the literature.

Mineral nutrients related to internal storage disorders in ‘Rocha’ pear Although some internal disorders in pear can affect all fruit flesh tissue, most frequently observed disorders develop in the medial and distal sections of a pear (Franck et al., 2007; Saquet and Almeida, 2017). The possible relationship between the mineral concentrations in the fruit flesh, sampled as described in the Fig. 3 and the occurrence of internal disorders in four orchards after 5 months in storage was examined. The incidence of internal disorders ranged from 25.0 to 61.7 % in fruit from the four orchards (Table 5). Nutrient concentrations differed in healthy and internal disordered fruit. No consistent trend was observed in the relationship between N concentrations of healthy and damaged tissue. Affected pear fruit from all orchards had same N concentrations than healthy fruit (Table 5).

Means were vertically compared within the same orchard between healthy and affectedorchard healthy fruit (Tukey the samepear between within compared Means were vertically HSD Test, alpha 0.05) Table ‘Rocha’ healthy in and disordred ratios and their concentration 5. Macronutrient storage. 5 months after fruit pear

The concentration of P was similar in affected and healthy pear fruit from all orchards. Low concentration of P is normally associated with storage disorders in apple (Amarante et al., 2012) and chilling injury if the susceptible apple cultivars ‘Bramley’s Seedling’ and ‘Cox Orange Pippin’ is aggravated in fruit with low P concentrations (Johnson, 1980), however in this case of ‘Rocha’ pear P seems not be decisively. A similar trend was observed in K concentrations (Table 5). The internal disordered fruit from all orchards showed similar concentrations of K. This macronutrient is also important during storage since an unbalanced K/Ca ratio may promote cork spot in ‘d’Anjou’ (Curtis et al., 1990) and ‘Alexander Lucas’ pears (Tomala and Trzak, 1994). Because pear trees generally show higher absorption and transport of Ca to the fruit than apple (Marcelle, 1995), the negative effect of K on quality of stored pear fruit is less frequent than in apple fruit (Brunetto et al., 2015). ‘Braeburn’ apple increases internal disorders with increasing K concentration or K/Ca ratio (Neuwald et al., 2014). Ca concentrations were significantly lower in affected pear fruit from three orchards (Table 5). Optimum fruit Ca concentration reduces storage related disorders (Bangerth, 1979; Kalcsits, 2016). Storage disorders such as internal breakdown in ‘Passe Crassane’, senescent breakdown in ‘Abbè Fetel’ and ‘Bosc’ (Gorini, 1988), cork spot in ‘d’Anjou’ (Curtis et al., 1990) and ‘Alexander Lucas’ pears (Tomala and Trzak, 1994) can be reduced by increasing concentration of Ca in the fruit. The dynamics and factors affecting Ca transport to the fruit are still not fully understood (Saure, 2005). Ca uptake by the fruit occurs only during the first part of fruit growth (Faust, 1989) or linearly until harvest (Zavalloni et al., 2001). Mg concentration on healthy and damaged tissue was not statistically different in fruit from all orchards investigated (Table 5). The equilibrium among nutrients are often expressed in ratios to better capture the balance or imbalance of the macronutrients (Curtis et al., 1990). The ratios N/Ca, K/Ca, Mg/Ca, (K+Mg)/Ca and K/Mg were analyzed in ‘Rocha’ pear in relation to the occurrence of internal disorders. The N/Ca ratio was higher in affected fruit from two orchards. The K/Ca, Mg/Ca and (K+Mg)/Ca ratios were higher in fruit affected by internal disorders from almost all orchards (Table 5). The K/Mg ratio, however, was similar in fruit from all orchards (Table 5). The N/Ca ratio was useful to predict cork spot incidence in ‘d’Anjou’ pear (Curtis et al., 1990). The (K+Mg)/Ca ratio has been used to predict the occurrence of bitter pit in ‘Cox Orange Pippin’ (Boon, 1980), ‘Gala’ and ‘Golden Delicious’ apples (Argenta and Suzuki, 1994; Nachtigall and Freire, 1998). The K/Mg ratio was the same in healthy and internally

65 damaged fruit and is less relevant than K/Ca or (K+Mg)/Ca in relation to occurrence of internal disorders in ‘Rocha’ pear (Table 5). These results however contrast with those of Seo et al. (2015), who found a positive linear correlation of K/Mg ratio with the occurrence of browning disorder severity in Asian pears (Pyrus pyrifolia Nakai) in which the K/Ca ratio is a reliable indicator to predict disorders. The nutrient imbalances reflected in the ratios K/Ca and (K+Mg)/Ca are related to increased susceptibility to physiological disorders such as lenticel spot (Perring et al., 1984), watercore (Perring et al., 1984), internal breakdown (Perring et al., 1985), bitter pit (Ferguson and Watkins, 1989) and carbon dioxide injury during controlled atmosphere storage of apple (Castro et al., 2007). Pear belongs to pome fruit group in which Zhang et al. (2004) have identified specific sucrose transporters related to active phloem unloading. It is reasonable to assume that pear share the same mechanism of fruit growth as apple, which maintains active phloem unloading during whole season (Morandi et al., 2014), while xylem becomes dysfunctional as the fruit develops (Lang, 1990; Lang and Ryan, 1994; Drazeta et al., 2004), resulting in a reduction in the rate of xylem inflow to the fruit (Lang, 1990). Calcium, as a xylem-mobile element, shows a progressive decline in its rate of accumulation during the season (Marschner, 2011). Furthermore, the sudden drop in fruit Ca concentration in apples coincides with the onset of rapid fruit growth and as a consequence Ca concentration dilute in the fruit. Once inside the fruit, Ca movement from the peduncle towards the distal fruit tissue define Ca concentration in the distal-end tissue (De Freitas and Mitcham, 2012). In fruit, in general, total tissue Ca concentration decreases from the peduncle towards to the distal end tissue (Nonami et al., 1995). Distal fruit tissue is more susceptible to Ca deficiency disorders than fruit tissue at the proximal region and Ca deficiency symptoms usually begin in the distal tissue, eventually spreading to the whole fruit in severe cases. The reasons for such fruit Ca distribution is not well understood, but different mechanisms can potentially be involved such as cell wall Ca- binding capacity, abundance of functional xylem vessels from peduncle to distal fruit tissue, as well as the driving force required for Ca translocation from peduncle to distal fruit tissue (De Freitas and Mitcham, 2012). While Ca transported is gradually restricted by the eventual collapse of the xylem vessels during the late stages of apple development (Drazeta et al., 2004), the supply of N, K and Mg still continue resulting in higher N/Ca, K/Ca e Mg/Ca ratios. In apple, both phloem and xylem fluxes to fruit contribute to growth in early and mid-stages of development, while phloem sap mainly contributes to enlargement close to harvest (Tagliavini et al., 2000). In

66 apple, the xylem-phloem balance explain why fruit Ca concentrations are lower in the fruit than in other plant organs (Lang and Ryan, 1994) and the relative sensitivity of cultivars to bitter pit disorder. The concentration of Ca in the inner tissue of ‘Rocha’ pear fruit (0.4 g kg-1) is only 2.5 % of the Ca present in leaves (17.1 g kg-1) (Table 1). K, which is a very mobile element within the plant, this percent value increases to 74.4 %. Research with some cultivars such as ‘d’Anjou’ seems to be promising in protecting fruit against some physiological disorders (Curtis et al., 1990; Raese and Drake, 2006). In the case of ‘Rocha’ pear, it was not found any literature about its responses to Ca treatments indicating a possibility in exploring this research area with this pear cultivar. Furthermore, considering the observed gradient in Ca concentrations in the fruit, it would be at least reasonable to test the effectiveness of this macronutrient under pre- and postharvest conditions. Similar gradient in Ca concentrations in ‘Conference’ pear was observed by Gastol and Domagała-Świątkiewicz (2009).

Micronutrients in healthy and internal disordered pear fruit The micronutrients in healthy and disordered ‘Rocha’ pear fruit tissues are shown in Table 6. The concentrations of Fe were different in fruit from only one orchard, in which disordered pear fruit had lower Fe concentration. The concentrations of Mn were similar in fruit from all orchards with no significant differences between healthy and disordered fruit. The concentrations of Zn had a similar trend like Fe and Mn with not significant differences in healthy and affected fruit from all orchards (Table 6). The concentrations of Cu were lower in fruit from the orchard 1 (6.1 to 6.2 mg kg-1), but they were statistically not different comparing healthy to disordered fruit in the same orchard (Table 6). The concentrations of Cu were gradually increasing from the orchard one to the others. Fruit from the orchard two showed Cu concentrations between 7.2 and 7.4 mg kg-1 in healthy and disordered fruit, respectively (Table 6).

Table 6. Micronutrients in healthy and internal disordered ‘Rocha’ pear fruit after storage.

-1 Affected Micronutrient (mg kg ) Orchard Class fruit (%) Fe Mn Zn Cu B Healthy 15.0 a 3.2 a 8.9 a 6.1 a 12.0 a 1 39.0 Affected 16.3 a 3.8 a 8.1 a 6.1 a 8.5 b Healthy 25.6 a 3.1 a 13.8 a 7.2 a 11.0 a 2 61.1 Affected 18.6 b 3.6 a 11.6 a 7.4 a 11.0 a Healthy 22.3 a 1.7 a 8.0 a 9.4 a 22.0 a 3 61.7 Affected 19.0 a 1.7 a 8.9 a 7.6 a 18.0 b Healthy 14.3 a 3.4 a 11.4 a 10.8 a 10.7 a 4 25.0 Affected 17.5 a 3.6 a 12.2 a 13.8 a 11.0 a Means were compared between healthy and affected fruit within the same orchard by the Tukey HSD Test at α=0.05.

Among all investigated micronutrients, B seems to play an important role in the development of internal disorders in ‘Rocha’ pear. Pears from orchards one and three had significant higher B concentrations in healthy pear fruit, while the orchards two and four there were no differences (Table 6). B concentration in healthy fruit was 30 and 19 % higher than the affected fruit in the orchards one and three, respectively. B favors fruit quality in pear (Wooldridge, 2002; Xuan et al, 2001, 2003 and 2005; Mielke and Chaplin, 2008), persimmon (Ferri et al., 2008) and sweet cherry (Nagy et al., 2008). The lower B concentration measured in the inner part of ‘Rocha’ pear fruit found radially in the present investigation (Table 3) overlaps with the same region that internal disorders occur during CA storage. From the same fruit flesh region were sampled tissue cylinders from healthy and internal disordered pear fruit after CA storage (Fig. 3). Preharvest B supply has been shown to prevent blossom blast and cork disorders in ‘Williams Bon Chretien’, ‘d’Anjou’ and ‘Bartlett’ pears (Peryea, 1994; Wooldridge, 2002). In Germany, ‘Conference’ pear has shown positive responses to B field sprays in protecting fruit against browning disorders during long-term CA storage (Xuan et al., 2001; 2003; 2005). In Poland, foliar application of B to ‘Conference’ pear increased fruit Ca concentration and consequently improved the fruit storability by reducing the permeability of cortex cells and reduced the incidence of internal disorders (Wójcik and Wójcik, 2003).

Preharvest B treatment has been shown to be beneficial in keeping quality of other fruit species. Low incidence of flesh browning in ‘Pink Lady’ apple was associated to higher B concentrations (Castro et al., 2007). The cracking of tomato fruit might occur due to deficiency of B (Lawler et al., 2002). Skin browning in ‘Fuyu’ persimmon was reduced during cold storage by preharvest B sprays (Ferri et al., 2008). However, sweet cherry fruit sensitivity to cracking was not influenced by B fertilization (Nagy et al., 2008). B did not show effect on internal breakdown in plum (Plich and Wójcik, 2002). The B benefits are not restricted to the prevention of physiological disorders. ‘Gala’ apple showed higher titratable acidity after treatment with 1 or 2 % of borax (Sestari et al., 2007). Asgharzade et al. (2012) measured higher total soluble solids and higher flesh firmness in apple followed by preharvest B treatment. The reported effects of B on pome fruit metabolism are inconsistent. While Xuan et al. (2005) observed lower respiration rate of ‘Conference’ pear after foliar B application, Brackmann et al. (2016) showed that B application promoted higher ethylene production and higher respiration rates in ‘Galaxy’ apple fruit.

Conclusions Ca and Mg concentrations decreased from the skin tissues toward the fruit center of ‘Rocha’ pear; P and K concentrations increased from the skin tissues to the fruit center of ‘Rocha’ pear; The five micronutrients showed decreasing concentrations from the skin tissues to the fruit center of pear; Lower Ca concentrations in fruit flesh was associated with internal disordered pear fruit from three orchards and Ca may be involved in the susceptibility of ‘Rocha’ pear to storage disorders; The K/Ca ratios were higher in fruit with physiological disorders from three orchards while (K+Mg)/Ca ratio were higher in affected fruit from all orchards investigated; B concentration was lower in fruit flesh of disordered pear fruit from two orchards and seems to play an important role in the development of storage disorders in ‘Rocha’ pear.

Chapter 6

Internal disorders of ‘Rocha’ pear affected by oxygen partial pressure and inhibition of ethylene action

Abstract: Current technologies allowing the use of extremely low pO2, the introduction of 1- 1-MCP, and the regulatory prohibition of diphenylamine are changing the conventional storage protocols for pear cultivars. Internal disorders, in particular, severely damage pear quality during CA storage. ‘Rocha’ pear (Pyrus cummunis L.) was stored for 136 d at -0.5 ºC in air or -1 under 3.0 and 0.5 kPa O2 with 0.6 kPa CO2. Fruit treated with 150 nL L 1-MCP were also stored at 3.0 and 0.5 kPa O2 after 32 d in air following the treatment. Internal disorders did not develop in fruit stored in air (20.8 kPa O2) or at 0.5 kPa O2 but affected 10.2 % of the fruit stored in 3.0 kPa O2 after 136 d. 1-MCP increased disorder incidence at 0.5 and at 3.0 kPa O2. Four types of internal disorders occurred: core browning, white cavity, necrotic cavity, and flesh browning. Low O2 reduced ethylene production and respiration rates, which were further reduced by the treatment with 1-MCP. ATP concentration and adenylate energy charge were higher in fruit stored in air than in those at 3.0 and were generally lowest in fruit at 0.5 kPa O2.

The effect of pO2 on energy metabolism prevailed over that of 1-MCP treatment. The linkage between ATP and AEC and incidence of internal disorders was not strong, since under the same pO2, 1-MCP enhanced the incidence of disorders with a negligible effect on adenylate nucleotides or AEC. It was not possible to establish a threshold of ATP concentration or AEC below which internal disorder develop. In conclusion, poststorage quality of ‘Rocha’ pear was better at the extremely low pO2 of 0.5 kPa than at 3.0 kPa. 1-MCP was detrimental to internal disorders and blocked poststorage softening of ‘Rocha’ pear stored at 0.5 kPa O2. There seems to be a risk zone of pO2 for internal disorder development in the range of pO2 ≤ 3.0 kPa O2 and

> 0.5 kPa O2.

Keywords: Adenylate energy charge, controlled atmosphere storage, fruit quality, physiological disorders, Pyrus communis.

Introduction The main European pear cultivars in the world market are suitable for long-term storage. At temperature of -1 to 0 ºC and relative humidity higher than 90 % pears can be stored in air for 3 to 6 months (Agar et al., 2000; Wang and Sugar, 2013), after which time postharvest life is limited by advanced ripening (Raese et al., 1999), decay (Spotts et al., 2007), and superficial scald (Calvo et al., 2015). CA storage significantly extends the storage period of pears. The recommended pO2 and pCO2 are specific for each cultivar and growing region. CA recommendations for pears are rapidly evolving as new technologies allow more precise control of pO2 and pCO2, and the dynamic control of gas concentration based on fruit physiological responses, namely changes in chlorophyll fluorescence (Prange et al., 2003), respiratory quotient (Gasser et al., 2008; Weber et al., 2015), and the ethanol accumulation in the fruit or its release into the atmosphere (Veltman et al., 2003a). CA storage extends the storage life of pear but alters the main causes of postharvest life termination: internal disorders become a major limiting factor under these conditions (Franck et al., 2007; Lum et al., 2016). ‘Rocha’ pear grown in warm climates is sensitive to superficial scald and internal disorders and both must be addressed to assure poststorage quality. Diphenylamine, a standard postharvest treatment until 2013, was effective in reducing the incidence and severity of both storage disorders in ‘Rocha’ pear (Silva et al., 2010; Almeida et al., 2016) and CA storage recommendations were developed for DPA-treated fruit. The recommended conditions were pO2 of 2.5 to 3 kPa, and pCO2 lower than 0.7 kPa (Silva et al., 2010; Almeida et al., 2016). However, under these CA-conditions the incidence of internal disorders incidence can be high in the absence of DPA (Almeida et al., 2016).

Pears are generally less tolerant than apples to very low pO2 (Streif et al., 2001; Thompson, 2010). This observation is consistent with the lower internal air volume, higher density, and higher resistance to O2 diffusion of pear in relation to apple (Ho et al., 2006). Low pO2 in storage rooms changes the energy status of pear fruit (Saquet et al., 2000; Veltman et al., 2003b), with detrimental consequences in membrane phospholipids and enhanced internal disorders in ‘Conference’ pear (Saquet et al., 2003a) and ‘Braeburn’ apple (Saquet et al., 2003b). Therefore, storage conditions that improve the levels of energy stored in the adenylate nucleotides and the overall cell energy status in pear have been related to lower incidence of internal disorders (Saquet, et al., 2000; Veltman et al. 2003b; Franck et al., 2007). The ethylene action inhibitor 1-MCP became an effective treatment to prevent superficial scald and extend storage life in pear (Argenta et al., 2003; Isidoro and Almeida, 2006; Villalobos-

Acuña et al., 2011b; Almeida et al., 2016). However, in contrast with apples, poststorage pear ripening can be impaired by 1-MCP (Chiriboga et al., 2011). The extension of storage period of ‘Rocha’ pear in the absence of DPA requires the mitigation of superficial scald and internal disorders. 1-MCP and ultra-low pO2 storage are two venues to achieve these goals. This study aimed to evaluate the effect of pO2 and 1-MCP on internal disorders and on overall quality maintenance of ‘Rocha’ pear during storage. ATP, ADP, and AMP were assessed under several storage conditions in fruit with and without ethylene action inhibition to address the relationship between cell energy status and internal disorders.

Materials and methods

Fruit material Pear (Pyrus communis L. ‘Rocha’) fruit were harvested at the mature-green stage from an orchard located in Cadaval, West Region, Portugal. The maturity stage at harvest was measured in 3 replicates of 15 fruits each. Fruit had uniform size (60 to 65 mm), a starch index of 8.2 (1 to 10 scale), flesh firmness of 52.4 N, TSS of 11.2 %, TA of 0.2 % expressed on malic acid equivalents, and a skin hue angle of 106.4 º. After harvest, fruit were drenched with fludioxonil at 580 mg L-1 (Scholar®, Syngenta, Basel, Switzerland) and cooled to -0.5 °C.

1-MCP treatment and storage conditions Fruit were stored in 0.55 m3 cabinets at -0.5 °C (±0.3 °C fluctuation) and 90 to 93 % relative humidity, in air or under two CA conditions: 0.5 kPa O2 or 3 kPa O2 with pCO2 below

0.6 kPa in each instance (balance N2). The pO2 was lowered by flushing the cabinets with N2 and the final pressure of 0.5 kPa O2 or 3.0 kPa O2 was reached within 26 and 18 h, respectively. 1-MCP generated from SmartFreshTM (Agrofresh, Inc., Springhouse, PA, USA) was applied at a dose of 150 nL L-1 for 24 h at -0.5 ºC (±0.3). After the treatment with 1-MCP, the fruit were maintained for 32 d in air before establishment of the CA conditions indicated above. The storage temperature and gas partial pressures inside the CA storage rooms were daily monitored and controlled by an automatic system (Isolcell, Laives, Italy).

Assessment of internal disorders Fruit were assessed for internal disorders after 44, 96 and 136 d in storage in 3 replicate batches of 60 fruit each. Fruit were assessed immediately after removal from storage at -0.5 ºC and after a subsequent 7-d shelf-life period at 20 ºC. Fruit were cut transversely at the level of the seed cavity in 3 sections and then cut longitudinally to visualize the extent of disorder development. Internal disorders were classified in four classes according to their symptomatology (Fig. 1): core browning, white cavity, necrotic cavity, and flesh browning. The occurrence of each disorder was expressed in percentage of affected fruit.

Fig. 1. Internal disorders observed in ‘Rocha’ pear. Core browning (A), white cavity (B),

necrotic cavity (C) and flesh browning (D).

Evaluation of fruit quality characteristics Skin color was measured in CIE L*a*b* color space with a tristimulus CR-400 chroma meter (Konica Minolta, Tokyo, Japan) with the C illuminant. One measurement was made in the widest part of the fruit in three replicates of 5 fruit each. Flesh firmness was assessed with a penetrometer equipped with an 8 mm probe (T.R. Turoni, Forli, Italy). The maximum force to insert the probe 8 mm into the fruit flesh without

73 skin was recorded. Firmness was measured twice on opposite sides of each fruit, in three replicated batches of 5 fruit each. Juice was extracted from a 10 mm thick cylinder excised from the equatorial region of the fruit and TSS and TA measured in the juice. TSS was measured with a digital refractometer (Hanna Instruments, Woonsocket, USA) and TA determined by titration of an aliquot of 10 mL of juice diluted in 90 mL of distilled water, with 0.1 M NaOH until pH 8.1.

Ethylene and respiration measurements Ethylene was measured in three replicate samples of 4 fruit each. Fruit were sealed inside 2.15 L glass jars at 20 ºC for 2 h. A headspace volume of 0.1 mL was removed with a glass syringe via a rubber septum and injected into a gas chromatograph (Trace 1300, Thermo

Fisher Scientific Inc., Marietta, USA) fitted with a TG bond alumina (Na2SO4) capillary column, 50 m long with 0.53 mm i.d. (Thermo Fisher Scientific Inc., Marietta, USA). Helium was used carrier gas at a flow rate of 15 mL min-1. The injector temperature was set at 160 ºC, the flame ionization detector at 180 °C, and the oven temperature held for 0.5 min at 100 °C, followed by an increase at 20 °C min-1 to reach 180 °C, and a holding of 0.5 min at 180 °C.

Respiration, expressed by the release of CO2, was measured in the same fruit samples used for ethylene determination. The headspace CO2 concentration was measured with the infrared sensor of an Oxycarb 6 gas analyzer (Isolcell, Laives, Italy) in a close circulation circuit at a flow rate of 100 mL min-1.

Determination of adenylate nucleotides concentration Tissue cylinders (18 mm thick and 40 mm length) were excised from the core region of the fruit and seeds removed. The cylinders were immediately frozen in liquid nitrogen and freeze-dried at -50 °C and -100 kPa. Samples were fine powdered and used for extraction. Three replicated extractions and measurements were performed per each treatment. Adenylate nucleotides (ATP, ADP and AMP) were extracted and their concentrations determined by bioluminescence as described by Saquet et al. (2003a) with adaptations (Saquet and Almeida, 2017). Briefly, 1 g of freeze-dried sample was extracted for 30 min at 4 °C with 10 mL of 5 % trichloracetic acid, 2 mM of ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA). After centrifugation at 21,000 g for 30 min at 4 ºC, the supernatant was diluted 30 fold with Tris-EDTA buffer (0.1 M Trizma Base and 2 mM EDTA, pH 7.75). ATP was determined by the emission of bioluminescence from 10 µL of extract mixed with 40 µL ATP monitoring reagent (BioThema AB, Handen, Sweden) and 150 µL of tris-EDTA buffer

(0.1 M Trizma Base and 2 mM EDTA, pH 7.75). Bioluminescence was measured at room temperature with a Synergy 2 Multi-Mode Reader (BioTek, Winooski, VT, USA). ADP was converted into ATP by incubation with 120 U mL-1 of pyruvate kinase (PK; Sigma-Aldrich, St. Louis, MO, USA) prepared in a PEP buffer for 30 min at room temperature. AMP was converted to ADP and ADP converted to ATP with a mixture of adenylate kinase (AK; Sigma- Aldrich, St. Louis, MO, USA) at 180 U mL-1 combined with PK (120 U mL-1; Sigma-Aldrich, St. Louis, MO, USA) in PEP buffer for 30 min at ca. 25 °C. Total ATP concentration was determined as described above and ADP and AMP calculated by difference. The concentrations of adenylate nucleotides were expressed on a fresh mass basis. AEC was calculated as [ATP] + 0.5 [ADP] / [ATP] + [ADP] + [AMP] (Atkinson and Walton, 1967).

Data analysis Data were subjected to one-way analysis of variance with the storage treatment as a fixed factor. Means were separated by the least significant difference (LSD) test at α=0.05. Analysis were made with the software Action Stat (2014, São Carlos, SP, Brazil).

Results and discussion

Incidence of disorders Superficial scald did not occur in any of the treatments. Even though ‘Rocha’ is a susceptible cultivar, seasonal variability of superficial scald incidence is high and orchard effects are strong. Internal disorders were absent from fruit stored under air for 136 d (Fig. 2).

Fruit stored under 3 kPa O2 developed internal disorders: 4.4 % were affected immediately after storage and the incidence increased to 10.2 % after 7 d in air at 20 °C (Fig.

2). Fruit stored at 0.5 kPa O2 did not develop internal disorders during storage or during -1 subsequent shelf-life period. The combined effect of 1-MCP at 150 nL L with low pO2 did not alter the incidence of internal disorder in fruit stored under 3 kPa O2 but increased their incidence under 0.5 kPa O2 (Fig. 2). It is not clear whether this effect is due to the inhibition of ethylene action per se or to the delayed CA regime in 1-MCP-treated fruit, although a delay in CA establishment is reported to reduce the occurrence of internal disorders in pear (Roelofs and De Jager, 1997).

Fig. 2. Occurrence of total internal disorders in ‘Rocha’ pear stored at -0.5 ºC for 136 d and following a 7 d shelf-life in air at 20 °C. Untreated fruit were stored in air or under 0.5 or 3.0 kPa O2 and 1-MCP-treated fruit were stored under 0.5 or 3.0 kPa O2. Vertical bars are standard deviation.

A potential dilemma arises in optimizing conditions for long-term CA-storage of

‘Rocha’ pear: If pO2 is reduced to levels that provide an effective control of superficial scald, the risk of internal disorder development increases. The inhibition of ethylene action by 1- MCP can mitigate the occurrence of superficial scald, even at 150 nL L-1 (Isidoro and Almeida, 2006; Almeida et al., 2016), a concentration that is 50 % of the commercially recommended dose for pear. However, an interaction is expected between 1-MCP treatment and CA storage (see 3.3. and 3.4). Moreover, the effect of 1-MCP on pear internal disorders is not clear: It has been shown to increase the occurrence of internal disorders during CA-storage in ‘Alexander Lucas’ (Hendges et al., 2015), but to alleviate it in ‘Abate Fetel’ (Vanoli et al., 2016), and in ‘Rocha’ pear (Almeida et al., 2016). When DPA was used as a postharvest treatment, storage of ‘Rocha’ pear was performed at pO2 of 2.5 to 3.0 kPa and pCO2 lower than 0.7 kPa (Silva et al., 2010; Almeida et al., 2016).

In the absence of DPA, internal disorders developed in fruit stored under pO2 of 3 kPa, but not in air (20.8 kPa O2) or at the lower pO2 of 0.5 kPa (Fig. 2). The absence of internal disorders under extremely low pO2 was reported for ‘d’Anjou’ pear maintained at 0.5 kPa O2 for 8 months (Mattheis and Rudell, 2011) and ‘Abbé Fétel’ pear stored for 7 months under dynamic controlled atmosphere with 0.8 kPa O2 and 0.45 kPa CO2 (Rizzolo et al., 2014). However, Mattheis et al. (2013) have subsequently reported an aggravation of an internal disorder described as pithy brown core in ‘d’Anjou’ pear stored at 0.5 kPa O2 in relation to 1.5 kPa O2.

These results hint at a risk zone of pO2 for internal disorder development, which is tentatively suggested to be between 0.5 and 3.0 kPa O2 for ‘Rocha’ pear.

Typology of internal disorders in ‘Rocha’ pear Symptomatology differed among the internal disorders observed in ‘Rocha’ pear. The confusing description, classification, and terminology regarding pear internal disorders is recognized (Franck et al., 2007) and there is still no consensus regarding their nature and etiology. However, an individual analysis of the effects of storage conditions on the distinct symptomatology is warranted since the damages were differentially affected by pO2 or 1-MCP. Four internal disorder symptomatologies were identified and characterized during the storage period of ‘Rocha’ pear under CA (Fig. 1): core browning, white cavity, necrotic cavity, and flesh browning. Core browning refers to brown parenchyma tissue with a maximum diameter of 15 mm restricted to the core region and brown patches with soft and wet appearance (Fig. 1 A). White cavity describes holes in the fruit flesh without brown surface, ranging in size from 2 mm to 35 mm, often with an elongate and narrow shape (Fig. 1 B and Fig. 3); these cavities were often located radially around the core region, but occasionally were also distributed longitudinally reaching, sometimes, the proximal neck region. Necrotic cavity consists of small cavities, not larger than 4 mm diameter and 3 mm deep, with necrotic brown surface, always located around the core region, with a typical ‘rosette’ appearance (Fig. 1 C). Flesh browning is brown tissue with firm and wet appearance without cavity formation; the brown tissue was frequently interspersed with bleached tissue and, in severe cases, affected more than 80 % of the fruit flesh (Fig. 1 D).

Fig. 3. Initial stages of visible white cavities in ‘Rocha’ pear showing the absence of necrotic tissues before the cavities enlarge to the symptom represented in Fig. 1 B.

Core browning, white cavity and flesh browning were the internal disorders most frequently observed in ‘Rocha’ pear after 136 d of storage, affecting 2.3, 3.7, and 2.7 % of the fruit, respectively (Fig. 4). The higher incidence of core browning, white cavity, necrotic cavity, and flesh browning was observed in fruit stored under 3.0 kPa O2, with or without 1- MCP treatment (Fig. 4).

Fig. 4. Occurrence of core browning (A), white cavity (B), necrotic cavity (C) and flesh browning (D) in ‘Rocha’ pear stored at -0.5 ºC for 136 d and following a 7 d shelf-life in air at

20 °C. Untreated fruit were stored in air or under 0.5 or 3.0 kPa O2 and 1-MCP-treated fruit were stored under 0.5 or 3.0 kPa O2. Vertical bars are standard deviation.

Core browning in pear has been described by the damage in the core region and is considered a senescence related disorder (Larrigaudière et al., 2004). Other authors use a generic term to classify all internal disorders observed in fruit stored under CA storage, such as ‘brown heart’ (Streif et al., 2001; Saquet et al., 2003a) or ‘core breakdown’ (Lammertyn et al., 2003), who used the same term to brown tissue and the cavities combined. However, core browning (Fig. 1 A) observed in ‘Rocha’ pear was restricted to the core region and seems to be unrelated to overall fruit senescence. Considering the firmness (Fig. 5 A) and skin color (Fig. 5 B) of the fruit with this disorder, core browning cannot be associated with senescent flesh breakdown. Core browning incidence was higher in fruit stored under 3 kPa O2 than 0.5

-1 kPa O2 and its incidence was aggravated by the treatment with 1-MCP at 150 nL L under both CA-conditions (Fig. 4 A). Two distinct symptomatologies included cavities. White cavities were more common than necrotic cavities (Fig. 4 B and C). The occurrence of white cavities in CA-stored fruit was higher under 3.0 kPa O2 than under 0.5 O2 and in both instances, the incidence increased in 1-

MCP-treated fruit. Cavity formation in apple is associated with high pCO2 during CA storage (Elgar et al., 1998; Clark and Burmeister, 1999), and symptom expression explained by dehydration of the affected tissue (Xuan et al., 2001; Lammertyn et al., 2003). However, inconsistencies persist regarding the etiology of the white cavity symptom. For example, large cavities were reported in ‘Conference’ pear stored under 0.5 kPa O2 with pCO2 lower than 0.5 kPa (Saquet et al., 2000). White cavities occurred in ‘Rocha’ pear stored at 3 kPa O2 even with pCO2 of 0.6 kPa (Fig. 4 B). Even though these pCO2 are one order of magnitude higher than those in air, 0.6 kPa CO2 is currently at the lowest range that can be practically obtained in commercial CA-storage. Whether pCO2 of about 0.6 kPa induce cavities remains unclear. Pear cavities are considered a late stage of development of the brown tissue (Lammertyn et al., 2000; Lammertyn et al., 2003; Franck et al., 2007). According to this hypothesis, disorder-inducing conditions during CA storage increase membrane permeability and the water leaked out of the cells diffuses and evaporates from the fruit surface resulting in dry, brown, gas-containing spaces that further dehydrate to form cavities (Xuan et al., 2001; Lammertyn et al., 2003). However, the white cavities observed in ‘Rocha’ pear (Fig. 3) were lined by white and surrounded by apparently healthy tissue. The early stages of white cavity formation occurred in white and not brown tissues (Fig. 3) and enlarged while remaining white (Fig. 1 B). Instead, we hypothesize that this type of cavity is caused by a programmed cell death mechanism similar to that of aerenchyma formation under hypoxic conditions in root tissues (Yamauchi et al., 2013). Necrotic cavities were less frequent (Fig. 4 C). The pattern of occurrence of this disorder was clearly defined, i.e., restricted and uniformly distributed around the core region. We were not able to associate the small and brown cavities around the core of the pear fruit to a specific storage condition (Fig. 4 C). Flesh browning (Fig. 4 D) was generally distributed in the flesh of ‘Rocha’ pear, always without cavities. This symptom may result from the development of core browning or coexist with it; the core region of fruit affected by flesh browning also had an intense brown coloration.

Poststorage fruit quality After 136 d at -0.5 ºC followed by 7 d at 20 ºC, fruit stored in air were soft with a firmness of 10 N (Fig. 5 A). Firmness was better retained during storage under low pO2 than in air, but subsequent softening after removal from storage was fast; at the end of a 7-d period in air at 20 ºC, the firmness of fruit stored under CA at 3.0 or 0.5 kPa O2 was similar to that of fruit previously stored in air (Fig. 5 A). 1-MCP slowed poststorage softening (Fig. 5 A), an effect that is very consistent in pear (Rizzolo et al., 2014; Almeida et al., 2016). The -1 combination of 1-MCP at 150 nL L with storage under 0.5 kPa O2 inhibited poststorage softening at 20 ºC for 7 d (Fig. 5 A).

Fig. 5. Flesh firmness (A), skin color (B), acidity (C) and total soluble solids (D) in fruit stored at -0.5 ºC for 136 d and following a 7 d shelf-life in air at 20 °C. Untreated fruit were stored in air or under 0.5 or 3.0 kPa O2 and 1-MCP-treated fruit were stored under 0.5 or 3.0 kPa O2. Vertical bars are standard deviation.

Fruit yellowed slightly during storage in air but not under CA (Fig. 5 B). Poststorage yellowing was reduced in fruit treated with1-MCP-treatment, and the combination of 1-MCP treatment and storage under 0.5 kPa O2 inhibited the subsequent decrease of hue angle during 7 d at 20 ºC (Fig. 5 B). Effects of CA storage and 1-MCP in maintaining green skin in pears are well documented (Mattheis and Rudell, 2011; Gago et al., 2015; Rizzolo et al., 2015; Vanoli et al., 2016).

Titratable acidity (Fig. 5 C) was lower in fruit stored in air than in fruit stored in CA, with or without 1-MCP treatment. TSS was similar in all storage conditions (Fig. 5 D). Taken together, the physicochemical properties of ‘Rocha’ pear after storage show that 3.0 and 0.5 kPa O2 prolong storage life while allowing poststorage ripening within 7 d at 20 ºC. However, an interaction between 1-MCP and pO2 effects is evident. Fruit treated with 1-MCP at 150 nL -1 L and stored under 3.0 kPa O2 ripened during shelf-life whereas poststorage ripening was inhibited during 7 d in fruit previously stored at 0.5 kPa O2. An examination of ethylene production and respiration rates may explain this observation.

Ethylene production and respiration rate Ethylene production was undetectable at harvest, increased slightly during the first 45 d in storage, and at a higher rate thereafter to reach a maximum at 96 d followed by a decline (Fig. 6 A). Ethylene production rate was highest in fruit stored in air with a peak rate of 31.4 -1 -1 -1 µg kg h . Lower peaks were observed under 0.5 and 3.0 kPa O2. 1-MCP at 150 nL L combined with CA further reduced the ethylene production rate; the peak rate of 1-MCP- treated fruit stored under 3.0 and 0.5 kPa O2 was of 52.1 and 15.5 % of that of untreated fruit under the same pO2, respectively. The stronger suppression of ethylene production was observed in 1-MCP-treated fruit stored under 0.5 kPa O2 (Fig. 6 A). Fruit ripening during storage in air lead to lower ethylene production rate during shelf- life in relation to that of fruit stored under 0.5 kPa O2 (Fig. 6 B). The combination of 1-MCP treatment and CA storage strongly reduced ethylene production during poststorage shelf-life, a suppression that was more intense in fruit stored under 0.5 than under 3.0 kPa O2 (Fig. 6 B). -1 Fruit treated with 150 nL L 1-MCP and stored under 0.5 kPa O2, failed to recover the ethylene emission remaining reaching 3.7 µg kg-1 h-1 even after 9 d shelf-life at 20 ºC (Fig. 6 B).

Fig. 6. Ethylene production rate of ‘Rocha’ pear during storage (A) and 9 d shelf-life at 20 °C after 136 d in storage (B). Untreated fruit were stored at -0.5 ºC in air or under 0.5 or 3.0 kPa

O2 and 1-MCP-treated fruit were stored under 0.5 or 3.0 kPa O2. Vertical bars are standard deviation.

Respiration reached the highest rate after 96 d in storage in all treatments, at the same moment of the maximum in ethylene production rate. Fruit stored in air for 96 d released CO2 -1 -1 at a higher rate (35.1 mg kg h ) than fruit stored under 3.0 or 0.5 kPa O2 which respired at 23.1 and 13.3 mg kg-1 h-1, respectively (Fig. 7 A). Treatment with 1-MCP further reduced pear fruit respiration at the end of storage when 1-MCP-treated fruit respired at a rate of 5.1 mg kg- 1 h-1 while air-stored fruit respired at 20.3 mg kg-1 h-1 (Fig. 7 A).

Fig. 7. Respiration rate of ‘Rocha’ pear during storage (A) and 9 d shelf-life at 20 °C after 136 d in storage (B). Untreated fruit were stored at -0.5 ºC in air or under 0.5 or 3.0 kPa O2 and 1-

MCP-treated fruit were stored under 0.5 or 3.0 kPa O2. Vertical bars are standard deviation.

In contrast with ethylene production, the respiration rate during shelf-life at 20 ºC was similar in fruit previously stored under low pO2 or in air (Fig. 7 B). No residual effect of CA storage on the poststorage respiration rate of ‘Rocha’ pear was observed. Respiration rate increased continuously during the shelf-life period at 20 ºC (Fig. 7 B). The rates were similar in fruit previously stored in air or under 0.5 or 3 kPa O2, but lower in pear treated with 1-MCP. Respiratory metabolism is source of chemical energy stored in the cellular pool of adenylate nucleotides.

Changes in adenylate nucleotides and energy charge Pear has a radial gradient in ATP concentration and AEC decreasing from the skin toward the core region (Almeida and Saquet, in press). Since the most internal disorders are located or originate at or surrounding the core region (Fig. 1) the adenylate nucleotides were quantified at the fruit core. ATP concentration at harvest was 965.7 nmol g-1, accounting for 68.6 % of the total adenylates. ATP decreased in the first 44 d of storage, and increased thereafter, when the effect of storage conditions was observed (Fig. 8 A). After 136 d fruit stored in air had higher ATP concentration than those stored in 0.5 or 3 kPa O2. Fruit treated with 1-MCP and stored at 0.5 or 3 kPa O2 had the lowest concentration of ATP at the end of storage but the effect of 1-MCP was negligible in relation that of pO2 (Fig. 8 A). Storage conditions affected ATP concentrations in a way that paralleled their effect on respiration rate (Fig. 7 A). A close relationship between ATP concentrations and fruit respiration rate was observed during storage of ‘Conference’ pear (Saquet et al., 2003a) and ‘Jonagold’ apple (Xuan and Streif, 2008), but not during ripening of ‘Rocha’ pear (Saquet and Almeida, 2017). 1-MCP has been shown to reduce ATP concentration during storage of ‘Jonagold’ apple (Xuan and Streif, 2008). ADP concentration at harvest was 249.1 nmol g-1 (17.7 % of total pool) and changed irregularly during storage (Fig. 8 B), as previously observed in pome fruit (Saquet et al., 2000; Saquet et al., 2003b). This erratic trend in ADP concentration is consistent with the possibility of conversion of ADP to ATP and AMP by the action of adenylate kinase or phosphorylated to ATP by ATP synthase (Igamberdiev and Kleczkowski, 2015). The concentration of AMP at harvest was 193.8 nmol g-1 (13.7 % of the total pool of adenylates) and decreased gradually during storage period with no clear effect of storage conditions or 1-MCP treatment (Fig. 8 C). The general decreasing trend in AMP concentration has been observed in during storage of ‘Conference’ pear (Saquet et al., 2003a) and in peach under chilling conditions (Jin et al., 2013), but AMP accumulated in banana after exposition for 8 to 12 h to hypoxia under

10 or 15 kPa O2 (Hill and ap Rees, 1995), and remained relatively unaltered during ripening of ‘Rocha’ pear (Saquet and Almeida, 2017).

Fig. 8. Changes in ATP (A), ADP (B) and AMP (C) in ‘Rocha’ pear during storage at -0.5 ºC.

Untreated fruit were stored at -0.5 ºC in air or under 0.5 or 3.0 kPa O2 and 1-MCP-treated fruit were stored under 0.5 or 3.0 kPa O2. Vertical bars are standard deviation.

The total pool of adenylates remained constant or declined during the first 94 d in storage and increased thereafter (Fig. 9 A). At the end of storage period, the total pool of adenylate nucleotides was highest in fruit stored in air, followed by fruit stored at 0.5 and 3 kPa O2 with intermediary values. The lowest concentrations were measured in 1-MCP-treated fruit (Fig. 9 A).

In fruit stored in air the AEC decreased from 0.78 at harvest to 0.67 after 44 d in storage to increase thereafter to a maximum of 0.87 and reached 0.81 at the end of the storage period

(Fig. 9 B). Fruit stored under low pO2 had lower AEC after the initial storage period, with minor and inconsistent effect of 1-MCP (Fig. 9 B). Overall, the AEC in ‘Rocha’ pear under low pO2 ranged from 0.58 to 0.78 (Fig. 9 B).

Fig. 9. Changes in total pool of adenylates (A) and AEC (B) in ‘Rocha’ pear during storage at

-0.5 ºC. Untreated fruit were stored at -0.5 ºC in air or under 0.5 or 3.0 kPa O2 and 1-MCP- treated fruit were stored under 0.5 or 3.0 kPa O2. Vertical bars are standard deviation.

Adenylates nucleotides and the development of internal disorders ATP concentration and AEC are frequently used to assess the energy status of plant cells (Geigenberger et al., 2009) and have been related to the development of internal disorders during CA storage of apple and pear (Saquet, et al., 2000, 2003a, 2003b; Veltman and Peppelenbos, 2003; Veltman et al. 2003b). Evidence that conditions improving fruit ATP concentration and AEC help maintain cell integrity and reduce the occurrence of internal

86 disorders during CA storage of pear was first presented by Saquet et al. (2000), and the argument was further supported by several studies (Saquet et al. 2001; Veltman and Peppelembos, 2003; Veltman et al., 2003b; Franck et al., 2003b). The argument that energy stored in the adenylate nucleotides is a factor in reducing internal disorders is partially, but not fully, supported by the results presented herein. Fruit stored in air (20.8 kPa O2) had higher AEC than fruit stored under at 3.0 kPa O2 (Fig. 8 B) and did not develop internal disorders (Fig. 2). However, two experimental conditions disrupt the linkage between ATP and AEC and disorder development. Fruit stored under the extremely low pO2 of 0.5 kPa did not develop internal disorders in contrast with fruit stored under 3.0 kPa O2 (Fig. 2) even if their ATP and AEC remained at relatively similar levels throughout storage (Fig. 7 A and 8 B). In addition, under the same pO2, 1-MCP enhanced the incidence of disorders (Fig. 2) with a negligible effect on adenylate nucleotides or AEC (Figs. 7 and 8). It is hypothesized that a pO2 of 0.5 kPa significantly reduces the production of reactive oxygen species so that the homeostatic balance between oxidative stress and repair capacity is maintained during storage. It was not possible to establish a threshold of ATP concentration or AEC below which internal disorder develop. Whether aspects of energy metabolism other than the equilibrium of the adenylate nucleotides play a role in unknown. The possible use of reducing power stored in NADPH (Lum et al., 2016) to better capture the cell energy status in relation to internal disorders is not likely since NADPH were not affected by experimental conditions during CA-storage of ‘Conference’ pear and ‘Jonagold’ apple, irrespective of disorder incidence (Saquet et al., 2000).

Conclusions Internal disorders in ‘Rocha’ pear did not develop after 136 d storage in air (20.8 kPa

O2) or under 0.5 kPa but 10.2 % of the fruit were affected after storage at 3.0 kPa O2;

A risk zone for internal disorder development is tentatively proposed between pO2 ≤ -1 3.0 kPa O2 and > 0.5 kPa O2. 1-MCP at 150 nL L combined with a 32 d delay in the pull down of pO2 enhanced the incidence of internal disorders under CA at 0.5 or 3.0 kPa O2; It was not possible to relate ATP concentrations or AEC with internal disorder development during CA storage of ‘Rocha’ pear.

Chapter 7

Responses of ‘Rocha’ pear to delayed controlled atmosphere storage depend on oxygen partial pressure

Abstract: Internal disorders hinder long-term CA storage of ‘Rocha’ pear. The delayed establishment of CA regime is often recommended to reduce internal disorders in pear. This investigation evaluated the effect of delayed CA at two pO2 at constant low pCO2 on internal disorders and on storage potential of ‘Rocha’ pear. Fruit were stored at -0.5 °C for 257 d under

0.6 kPa CO2 combined with 0.5 or 3.0 kPa O2. The pull down of pO2 was imposed at the beginning of cold storage (immediate CA) or after 46 d (delayed CA). ‘Rocha’ pear tolerated immediate storage at 0.5 kPa O2 without developing internal disorders for 257 d storage.

However, 63.3 % of fruit immediately stored at 3.0 kPa O2 developed internal disorders.

Delayed pull down of pO2 for 46 d reduced internal disorders in fruit at 3.0 kPa O2 to 35.5 %, but increased the disorder incidence in fruit at 0.5 kPa O2 to 27.3 %. Poststorage ethylene production rate was lower in fruit stored under delayed CA at 3.0 and 0.5 kPa O2. In conclusion, the immediate storage of ‘Rocha’ pear at 0.5 kPa O2 prevented the development of internal disorders for 257 d, while allowing adequate poststorage ripening and maintenance of quality traits. The effect of delayed CA on internal disorders depended on pO2. Delaying CA for 46 d did not benefit ‘Rocha’ pear during long-term storage.

Keywords: Fruit quality, immediate controlled atmosphere storage, physiological disorders, Pyrus communis, ripening.

Introduction Long-term storage of pears extends the marketing period, improves packinghouse management, and facilitates the amortization of facilities and equipments. Several pear cultivars are suitable for long-term storage, including ‘Rocha’ that can be maintained at -1 ºC under CA storage for up to 10 months (Almeida et al., 2016). However, internal disorders characterized by brown discolorations and cavities in the flesh and core can develop during CA-storage (Franck et al., 2007) leading to supply chain inefficiencies. Moreover, pear internal disorders cannot be detected by external examination causing consumer disappointment when fruit with good appearance reveal internal damage. Prevention of internal disorders remains a challenge and is the major determinant of storage life termination in ‘Rocha’ pear under CA. Delayed CA is a storage management practice in which fruit are maintained in air at low temperature for a period at the beginning of storage, often two to eight weeks, before the CA gas regime is imposed. This technique contrasts with immediate CA storage in which the pull down of pO2 starts immediately after the fruit are cooled. Delayed CA effectively reduces the incidence of internal disorders in ‘Conference’ pear (Höhn et al., 1996). This effect was first investigated by Höhn et al. (1996), who observed a reduction in the incidence of cavities in ‘Conference’ pear when the CA regime was delayed. A 21-d delayed CA reduced cavity formation by 50 to 90 %, depending on the orchard region (Höhn et al., 1996). The beneficial effect of a 21-d delayed CA in reducing internal disorders in ‘Conference’ pear was subsequently confirmed (Roelofs and de Jager, 1997; Saquet et al., 2001; Saquet et al., 2003a). Delayed CA is now a standard commercial practice for successful long-term CA storage of ‘Conference’ pear and is also effective in reducing the occurrence of internal disorders in apples (Streif and Saquet, 2003; DeEll and Ehsani-Moghaddam, 2012; Neuwald et al., 2014). The physiological and biochemical effects underlying the benefits of delayed CA in reducing internal disorders have been explored in ‘Conference’ pear. Compared with immediate CA, fruit stored under delayed CA have higher ethylene production, higher respiration rate, and higher adenylate energy charge during the first three months in storage (Saquet et al., 2001) and maintain higher levels of oleic, linoleic, and linolenic fatty acids during the initial storage period (Saquet et al., 2003a). Higher adenylate energy charge and better maintenance of membrane integrity are the proposed mechanisms to explain the effect of delayed CA on the reduction of internal disorders in pear and apple (Saquet et al., 2003a; 2003b). Together, these results and the industry practice with ‘Conference’ pear show that delayed CA is an effective pre-conditioning procedure to reduce internal disorder development

89 during long-term CA storage of pears in Northern Europe. However, the few trials with ‘Rocha’ pear grown under warm summer conditions showed little or no benefit of delayed CA (Almeida et al., 2016). This study evaluated the effect of delayed CA on internal disorders development and on fruit quality maintenance during long-term storage of ‘Rocha’ pear under 3.0 and 0.5 kPa

O2.

Material and methods

Fruit material Mature-green fruit of ‘Rocha’ pear (Pyrus communis L.) were harvested in an orchard located in Cadaval, Oeste Region of Portugal. At harvest, fruit with a size of 60 to 65 mm had an average (n=45) starch pattern index of 8.2 (1 to 10 scale), flesh firmness of 52.4 N (±6.1), total soluble solids of 112 g kg-1 (±6.3), titratable acidity, expressed as malic acid equivalents, of 2 g kg-1 (±0.2), and skin hue angle (hº) of 106.4º (±3.2).

Storage conditions Uniform unblemished fruit were placed into plastic crates and stored in experimental CA cabinets within cold rooms previously cooled at -1 ºC (±0.3). Three replicated fruit batches containing of 30 kg each (total of 90 kg) were stored in each cabinet.

The CA cabinets were sealed, the pO2 was lowered by N2 flushing and the pull down monitored by gas analyzers (Isolcell Italia, Laives, Italy). Temperature inside the cabinets was maintained at -0.5 °C (±0.3) and relative humidity at 92 to 93 %. CA pO2 were 3.0 or 0.5 kPa

O2 with 0.6 kPa CO2 in both conditions (balance with N2). CO2 above 0.6 kPa was scrubbed by an automatic system (Isolcell Italia, Laives, Italy) using a 30 % KOH solution. CA conditions were established within 48 h after harvest or, in delayed CA, the fruit maintained in air for 46 d at -0.5 °C (±0.3) before the pO2 was reduced to 3.0 and 0.5 kPa O2.

Pull down of pO2 required 18 and 26 h to reach 3.0 and 0.5 kPa O2, respectively.

The storage temperature, relative humidity, pO2 and pCO2 were continuously monitored and controlled automatically (Isolcell Italia, Laives, Italy). After 257 d in storage, fruit were evaluated during 7 or 8 d of shelf life at 20 °C for quality traits or for ethylene production and respiration rates, respectively.

Assessment of fruit quality Poststorage fruit quality was assessed by changes in flesh firmness, skin color, juice total soluble solids and titratable acidity. Flesh firmness was measured after the skin removal with a handheld penetrometer (T.R. Turoni, Forli, Italy) equipped with an 8 mm probe. The maximum force required to insert the probe into the fruit flesh without skin was recorded. Two measurements were performed per fruit, on opposite sides of the equatorial region in 3 replicated lots of 15 fruit each. Skin color was measured in CIE L*a*b* color space with a tri-stimulus CR-400 chroma meter (Konica Minolta, Tokyo, Japan) with the C illuminant. The measurements were performed on widest part of the fruit in 3 replicated blocks of 15 fruit each. Juice extracted from pears (3 replicates of 15 fruit each) was used for TSS and TA measurements. TSS were measured with a refractometer (Hanna Instruments, Woonsocket, USA). A volume of 10 mL juice was diluted in 90 mL distilled water before titration with 0.1 M NaOH until pH 8.1 and TA expressed in malic acid equivalents.

Internal disorders The occurrence of internal disorders was monitored immediately after removal of fruit from storage in three replicated batches of, at least, 60 fruit each, after 91, 134, 187 and 257 d of storage. Fruit were cut transversely and longitudinally in three sections to assure the detection of disorders. Symptoms of core browning and necrotic cavities (Saquet and Almeida, 2017a) were observed, were combined and damage incidence is expressed as the percentage of fruit affected.

Analysis of ethylene production and CO2 release Ethylene production rate was measured in three replications of 4 fruit each. Fruit samples were placed inside of 2.15 L sealed glass jars and maintained at 20 ºC for 2 h. A headspace volume of 0.1 mL was removed from the jars through a rubber septum and injected into a gas chromatograph (Trace 1300, Thermo Fisher Scientific Inc., Marietta, USA) fitted with a capillary column TG bond alumina (Na2SO4) 50 m length and 0.53 mm i.d. (Thermo Fisher Scientific Inc., Marietta, USA) as described (Saquet and Almeida, 2017b).

The respiration rate, expressed as release of CO2, was determined in the same fruit samples used for ethylene measurements, immediately after ethylene determination.

Headspace CO2 concentration was measured with an infrared gas analyzer (Oxycarb 6, Isolcell,

Laives, Italy) with a continuous flow rate of 100 mL min-1 and the respiration rate calculated on a fresh mass basis.

Data analysis Data on the percentage of affected fruit were subjected to the arcsine square root transformation (McDonald, 2014) before analysis of variance (ANOVA). Data were subjected to one-way ANOVA with the storage treatment as a fixed factor according to a randomized block design with 3 replicates. Means were separated by the least significant difference (LSD) test at α=0.05. Statistical analysis was made with the software Action Stat (2014, São Carlos, SP, Brazil).

Results and discussion

Physicochemical fruit characteristics Successful long-term storage of pear must assure firmness maintenance during storage, but allow softening during subsequent ripening in shelf life. Fruit firmness was lower after 257 d in storage at 3.0 kPa O2 than at 0.5 kPa O2 (Fig. 1). Fruit softened during the 7-d shelf life at 20 ºC after removal from storage to final firmness values of 15.1 to 23.6 N, an ideal range for consumption (Kappel et al., 1995; Villalobos-Acuña et al., 2011a), with no significant differences between storage conditions (Fig. 1). Delayed CA of 21 d has been reported to have no detrimental effect on firmness retention in ‘Conference’ pear (Höhn et al., 1996; Saquet et al., 2001), but accelerated softening in ‘Elstar’ apple (Streif and Saquet, 2003).

Fig. 1. Flesh firmness of ‘Rocha’ pear after 257 d storage at -0.5 ºC and 0.6 kPa CO2 under different O2 partial pressure regimes followed by 7 d shelf life at 20 °C. Vertical bars are the standard deviations. 92

Hue angle decreased during storage when pO2 was 3.0 kPa but not under 0.5 kPa O2

(Fig. 2). The effect of pO2 on color prevailed over that of the delay period. After removal from storage, pear from all storage conditions yellowed and the initial effect of storage pO2 on hue angle did not persist after 7 d in shelf life (Fig. 2). The effect of low pO2 in keeping green color is well documented in pear (Mattheis and Rudell, 2011; De Martin et al., 2015). No color differences were observed in ‘Conference’ pear subjected to a 21-d delay in CA establishment in relation to those immediately stored in CA (Saquet et al., 2003a). The color of ‘Rocha’ pear pre-conditioned for 60 d before CA storage did not differ after 9.4 months from that of fruit stored under immediate CA (Almeida et al., 2016).

Fig. 2. Skin color of ‘Rocha’ pear after 257 d storage at -0.5 ºC and 0.6 kPa CO2 under different

O2 partial pressure regimes followed by 7 d shelf life at 20 °C. Vertical bars are the standard deviations.

TSS increased during 257 d in storage from 112 g kg-1 to the range 116 to 122 g kg-1 with no significant differences of pO2 of the delay in the atmosphere regime (Fig. 3). After 7 d of shelf life, no significant differences in TSS were found between treatments (Fig. 3), consistent with the complete breakdown of starch.

Fig. 3. Total soluble solids of ‘Rocha’ pear after 257 d storage at -0.5 ºC and 0.6 kPa CO2 under different O2 partial pressure regimes followed by 7 d shelf life at 20 °C. Vertical bars are the standard deviations.

The 2 g kg-1 TA measured at harvest decreased to 0.7 to 0.8 g kg-1 during 257 d storage, with no significant effect of storage regime (Fig. 4). TA remained in the range of 0.6 g kg-1 after 7 d shelf life in fruit from all storage treatments (Fig. 4). A relative stability of TA during CA storage and subsequent shelf life has been reported for ‘Rocha’ pear (Almeida et al., 2016).

Fig. 4. Titratable acidity of ‘Rocha’ pear after 257 d storage at -0.5 ºC and 0.6 kPa CO2 under different O2 partial pressure regimes followed by 7 d shelf life at 20 °C. Vertical bars are the standard deviations.

Ethylene production and respiration rate Ethylene production rate after 187 or 257 d in storage was lower in fruit previously stored under delayed CA at 3.0 or 0.5 kPa O2 than those under immediate CA (Fig. 5 A and C). The evolution of ethylene production rate during shelf-life in air at 20 ºC differed in fruit previously stored under 3 or 0.5 kPa O2: declined sharply in the former but increased for 3 d before decreasing in the latter conditions (Fig. 5 A and C). After 187 d storage, the respiration rate of fruit from immediate CA storage was 40 % or 24 % lower than those subjected to delayed storage at 3.0 or 0.5 kPa O2, respectively, after 8 d shelf life (Fig. 5 B). Respiration rate increased during poststorage shelf life at 20 ºC either after 187 or 257 d storage showing including significant differences in their intensities according to storage conditions (Fig. 5 B and D). Pear stored in immediate CA respired at significantly lower rates after storage than the fruit stored in delayed CA. However, after 257 d in storage, no significant differences were observed between both delayed CA conditions (Fig. 5 D). A previous study in ‘Rocha’ pear reported statistically similar respiration rate of fruit stored at 3 or 0.5 kPa O2 after 136 d and similar levels of ATP and adenylate energy charge (Saquet and Almeida, 2017a). Furthermore, even under immediate 0.5 kPa O2 storage, ‘Rocha’ pear fruit had the ability to recover the respiration rate during the 8-d shelf life period in air at 20 °C (Fig. 5 B and D). This recovery capacity of respiratory rate of ‘Rocha’ pear in response to delayed CA differs from that reported for various apple cultivars, in which a strong residual effect of CA conditions is observed in fruit metabolism during 7-d shelf life at room temperature (Patterson et al., 1974; Brackmann et al., 1993; Saquet et al., 2003c).

Fig. 5. Ethylene production (A, C) and respiration (B, D) rates of ‘Rocha’ pear during 8 d shelf life at 20 ºC after storage at -0.5 ºC and 0.6 kPa CO2 under different O2 partial pressure regimes for 187 d (A, B) and 257 d (C, D). Vertical bars are the standard deviations.

The response of respiration to ethylene production in climacteric fruit is well- established (Lelièvre et al., 1997; Pech et al., 2008), but ‘Rocha’ pear fruit respiration was not synchronized with ethylene production during the shelf life period (Fig. 5 A, B, C and D). The increase in respiration measured during shelf life of ‘Rocha’ pear independent of previous or concomitant increase in ethylene production rate suggests a postclimacteric stage accelerated by the fruit exposure to 20 ºC.

Occurrence of internal disorders Internal disorders in ‘Rocha’ pear were first detected after 134 d in storage. Fruit stored under 0.5 kPa O2 after a 46 d delay in the pO2 pull down had 23.6 % of internal disorders, an incidence that increased slightly to 27.3 % at the end of storage period of 257 d (Fig. 6). The immediate pull down of pO2 to 0.5 kPa O2 prevented the development of internal disorders for 257 d storage (Fig. 6).

Fig. 6. Occurrence of internal disorders in ‘Rocha’ pear during 257 d storage period at -0.5 ºC and 0.6 kPa CO2 under different O2 partial pressure regimes. Vertical bars are the standard deviations.

Storage under immediate CA at 3.0 kPa O2 induced damage in 4.4 % of fruit after 134 d, a proportion that increased to 15.6 % after 187 d and to 63.3% after 257 d storage (Fig. 6).

The delayed establishment of 3.0 kPa O2 reduced the proportion of affected fruit in relation to the immediate CA: 2.3 % after 187 d and 35.5 % after 257 d in storage (Fig. 6).

Clearly, a 46 d delay in pO2 pull down reduced the internal disorders in fruit stored at th 3.0 kPa up to the 187 d in storage, but strongly increased the disorders at 0.5 kPa O2. The

96 development of internal disorders in ‘Rocha’ pear after long-term storage with pO2 between

2.5 and 3.0 kPa and pCO2 below 0.7 kPa is consistently reported (Silva et al., 2010; Almeida et al., 2016). The results presented herein reveal a remarkable tolerance of ‘Rocha’ pear to the extremely low 0.5 kPa O2 and the advantage of the immediate pull down in pO2 to 0.5 kPa O2 to prevent the development of internal disorders during the 257 d storage period. A slight and inconsistent reduction in the occurrence of internal disorders was previously observed in ‘Rocha’ pear subjected to 56 d delayed CA followed by 9.4 months storage at 3.0 kPa O2 and 0.7 kPa CO2 in relation to immediate CA under the same conditions (Almeida et al., 2016), consistent with the results presented herein (Fig. 6). However, delaying the pull down of pO2 to 0.5 kPa was clearly detrimental to quality maintenance of ‘Rocha’ pear. The results of this study with ‘Rocha’ pear grown in the Oeste Region of Portugal contrast with the consistently reported positive effects of delayed CA on ‘Conference’ pear and apples produced in cooler summers of Northern Europe and North America. Delayed CA reduced the internal disorders in ‘Conference’ pear (Höhn et al., 1996; Saquet et al., 2001; Saquet et al., 2003a), and in the apple cultivars ‘Fuji’ (Argenta et al., 2000), ‘Elstar’ (Streif and Saquet, 2003), ‘Pink Lady’ (Castro et al., 2007), ‘Empire’ (DeEll and Ehsani-Mogaddam, 2012), and ‘Braeburn’ (Saquet et al., 2003b; Hatoum et al., 2014; Neuwald et al., 2014). However, in ‘Santana’ (Neuwald et al., 2015b) and ‘Kanzi’ (Kittemann et al., 2015) apples, which are susceptible to internal disorders during CA-storage, delaying CA for 21 d aggravated the occurrence of damaged fruit.

The differential effect of delayed CA depending on storage pO2 has not been studied in other pear cultivars; it is therefore unclear whether the detrimental effect of delayed on internal disorders under extremely low pO2 is specific of the cultivar and growing region or is a general response of pome fruit.

Practical implications of the relationship between delayed CA and pO2 Until the recent ban on diphenylamine as a postharvest treatment the recommended

CA-storage conditions of ‘Rocha’ pear were 2.5 to 3.0 kPa O2 and less than 0.7 kPa CO2 (Silva et al., 2010; Almeida et al., 2016). Despite the positive effect of diphenylamine, ‘Rocha’ pear stored under these CA-conditions develop internal disorders (Silva et al., 2010; Almeida et al.,

2016). Current nitrogen generators, CO2 scrubbers, gas sensing and control system allow the maintenance of very low pO2 and pCO2 with a precise control of gas concentrations. Therefore, the reassessment of storage protocols is warranted.

The high incidence of internal disorders in ‘Rocha’ pear after 257 d in storage at 3.0 kPa (63.3 %) was reduced to 35.5 % when the pull down in pO2 was delayed (Fig. 6). Despite this effect, the pre-conditioning protocol was not sufficient to assure enough poststorage quality. In contrast, the immediate storage at 0.5 kPa O2 prevented the development of internal disorders during 257 d storage (Fig. 6) and allowed normal poststorage ripening (Fig. 1 and 2).

Delaying the pull down to 0.5 kPa O2 enhanced the development of internal disorders (Fig. 6) and did not keep the fruit physicochemical characteristics at a commercially acceptable level.

Current technologies allow the operation of CA storage with the extremely low pO2 of

0.5 kPa and pCO2 of 0.6 kPa, conditions which prevented the development of internal disorders and maintained quality of ‘Rocha’ pear. Under extremely low pO2, delayed CA storage was detrimental for the quality of ‘Rocha’ pear grown in the Oeste Region of Portugal.

Conclusions

The responses of ‘Rocha’ pear to delayed CA storage was differently affected by pO2.

Delaying the pull down of pO2 for 46 d enhanced internal disorders in fruit stored under 0.5 kPa O2, but delayed their development in fruit stored at 3.0 kPa O2 until 187 d storage;

‘Rocha’ pear could be successfully stored under immediate 0.5 kPa O2 combined with

0.6 kPa CO2 for 257 d without development of internal disorders and fruit ripened to an adequate firmness and skin color during subsequent 7-d shelf life at 20 °C.

Chapter 8

General discussion During two consecutive seasons (2014/2015 and 2015/2016) experiments were carried out on the physiology and biochemistry of ripening and on the storability of ‘Rocha’ pear under various storage conditions. The storage temperature for pear cultivars including ‘Rocha’ has been well established between -1 and 0 °C (Porritt, 1964; Knee, 1987). The tolerance of pear to pCO2 during long-term CA storage is also defined and should be maintained below 0.7 kPa CO2 (Saquet et al., 2000; Streif et al., 2003; Pedreschi et al., 2008; Rizzolo et al., 2015; Isidoro and Almeida,

2006; Almeida et al., 2016). Therefore, focus was placed on determining the optimal pO2 during CA storage and investigate the effect of ethylene inhibition on fruit quality maintenance during and after long-term storage. The effect of delaying the pull down of pO2 in keeping quality of ‘Rocha’ pear during further CA storage was also investigated. In addition to the storage conditions, knowledge gaps regarding ripening of ‘Rocha’ pear were addressed.

The physiology and biochemistry of ‘Rocha’ pear ripening The investigation on postharvest ripening physiology and biochemistry of ‘Rocha’ pear treated with 1-MCP allowed new findings. One of the main outcomes was that ‘Rocha’ pear, in contrast with many other European pear cultivars (Villalobos-Acuña and Mitcham, 2008), ripened immediately after harvest without exposure to chilling exposition or exogenous ethylene application with a typical climacteric pattern of ethylene production and respiration rates (Chapter 3). ‘Rocha’ pear ripened to a buttery-juicy texture without chilling or exogenous ethylene treatment. Inhibition of ethylene action by 1-MCP treatment induced a transient reduction of ATP concentrations and AEC during ripening, while AEC of control fruit slightly increased during the first week and decreased to a stable value of 0.7 toward the end of ripening. ‘Rocha’ pear was able to adjust the AEC during ripening even when respiration rate was reduced by 1-MCP treatment. This specific study on aspects of energy metabolism in ripening ‘Rocha’ pear is a new contribution for the knowledge of postharvest science. Postharvest physiology and technology serve consumer satisfaction. Therefore, the characterization of ripening must address sensory perceptions in complement of instrumental quality evaluation. The similarities and differences in sensory and instrumental assessment of quality characteristics during ripening study were addressed in Chapter 4. Results showed that1- MCP used after a period of cold storage can modulate ripening without impairing it, a condition often reported in pear as evergreen (Chiriboga et al., 2011; Folchi et al., 2015). ‘Rocha’ pear

99 treated with 300 nL L-1 1-MCP was perceived by taste panelists as juicier, sweeter, and with higher flavor intensity compared to control fruit ripened at 10 or 20 °C. The sensory effects of 1- MCP on pear remain contradictory (Rizzolo et al., 2014; Escribano et al., 2016), but 1-MCP can be clearly be used to modulate the sensory profile of ripe ‘Rocha’ pear using the protocol adopted in this thesis. Positive correlations between instrumentally assessed skin color and flesh firmness with the sensory evaluated color and hardness were observed. For the pear industry, which currently does not resort to sensory evaluation due to high costs and time expenditure, this finding indicates that the instrumental measurements of skin hue angle and firmness are reliable indicators of the human perception of color and hardness.

Energy and mineral gradients within the fruit The detailed characterization of the radial and longitudinal gradients of adenylate nucleotides and AEC within ‘Rocha’ pear fruit showed a significant radial gradient in AEC decreasing from the skin tissues toward the fruit center (Chapter 2). However, no significant gradient from the proximal to the distal fruit sections was found. Radial AEC values in ‘Rocha’ pear were 0.80, 0.72, and 0.69 in the skin tissues, outer and inner flesh tissues, respectively. The occurrence of internal disorders during CA storage of pears begins around the core region (Franck et al., 2007), and the lower AEC measured in the fruit center is consistent with a role of cellular energy status on disorder development. The possible involvement of ATP and AEC in the development of internal disorders in ‘Conference’ pear was reported, and the energy shortage during CA storage was proposed as one possible reason associated to the development of internal disorders (Saquet et al., 2000; Saquet et al., 2001; Veltman et al., 2003). However, the association between energy status and internal disorders is not absolute; ‘Rocha’ pear seem to maintain an adequate level of AEC during long-term storage under which disorders may or may not occur (Chapter 6). The detailed mapping of macro- and micronutrients within ‘Rocha’ pear (Chapter 5) showed significant decreasing radial gradients in B and Ca concentrations from the skin tissues to the fruit center. Fruit affected by internal disorders had lower Ca and B concentrations than healthy fruit. B fertilization been shown to reduce the incidence of internal disorders in ‘Conference’ (Wójcik and Wójcik, 2003; Xuan et al., 2001; 2003; 2005) and ‘Concorde’ pear (Mielke and Chaplin, 2008). Ca plays an important role in keeping pear fruit quality related to storage disorders (Gorini, 1988; Curtis et al., 1990; Brunetto et al., 2015). According to these results, pear growers should manage the Ca and B supply in pear fruit to reduce the incidence of internal disorders.

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Internal disorders in ‘Rocha’ pear and long-term storage Storage trials evaluated the storability of ‘Rocha’ pear during 136 d at -0.5 ºC in air or -1 under 3.0 and 0.5 kPa O2 combined 0.6 kPa CO2, treated or not with 150 nL L 1-MCP (Chapter

6). The results demonstrate the high tolerance of ‘Rocha’ pear to the extremely low pO2, as low as 0.5 kPa O2, without occurrence of storage disorders. 1-MCP treatment slightly increased disorders incidence at 0.5 and at 3.0 kPa O2. The high tolerance of ‘Rocha’ pear to the extremely low 0.5 kPa O2 during the full storage period without internal disorders indicates that it is possible to maintain healthy fruit during long-term storage without chemical postharvest treatments with diphenylamine, ethoxyquin, or 1-MCP. The linkage between ATP and AEC and the incidence of internal disorders was not strong, since under the same pO2 1-MCP enhanced the incidence of disorders with a negligible effect on adenylate nucleotides or AEC. Furthermore, it was not possible to establish a threshold of ATP concentration or AEC below which internal disorders develop during storage. According to the results presented in this thesis, ‘Rocha’ pear is more tolerante than ‘Conference’ to the low pO2. The literature of pears in general stress the need to store the fruit at pO2 higher than 2 kPa to avoid internal disorders (Höhn et al., 1996; Saquet et al., 2003a; Nguyen et al., 2007; Pedreschi et al., 2008). In specific case of ‘Rocha’ pear there seems to be a risk zone of pO2 for internal disorders development in the range of pO2 ≤ 3.0 kPa

O2 and > 0.5 kPa O2. A further storage trial (Chapter 7) investigated the tolerance of ‘Rocha’ pear to immediate storage at 0.5 kPa O2 during 257 d at 0.5 kPa O2. ‘Rocha’ pear lasted 257 d without occurrence of storage disorders. Moreover, this storage condition allowed pear fruit with adequate firmness and skin color after 7 d shelf life at 20 °C. The effect of delayed pull down in pO2 on internal disorders depended on pO2 during storage period did not benefit long-term storage of ‘Rocha’ pear. This result differed from those reported with ‘Conference’ pear (Höhn et al., 1996; Saquet et al., 2003a; Verlinden et al., 2002), and various apple cultivars (Argenta et al., 2000; Streif and Saquet, 2003; Castro et al., 2007; Neuwald et al., 2014). Based on the results of this thesis, internal disorders of ‘Rocha’ pear can be minimized during long-term storage in the absence of chemical postharvest treatments if the fruit are stored at – 0.5 ºC with an immediate pull down of pO2 to 0.5 kPa.

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Conclusions

The main conclusions of this thesis are the following:

1. There was a radial gradient in AEC values within ‘Rocha’ pear fruit decreasing from 0.80 in the skin tissues to 0.69 in the inner fruit flesh tissues; 2. Immediately after harvest, ‘Rocha’ pear could ripen without exposure to chilling or exogenous ethylene application; 3. ‘Rocha’ pear, whether or not treated with 150 or 300 nL L-1 1-MCP, adjusted the AEC at a stable value around 0.7 and sustained ripening during 14 d shelf life at 20 °C; 4. Ripening temperature had a stronger effect on sensory profile of pear ripening than 1- MCP treatment, which can be used to modulate the sensory profile of ripe pears; Pears treated with 300 nL L-1 1-MCP were perceived as juicier, sweeter, and higher flavor intensity compared to control fruit; Instrumentally measured skin color and firmness correlated significantly with sensory scores in color and hardness, but sweetness and acidity were not significantly correlated with total soluble solids and titratable acidity; 5. There was a significant radial gradient in B and Ca concentrations within ‘Rocha’ pear decreasing toward the fruit center; Fruit with internal storage disorders had lower B and Ca concentrations in the flesh than healthy fruit; 6. ‘Rocha’ pear was very tolerant no hypoxia and was successfully stored at immediate

0.5 kPa O2 for 257 d without occurrence of storage disorders, after which time it ripened with suitable quality during 7 d shelf life at 20 °C;

7. Delaying the pull down of pO2 in 46 d was not effective in alleviating or preventing internal disorders during 257 d CA storage of ‘Rocha’ pear.

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Future research directions

Science brings us too many new questions. As new findings emerge, new research questions arise in the same field. The results presented herein contribute significantly for understanding of ‘Rocha’ pear postharvest physiology and support practical storage recommendations. However, new issues have emerged. The main research venues that, in our opinion, might further our understanding of the physiological behavior of ‘Rocha’ pear during storage and ripening are: - To elucidate the metabolism underlining the ability of ‘Rocha’ pear to ripen at stable AEC even under lower respirations rates; - The ability of ‘Rocha’ pear to overcome the blockage effect that 1-MCP normally induce in pears remains unclear and deserves in-depth biochemical analysis in relation to ethylene sensing and action in the coordination of ripening; - A detailed investigation on the sensory profile of ‘Rocha’ pear during ripening, particular the changes in mechanical properties such as the texture as affected by storage conditions and postharvest treatments are important to allow the modulation of texture according to market preferences; - To investigate the role of Ca and B in the development of storage disorders; - The present investigation regarding the energy metabolism was not enough to clarify

the capacity of ‘Rocha’ pear to stand storage for 8.5 months at 0.5 kPa O2 without internal disorders. New investigation involving the energy status and fermentative metabolism could help to improve the knowledge about the fruit metabolism under severe hypoxia; - Gas diffusion properties within pear fruit and possible changes in gas diffusion during long-term storage must be clarified, as they influence decisively the fruit metabolism during storage period.

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