P
HYSIOLOGY ANDB
IOCHEMISTRY OF‘R
OCHA’
P
EARD
URINGR
IPENING ANDL
ONG-
TERMC
ONTROLLEDA
TMOSPHERES
TORAGEADRIANO ARRIEL SAQUET
ORIENTADOR: Professor Doutor Domingos Almeida COORIENTADOR: Doutor Josef Streif
TESE ELABORADA PARA OBTENÇÃO DO GRAU DE DOUTOR EM ENGENHARIA AGRONÓMICA
P
HYSIOLOGY ANDB
IOCHEMISTRY OF‘R
OCHA’
P
EARD
URINGR
IPENING ANDL
ONG-
TERMC
ONTROLLEDA
TMOSPHERES
TORAGETESE 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).
ii
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)
iii
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
iv
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
v
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
vi
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
vii TCA Trichlor acetic acid
TCA cycle Tricarboxylic acid cycle TSS Total soluble solids
viii 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.
ix 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.
x 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.
xi
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
com 150 nL L-1 de 1-MCP também foram armazenados em 3.0 e 0.5 kPa de O
2 após 32 dias
xii
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.
xiii
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.
1
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
2
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).
3
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,
4
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).
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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
6
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
7
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
8
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
9
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
10
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
11
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
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‘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.
13
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
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(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.
15
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
16
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.
17
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.
18 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
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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
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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.
Nucleotide or AEC
Adenylate nucleotides (nmol g-1 FM) in the radial section of the fruit
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.
Nucleotide and AEC Adenylate nucleotides (nmol g
-1 FM) in the axial section of the fruit
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)
