Biochemical and physiological changes in Combretum leprosum Mart. seeds during storage

BIOCHEMICAL AND PHYSIOLOGICAL CHANGES IN

Combretum leprosum Mart. SEEDS DURING STORAGE

BRUNO SILVA GUIRRA

Macaíba/RN May of 2020

Nº 073 MINISTÉRIO DA EDUCAÇÃO

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE

PRÓ-REITORIA DE PÓS-GRADUAÇÃO

UNIDADE ACADÊMICA ESPECIALIZADA EM CIÊNCIAS AGRÁRIAS - UAECIA ESCOLA AGRÍCOLA DE JUNDIAÍ - EAJ

ii BRUNO SILVA GUIRRA

BIOCHEMICAL AND PHYSIOLOGICAL CHANGES IN

Combretum leprosum Mart. SEEDS DURING STORAGE

Dissertation presented to Programa de Pós-Graduação em Ciências Florestais da Universidade Federal do Rio Grande do Norte to obtain the degree of Master in Forest Science (Area of Concentration in Forest Sciences - Research Line: Seeds, Propagation and Physiology of Forest Species).

Advisor:

Prof. Dr. Salvador Barros Torres

Co-Advisor:

Prof. Dr. Caio César Pereira Leal

Macaíba/RN May of 2020

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Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI Catalogação de Publicação na Fonte. UFRN –

Biblioteca Setorial Prof. Rodolfo Helinski - Escola Agrícola de Jundiaí – EAJ

Elaborado por Valéria Maria Lima da Silva - CRB-15/451

Guirra, Bruno Silva

Biochemical And Physiological Changes In Combretum leprosum Mart. Seeds During Storage / Bruno Silva Guirra. - 2020.

53f.: il. –

Dissertation (Masters) - Universidade Federal do Rio Grande do Norte, Unidade Acadêmica Especializada em Ciências Agrárias, Programa de Pós- Graduação em Ciências Florestais, Macaíba, RN, 2020.

1.Forest Seeds – Dissertation. 2. Seed Physiological Quality – Dissertation. 3. Seed Metabolic Responses – Dissertation. 4. Viability – Dissertation. 5. Vigor – Dissertation. I. Torres, Salvador Barros. II. Leal, Caio César Pereira. III. Título.

iv

BIOCHEMICAL AND PHYSIOLOGICAL CHANGES IN Combretum leprosum Mart. SEEDS DURING STORAGE

Bruno Silva Guirra

Dissertation presented to Programa de Pós-Graduação em Ciências Florestais da Universidade Federal do Rio Grande do Norte to obtain the degree of Master in Forest Science (Area of Concentration in Forest Sciences - Research Line: Seeds, Propagation and Physiology of Forest Species) and approved by the examining board on May 29, 2020.

Examining Board

Prof. Dr. Salvador Barros Torres EMPARN/UFERSA/PPGCFL-UFRN

President

Prof. Dr. Mauro Vasconcelos Pacheco PPGCFL/EAJ/UFRN

Internal Examiner

Prof. Dr. Alek Sandro Dutra DF/CCA/UFC External Examiner

Macaíba/RN May of 2020

v To my friends, teachers and alumni, from the Escola Família Agrícola de Sobradinho-BA, for making me see that “good students prepare to receive a diploma, but fascinating students prepare for life, they are thinkers, close to the art of doubt and criticism”.

vi

ACKNOWLEDGMENTS

__________________________________________________________________________ To God, for the strength and graces achieved.

To my family - parents, brothers, nephews and brothers-in-law, especially my sister Keylan Guirra and her husband Eduardo Santos, who are always present supporting me during the academic journey.

To all professors of Programa de Pós-Graduação em Ciências Florestais of Universidade Federal do Rio Grande do Norte (PPGCFL/UFRN), for providing students with new experiences, which are essential for qualified professional training.

To colleagues at PPGCFL/UFRN, especially João Henrique, Eudes Silva, Janekely Buriti, Patrícia Baulduíno, Felipe Gomes, Raiane Sales and Adriana Ferreira, who were with me during this difficult and intense journey, but full of learning.

To my advisor, prof. Salvador Torres, and coadvisor, prof. Caio Leal, for the availability in which they have always shown themselves.

To colleagues at Laboratório de Análise de Sementes(LAS) of Universidade Federal Rural do Semi-Árido (UFERSA), in particular, Simara Melo, Washington Brito, Bruna Lima, Kleane Pereira, Tatiane Alves and Lília Souza, who helped me during the physiological and biochemical. Also to Gutierres Aquino, Ana Letícia Rêgo and Assis Nogueira, for making themselves available for the collection of mofumbo seeds.

To laboratory technicians from UFERSA, Sara Carvalho, from LAS; Vilma Amâncio, from Laboratório de Ciência Animal; Naama Melo, from Laboratório de Fitopatologia II; and Tatiana Barreto, from the Laboratório de Bioquímica, I thank you for the help and availability that were essential for the construction of this work.

To teachers Clarisse Benedito and Emanoela Paiva, who were part of the qualification panel and suggested very significant contributions. Also to the defense banking professors, Mauro Pacheco and Alek Dutra, who added even more to the work.

vii

GENERAL ABSTRACT

__________________________________________________________________________

BIOCHEMICAL AND PHYSIOLOGICAL CHANGES OF

Combretum leprosum MART. SEEDS DURING STORAGE

Combretum leprosum Mart., Combretaceae family, has exclusive distribution in South America. It is a species that significantly contributes to conservation and improvement of soil quality, as it facilitates the processes of recovery of degraded areas and succession in dry forests. Despite the ecological importance, there are still no basic studies aimed at the storage of its seeds. Thus, the objective was to evaluate the biochemical and physiological changes in C. leprosum seeds, based on the degradation of reserve tissues and accumulation of soluble metabolites during the storage period. The experimental design was completely randomized, in a factorial scheme (2 x 2 x 7), with two storage conditions x two forms of seed processing x seven storage periods (0; 60; 120; 180; 240; 300 and 360 days) and four replicates per treatment. After collecting C. leprosum fruits, half of the lot was manually processed by removing the external protection that covered the seed, while in the other half, the diaspores were kept. For storage, all seeds, both naked and protected with diaspores, were placed in kraft paper bags and kept under two storage conditions: natural environment and air-conditioned chamber (10±1°C; 53±7% relative humidity). At the beginning and at intervals of 60 days, for 360 days, the seeds were analyzed for moisture content, physiological quality (emergence, emergence speed index, and seedling length and dry mass) and biochemical quality (neutral lipids, total and reducing sugars, total free amino acids, starch and lipid peroxidation). Physiological variables showed better results under the condition of natural environment, regardless of the form of seed processing. This environment also promoted less degradation of lipid reserve. Regarding starch, there was hydrolysis of this polysaccharide throughout the storage period. Finally, it was found that seed deterioration after 180 days of storage is associated with the attack of reducing sugars against amino acids, reducing viability and vigor.

Key words: forest seeds, seed physiological quality, seed metabolic responses, viability,

viii

RESUMOGERAL

__________________________________________________________________________

ALTERAÇÕES BIOQUÍMICAS E FISIOLÓGICAS DE SEMENTES DE

Combretum leprosum Mart. DURANTE O ARMAZENAMENTO

Combretum leprosum Mart., família Combretaceae, possui distribuição exclusiva na América do Sul. Trata-se de uma espécie que contribui expressivamente para conservação e melhoria da qualidade do solo, pois é facilitadora no processo de recuperação de áreas degradadas e sucessão em florestas secas. Apesar da importância ecológica, ainda não se tem estudos básicos voltados ao armazenamento de suas sementes. Dessa maneira, objetivou-se avaliar as alterações bioquímicas e fisiológicas em sementes de C. leprosum, com base na degradação dos tecidos de reservas e acúmulo de metabólitos solúveis, durante o período de armazenamento. O delineamento experimental adotado foi inteiramente casualizado, em esquema fatorial (2 x 2 x 7), com duas condições de armazenamento x duas formas de beneficiamento da semente x sete períodos de armazenamento (0; 60; 120; 180; 240; 300 e 360 dias) e quatro repetições por tratamento. Após a coleta dos frutos de C. leprosum, metade do lote foi beneficiado manualmente, removeu-se a proteção externa que recobria a semente e, a outra parte, manteve-se os diásporos. Para realização do armazenamento, todas as sementes nuas e as protegidas com os diásporos foram acondicionadas em sacos de papel kraft e mantidas em duas condições de armazenamento: ambiente natural e câmara climatizada (10±1°C; 53±7% de umidade relativa). No início e em intervalos de 60 dias, durante 360 dias, as sementes foram analisadas quanto à determinação do teor de água, qualidade fisiológica (emergência, índice de velocidade de emergência, comprimento e massa seca de plântulas) e bioquímica (lipídios neutros, açúcares totais e redutores, aminoácidos livres totais, amido e peroxidação de lipídios). As variáveis fisiológicas expressaram melhores resultados na condição de ambiente natural, independente da forma de beneficiamento da semente. Esse ambiente também proporcionou menor degradação da reserva de lipídios. No tocante ao amido, houve hidrólise deste polissacarídeo ao longo do período de armazenamento. Por fim, verificou-se que a deterioração das sementes após 180 dias de armazenamento está associada ao ataque dos açúcares redutores aos aminoácidos, reduzindo a viabilidade e vigor.

Palavras-chave: sementes florestais, qualidade fisiológica de sementes, respostas

ix SUMMARY __________________________________________________________________________ Page 1.GENERAL INTRODUCTiON ... 1 2.GENERAL OBJECTIVE ... 4 3. LITERATURE REVIEW ... 6

3.1. General aspects about the species ... 6

3.2. Storage of seeds ... 7

3.2.1. Deterioration of seeds and biochemical responses during storage ... 11

3.3. Maintenance of seed viability through morphological structures. ... 12

4. MATERIAL AND METHODS ... 14

4.1. Obtaining of seeds ... 14

4.2. Storage conditions ... 14

4.3. Determination of seed moisture content... 14

4.4. Physiological evaluations ... 14

4.4.1. Emergence ... 14

4.4.2. Emergence speed index (ESI) ... 15

4.4.3. Seedling length ... 15

4.4.4. Seedling dry mass ... 15

4.5. Biochemical evaluations ... 15

4.5.1. Neutral lipids (NL) ... 15

4.5.2. Lipid peroxidation (LP) ... 15

4.5.3. Total soluble sugars (TSS), reducing sugars (RS) and total free amino acids (TFAA) ... 16

4.5.4. Starch ... 16

4.6. Statistical analysis and experimental design ... 16

5. RESULTS AND DISCUSSION ... 18

6. CONCLUSIONS... 29

7. ACKNOWLEDGMENTS ... 31

x

FIGURE LIST

__________________________________________________________________________

Figure 1. Emergence of Combretum leprosum Mart. seeds as a function of environment

conditions and storage periods... ... 19

Figure 2. Emergence speed index of Combretum leprosum Mart. seeds as a function of the

environment conditions and storage periods. ... 20

Figure 3. Length of Combretum leprosum Mart. seedlings as a function of storage periods.21 Figure 4. Total dry mass of seedlings of Combretum leprosum Mart. as a function of storage

periods. ... 21

Figure 5. Neutral lipids of Combretum leprosum Mart. seeds as a function of forms of seed

processing, environment conditions and storage periods. ... 23

Figure 6. Lipid peroxidation in Combretum leprosum Mart. seeds as a function of forms of

seed processing, environment conditions and storage periods. ... 24

Figure 7. Total soluble sugars (A) and reducing sugars (B) of Combretum leprosum Mart.

seeds as a function of the storage periods ... 25

Figure 8. Starch content in Combretum leprosum Mart. seeds as a function of storage

periods ... 26

Figure 9. Total free amino acids of Combretum leprosum Mart. seeds as a function of

xi

TABLES LIST

__________________________________________________________________________

Table 1. Moisture content in Combretum leprosum Mart. seeds as a function of forms of seed

processing, environment conditions and storage periods ... 18

Table 2. Summary of analysis of variance for emergence (E), emergence speed index (ESI),

total seedling length (TSL), total seedling dry mass (TSDM) of Combretum leprosum Mart. as a function of the forms of seed processing, environment conditions and storage periods... 19

Table 3. Total dry mass of seedlings of Combretum leprosum Mart. as a function of the

environment conditions ... 22

Table 4. Summary of analysis of variance for neutral lipid (NL), lipid peroxidation (LP), total

soluble sugar (TSS), reducing sugar (RS), starch content (SC) and total free amino acids (TFAA) of Combretum leprosum Mart. seeds as a function of forms of seed processing, environment conditions and storage periods ... 22

Table 5. Total free amino acids of Combretum leprosum Mart. seeds as a function of the

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ABBREVIATIONS LIST

__________________________________________________________________________

B.O.D. - Biochemical oxigen demand ESI - Emergence speed index

LP - Lipid peroxidation MDA - Malonaldehyde NL - Neutral lipids

ROS - Reactive Oxygen Species RS - Reducing sugars

TFAA - Total free amino acids TSS - Total soluble sugars

xiii

General Introduction

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1

1. GENERALINTRODUCTION

__________________________________________________________________________ Combretum leprosum Mart. is a species popularly known in Portuguese as ‘mofumbo’, belonging to the Combretaceae family, whose distribution is exclusive to South America, with records specifically in Paraguay, Bolivia and Brazil (LOIOLA, 2009). In Brazil, it occurs in several ecosystems such as the Amazon Forest, Cerrado, Pantanal, Atlantic Forest and Caatinga, covering the North, Midwest, Northeast and Part of the Southeast (MARQUETE and VALETE, 2010).

This species has great importance for the Caatinga and for other Brazilian ecosystems, as it has numerous potentialities, from medicinal, beekeeping and timber applications, besides contributing to soil conservation (VIEIRA et al., 2013). In addition, C. leprosum is a facilitating agent during the processes of recovery of degraded areas and succession of dry forests (HORINOUCHI et al., 2013).

The demand and pressure on native species have increased sharply due to plant extractivism, leaving them at risk of joining the list of endangered species. In this context, C. leprosum is a threatened species in the face of the practices used for exploitation (MING et al., 2003).

Brazilian environmental policies encourage ecological restoration, increasing the demand for seeds of native species, which are basic inputs in ecosystem recovery and conservation programs (CARVALHO et al., 2006). Generally, the seeds are stored to be used in the same harvest year or even in the following years, because the production of seeds of these species is cyclical, characterized by a period of high production, followed by one or two of low production (GRUNENNVALDT et al., 2014).

Maintenance of seed viability and minimization of deterioration rate are objectives of storage, but these goals are achieved through conservation technologies that are appropriate for each species. Seed deterioration is identified from changes in respiratory activity and cell membrane degradation, which leads to the loss of cellular compounds and accumulation of toxic substances (MARCOS-FILHO, 2015). In addition, the reduction in seed quality can be verified by the decrease in germination, increase in the number of abnormal seedlings and reduction in seedling vigor (BILAL and ABIDI, 2015).

In this context, the possible physiological and biochemical changes that occur after physiological maturity are important for maintaining seed viability and vigor during storage (GUEDES et al., 2012).

Several factors cause effects on seed viability and vigor during storage, such as initial quality, moisture content, temperature, relative humidity, degree of infection by microorganisms and insects, type of package and duration of storage period (KOCSY, 2015).

2 Among these factors, temperature directly influences the speed of chemical reactions and respiratory activities of seeds, as it accelerates metabolism and directly contributes to the development of microorganisms, which consequently lead to deterioration (MARCOS-FILHO, 2015).

Loss of physiological quality in seeds may also be associated with their adaptive structure of maintenance and dispersion. Studies related to the storage of seeds protected by the fruit as an alternative for conservation are still scarce, but those with Caesalpinia leiostachya (BIRUEL et al., 2007), Crambe abyssinica (COSTA et al., 2012) and Ocotea puberula (VICENTE et al., 2016) stand out.

3

General Objective

_____________________________

4

2. GENERALOBJECTIVE

__________________________________________________________________________

In view of the above, the objective was to evaluate the biochemical and physiological changes in C. leprosum seeds, based on the degradation of reserve tissues and accumulation of soluble metabolites, during the storage period.

5

Literature Review

_____________________________

6

3. LITERATUREREVIEW

__________________________________________________________________________

3.1. General aspects about the species

The Caatinga is an exclusively Brazilian ecosystem, concentrated in the Northeast region, characterized as dry forest. Its vegetation has multiple forms of use, as food, medicinal, energy and timber sources, besides being important for the maintenance of local biodiversity (PAREYN, 2010).

Native species of the Caatinga have been suffering from predatory exploitation, which put them at risk of extinction. Agricultural production models that use inappropriate technologies for soil preparation (such as fire) are the main responsible for anthropic pressure (SÁ et al., 2010).

In this context, studies indicate that only 40.56% of the Caatinga area still have remnants of native vegetation (SÁ et al., 2010). It is worth pointing out that one of the species found in greatest proportion in this ecosystem is popularly known in Brazil as ‘mofumbo’ (Combretum leprosum Mart.) (COSTA et al., 2009).

C. leprosum, belonging to the Combretaceae family, is a neotropical plant species with exclusive distribution in South America, recorded in Bolivia, Paraguay and Brazil (LOIOLA, 2009). In Brazil, it is found in the Amazon Forest, Atlantic Forest, Caatinga, Cerrado and Pantanal, in the states of Pará, Amazonas, Tocantins (North); Maranhão, Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Bahia (Northeast); Mato Grosso, Goiás, Mato Grosso do Sul (Midwest); and in Minas Gerais (Southeast) (MARQUETE and VALETE, 2010).

Considered a scandent shrub, C. leprosum can reach up to 15 m in height and has semideciduous, heliophytic and selective xerophytic characteristics (LORENZI, 2009). Its leaves are opposite and petiolate; chartaceous to sub-coriaceous; elliptical, broadly elliptic to round; and abaxially prominent veins. The inflorescences are panicles of racemes, dense, congested, terminal and axillary with subsessile flowers, with flowering in the rainy season and fruiting in the dry season (AMORIM et al., 2009; LOIOLA, 2009).

The species C. leprosum is anemochorous, with the fruit being the unit of dispersion. The fruits are of the betuloid type, broadly elliptical, dry, indehiscent, winged and monospermic. Their epicarp is light green in color when unripe and reaches a reddish brown or yellowish brown color as they ripen (BARBOSA et al., 2003; BARROSO et al., 2004).

The seeds accompany the shape of the fruit, being ovoid or ellipsoid, with rough appearance and brown color. The species is propagated sexually, after the removal of the seeds from the fruit. Germination is hypogeal, crypto-cotyledonary with reserve cotyledons,

7 radicle protrusion usually occurs four days after sowing (LIMA et al., 2009; PAULINO et al., 2013), and the formation of normal seedlings is stabilized at 19 days after sowing (PACHECO et al., 2014).

Studies related to C. leprosum have shown that the species has great forest importance for the various ecosystems, considering that its presence significantly contributes to conservation and improvement of soil quality, as it facilitates the recovery of degraded areas and succession in dry forests (VIEIRA et al., 2013). In addition, it has timber potential, being used in the manufacture of wooden boxes and plywood and for firewood. It also presents itself as an alternative to beekeeping production (LORENZI, 2009). Flowers and leaves are used by folk medicine to control bleeding and prevent rashes, besides acting as sedative, expectorant and anti-inflammatory drugs (HORINOUCHI et al., 2013).

Also in relation to the use of C. leprosum in medicine, pharmacological studies use the bark of its trunk and root to produce ethanol extracts that have gastroprotective and chest- relaxing activity (ALVES FILHO et al., 2015). When removed from dry roots, ethanol extract has 65% arjunolic acid, with anti-inflammatory, antinociceptive and anticholinesterastic activities, revealing a new class of natural products in the treatment of Alzheimer’s disease (FACUNDO et al., 2005). Results of the study conducted by Fernandes et al. (2014) provide scientific support for the popular use of this extract in the treatment of snake bites, with emphasis on the arjunolic acid component as a promising anti-venom substance.

3.2. Storage of seeds

Seeds reach maximum germination and vigor close to physiological maturity, a stage in which there is the greatest accumulation of dry mass, consequently leading to complete formation of biochemical, morphological and structural systems. This is an event that is not always noticeable, as there is variation between species and even within the same species (CARVALHO and NAKAGAWA, 2012).

Seeds should be collected from trees immediately after harvest point, otherwise they will be exposed to climatic variations of the environment, which accelerate the deterioration process. During seed storage under controlled environmental conditions, the process of deterioration can be delayed, but in the case of C. leprosum, there are no studies on physiological and biochemical changes as a function of storage conditions.

Typically, seeds of forest species are not used immediately after collection. Therefore, it is important to store them for future use, considering that the native species have a cyclic production of seeds, characterized by a period of high production, followed by another of low production. Therefore, it is necessary to minimize the rate of seed deterioration, and it is essential to use appropriate conservation technologies for each species (KISSMANN, 2009).

8 Thus, for providing continuous availability of viable seeds, seed storage directly contributes to ecological studies with practical applications of biodiversity management and conservation (FLORIANO, 2014).

Regarding the essential conditions to conserve the viability of forest sees during storage, Carvalho and Nakagawa (2012) pointed out some factors such as the moisture content of the seeds, relative humidity of the environment and temperature. According to Cardoso et al. (2012), any seed stored is subject to deterioration, but this can be delayed depending on the environmental conditions and characteristics of the seed itself.

Temperature is responsible for accelerating seed metabolism and directly contributes to the development of microorganisms, consequently leading to deterioration. In this context, the best way to conserve seed quality is usually storage under conditions of low relative humidity and low temperature, because the embryo will be kept at low metabolic activity and the chance of microorganism development is minimized (MARCOS-FILHO, 2015). However, the responses vary depending on species and storage time (GONÇALVES, 2015).

A study with seeds of Acca sellowiana O. Berg. (GOMES et al., 2013) reports on its tolerance to drying, but warns about its intolerance to low storage temperature for periods longer than 60 days. According to the authors, reduction in seed quality during storage may be related to moisture content and the high incidence of pathogens such as Aspergillus sp., Rhizopus sp. and Pestalotia sp.

In study with Libidibia ferrea Mart. seeds, placed in polyethylene terephthalate (PET) bottles and subjected to storage in different environments and periods, it was found that seed germination evaluated at nine months (34%) was lower than the germination obtained at three months (60%) and in freshly harvested seeds (67%) (GONÇALVES, 2015). According to this author, the laboratory environment exposed the seeds to a more pronounced increase in moisture content, which consequently affected the germination of seeds stored under this environment condition, when compared to those kept in a cold chamber.

Storage of Psidium friedrichsthalianum O. Berg. seeds in hermetic containers for three months, with moisture content of 15.4%, led to a decrease in physiological quality, which resulted in the death of the seeds at six months of storage (GENTIL et al., 2018). It is should be noted that the waterproof package probably favored deterioration.

In seeds of Tabebuia roseoalba Ridl., Abbade and Takaki (2014) verified that the storage period significantly compromised germination and emergence, which were initially 99 and 88% in freshly harvested seed, respectively, and changed to 52 and 14% at 24 months. Therefore, knowledge about the physiology and characteristics of seeds of native species involving germination and storage is indispensable for the generation of appropriate technologies for propagation, conservation of phytogenetic resources and rational management (GENTIL et al., 2018).

9 Therefore, storage should always be carried out under appropriate conditions for the species to be used, as this ensures the maintenance and physiological viability of the seeds, through the reduction of metabolic processes. Thus, storage becomes a very important practice when the goal is to preserve the physiological characteristics of seeds for a certain period, because it acts directly in the control of deterioration processes (SRAVANTHI et al., 2013).

Other problems related to seed storage are directly associated with the use of inappropriate packages, which facilitate the attack of insects and fungi, contributing to changes in the physiological quality of the stored material. Therefore, attention should be paid to the care with the propagation material to be stored, especially with factors related to moisture content, temperature and choice of package for the packaging, conservation and maintenance of quality of seeds during the storage period (CARVALHO and NAKAGAWA, 2012).

Packages for seed storage are classified according to the degree of permeability to water vapor as permeable, semipermeable and impermeable (BAUDET and VILLELA, 2006). Permeable packages allow water vapor exchange between seeds and atmospheric air and can be made from cotton fabric, jute, paper and cardboard. Semipermeable packages offer some resistance to moisture exchange and are produced from material of thin plastic bags or polyethylene. Impermeable packages, on the other hand, do not allow air humidity to exert influence on the seed, being mostly produced based on metal (can), plastic and glass (BRASIL, 2009).

The adequate choice of packages is an important factor to be considered because, when seeds are stored in packages that allow exchange of water vapor with the atmospheric air, there may be absorption of moisture in places with high relative humidity, consequently leading to seed deterioration more quickly (MARCOS-FILHO, 2015).

Regardless of the species, seeds stored in permeable packages may be more influenced by the climatic conditions of the storage site than those stored in semi-permeable and impermeable packages (SILVA et al., 2010).

In a study with Handroanthus heptaphyllus Vell. seeds, storage in plastic bags kept in an air-conditioned room or in a cold chamber was the most appropriate method for seed preservation for a period of 300 days (TONETTO et al., 2015). According to the authors, the package and storage environment in cold chamber promoted lower incidence of fungi.

In the experiment conducted by Lisboa et al. (2014), the storage of Cajanus cajan L. seeds was evaluated. These authors found that the packages of PET bottles and polyethylene bags were more efficient than Kraft paper for conservation, because they maintained the vigor and viability of the seeds throughout the ten months of storage, regardless of the tested environments.

10 In another case, with research focused on storage of Talisia esculenta (A. St. Hil.) Radlk. seed, Sena et al. (2016) concluded that seeds should be stored until 25 days in a cold chamber environment (18 °C, 50% relative humidity) or natural laboratory environment (28 ± 5 °C, 65% relative humidity) and packed in polyethylene bags. These same authors also found that neither the Kraft paper bag nor the PET bottle should be used for storage of T. esculenta seeds, as they caused reduction in seedling vigor.

The physiological quality of Amburana cearensis (Allemão) AC Sm. seeds was also affected by the storage environment only when permeable package was used (LUCIO, 2016). Paper bag package resulted in lower physiological quality of seeds after nine months of storage under cold chamber condition.

Temperatures during storage can significantly influence longevity and viability, even in species with orthodox characteristics, such as Tabebuia aurea Benth. This species maintained viability for 360 days when stored under refrigeration conditions at 13 °C, both in paper and plastic packages. However, if stored under ambient conditions, the longevity and maximum viability of its seeds was 150 days (NEVES et al., 2014).

For storage of seeds in controlled environment systems, the cold chamber is the most used, as it allows the maintenance of temperatures below 10 °C, besides providing control of air humidity inside the chamber (ZONTA et al., 2014). In general, the interaction of the factors temperature and type of package is the reason why seed longevity may vary (MARCOS-FILHO, 2013). This occurs mainly due to the exchange of moisture and oxygen from the environment with seed tissues (NERY et al., 2014).

This interaction between package and temperature can be verified in the study conducted by Nery et al. (2017), in which the seeds of Calophyllum brasiliense Camb. did not show significant losses of viability and vigor when stored for nine months under low temperature conditions. In relation to the germination speed index, there were also no significant differences between seeds kept in cold chamber and those kept at ambient temperature, despite a difference for polyethylene package in the three-month storage. On the other hand, there were no significant differences for paper and aluminum packages over the storage period.

Seeds kept in a cold chamber and at ambient temperature varied in terms of moisture content, especially when considering the type of package used. Under cold chamber conditions, it was also found that there was a reduction in the initial moisture content of seeds packed in polyethylene bags, compared to other packages in all treatments evaluated (NERY et al., 2017).

11 3.2.1. Deterioration of seeds and biochemical responses during storage

In germplasm banks, seed viability is routinely evaluated only through the germination test. However, this test does not make it possible to detect the progress of the deterioration, because it only indicates the final stages of the process. However, the initial stages of deterioration could be detected throughout storage, which would contribute to the understanding of the physiological condition of the seeds of certain species during the conservation period (DONA et al., 2013).

For forest species, there are almost no specific studies regarding the physiological and biochemical responses of seeds during storage, especially related to long-term conservation, which makes it difficult to establish adequate methods for the conservation of these species (GUEDES et al., 2012). Knowledge about the performance of forest seeds, when subjected to different storage conditions is important for the rational management of the species, as it ensures a longer period of availability of seeds with high germination rate and vigor.

Storage environments with high relative humidity and high temperature are factors that directly affect the physiological quality of seeds, because they influence metabolic processes, especially respiratory rate, leading to faster deterioration (MARCOS-FILHO, 2015). To maintain the physiological and biochemical quality of seeds during storage, several conservation techniques have been developed and improved (BEWLEY et al. 2013). However, there is still no concrete information about the mechanisms responsible for deterioration during the natural ageing of seeds. It is known that, at first, seed deterioration is identified from the degradation of the cell membrane, which leads to the loss of cellular compounds.

Therefore, it is possible to infer that the reduction in the internal quality of seeds begins through several deterioration reactions that damage the biomolecules, including peroxidative, oxidative and hydrolytic reactions (BLACK et al., 2006; BEWLEY et al., 2013). Peroxidative reactions occur through the insertion of oxygen atoms into biomolecules; and oxidative reactions involve the removal of electrons from carbon chains. Hydrolytic reactions can occur in seeds with higher moisture content and involve actions of enzymatic and non-enzymatic degradation of reserves such as hydrolysis of sucrose and oligosaccharides, de-esterification of triacylglycerols and depolymerization of starches (BLACK et al., 2006).

During the deterioration process, it is also noted that the activity of the enzyme is reduced, the functionality of the membranes is altered, and there are lipid peroxidation, enzyme inactivation and nucleic acid degradation (BEWLEY et al., 2013). The reduction in the activity of enzymes that capture Reactive Oxygen Species (ROS) increases the sensitivity of seeds to oxidative stress, since ROS directly influence seed aging (BAILLY et al., 2008; WALTERS et al., 2010). The enzymes superoxide dismutase, catalase and

12 peroxidase eliminate ROS and peroxides, as well as malate dehydrogenase, acid phosphatase and glutamate dehydrogenase, which are indicative of deterioration, because they are involved in cellular metabolism (BERJAK, 2006). Therefore, it is pertinent to emphasize that information on the activity of certain enzymes, added to information on the degradation of reserves and/or biosynthesis of new tissues, can be used in studies related to seed deterioration (TAVEIRA et al., 2012).

Degradation in orthodox seeds during storage is also visualized through non-enzymatic hydrolysis of carbohydrates, which is considered one of the main mechanisms in the aging process. The release of reducing sugars and Maillard reactions culminate in the progressive loss of viability and vigor (VESELOVA et al., 2015).

In a study with Tabebuia roseoalba Ridl. seeds, Abbade and Takaki (2014) found that lipid content was reduced during storage and that the higher the peroxidase activity, the better the preservation of the physiological quality of the seeds. However, the longer the storage period, the lower the activity of peroxidase, which favors deterioration. The authors also revealed that the stabilization of enzyme activity after twelve months of storage coincided with that of lipid content, whose reduction is also stabilized after the same period.

3.3. Maintenance of seed viability through morphological structures

Information on the mechanisms that lead to losses of physiological quality of seeds during storage is not yet fully described in the literature. Thus, seed deterioration may also be associated with adaptive structures of seed dispersal. In some studies, the structures are cited as being extremely important for the survival of certain species in the habitat (BICALHO et al., 2016).

The forms of maintenance of diaspores may have palisade layers that influence germination aspects, as they act as a physical barrier that regulates water entry, induce seed dormancy and influence germination time (BASKIN and BASKIN, 2014).

With regard to the adaptive structure to maintain seed viability during storage, Randi (1982) working with Ocotea puberula Rich recommends that the seeds be stored with the fruits, as they contain substances that inhibit germination and induce dormancy during storage, hence enabling a longer period of viability.

With Caesalpinia leiostachya Benth. seeds stored within the fruit, BIRUEL et al. (2007) verified the efficiency of this method in maintaining the physiological quality, because the seeds showed greater viability and longevity compared to those stored without fruit.

13

Material and Methods

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14

4. MATERIALANDMETHODS

__________________________________________________________________________ The experiment was conducted at the Seed Analysis Laboratory of the Center for Agricultural Sciences of the Universidade Federal Rural do Semi-Árido (UFERSA), Mossoró, RN, Brazil, from January 2019 to February 2020.

4.1. Obtaining of seeds

Diaspores of C. leprosum were collected between August and December 2018 from different parent plants in the vicinity of BR-405, municipality of Apodi, RN, when the fruits already showed visible signs of having reached physiological maturity, with brown-colored epicarp. After collection, half of the lot was manually processed by removing the external protection of the fruit that covered the seed, while in the other half the diaspores were kept intact.

4.2. Storage conditions

For storage, both naked seeds and diaspores were packed in kraft paper bags and kept under two environment conditions: natural condition (26 ± 3 °C; 55 ± 12% relative humidity) and air-conditioned chamber (10 ± 1 °C; 53 ± 7% relative humidity), for 360 days. Before storage and at 60-day intervals, the seeds were evaluated for moisture content and physiological and biochemical quality.

4.3. Determination of seed moisture content

Prior to the installation of the experiment, the moisture content of the seeds was determined using the oven method at 105 ± 3 °C for 24 h (BRASIL, 2009), with two samples of approximately 4.5 g of seeds each, and the results were expressed as a percentage (wet basis).

4.4. Physiological evaluations

4.4.1. Emergence – four replicates of 25 seeds each were distributed in aluminum trays containing as substrate washed sand, previously sterilized in autoclave at 150 °C for 2 h. Substrate moisture was checked every day and irrigation was performed according to the need of the seedlings. The final counts of seedlings were performed 19 days after sowing, when seedling emergence was stabilized, and as recommended by Pacheco et al. (2014) for

15 the final evaluation in germination tests. For this, the seeds that originated normal seedlings were considered as emerged (BRASIL, 2009).

4.4.2. Emergence speed index (ESI) – conducted together with the emergence test, with the daily counts of emerged seedlings, according to the methodology recommended by Maguire (1962).

4.4.3. Seedling length – at the end of the emergence test, the normal seedlings of each repetition were measured with a ruler graduated in centimeters, and the results were expressed in cm seedling-_{¹, based on the recommendation of Nakagawa (1999).}

4.4.4. Seedling dry mass – normal seedlings, previously measured, were packed in paper bags and dried in a forced ventilation oven at 65 ºC for 72 h. The dried material was weighed on a precision analytical scale (0.001 g), with results expressed in mg seedling-_¹.

4.5. Biochemical evaluations

4.5.1. Neutral lipids (NL) - quantified by the gravimetric method (SOXHLET, 1879). For this, 1 gram of macerated seed was placed in a filter paper cartridge, transferred to the Soxhlet set and kept for six hours. The extraction was performed in the presence of hexane and, after complete evaporation and recovery of this solvent, the samples were weighed and the lipid content was determined.

4.5.2. Lipid peroxidation (LP) – determined based on the amount of malonaldehyde (MDA) present in the seeds, from the thiobarbituric acid reaction, according to the methodology proposed by Heath and Packer (1968). For this quantification, 0.2 g of seeds macerated in 2 mL of trichloroacetic acid (0.1%) was used and, immediately after, the material was centrifuged at 10000 rpm for 5 min. After centrifugation, 0.25 mL of the supernatant was collected, mixed with 1 mL of solution containing 0.5% thiobarbituric acid (w/v) + 20% TCA (w/v), kept incubated for 30 min in a water bath at 95 ºC and then put on ice to stop the reaction. After cooling, the samples were again centrifuged at 10000 rpm for 10 min, in order to separate any residue that may have been generated during heating and lighten the samples. Readings were performed in a spectrophotometer at 535 nm and 600 nm, and the results were later subtracted. MDA concentrations were estimated using an extinction coefficient of 155 (mmol L-1 _cm-1_{) as described by Meriga et al. (2004).}

16 4.5.3. Total soluble sugars (TSS), reducing sugars (RS) and total free amino acids (TFAA) – after the extraction of TSS, RS and TFAA, the samples with about 0.2 g of fresh mass of seeds were macerated in 2 mL of 80% ethanol (v/v). The extract was centrifuged at 10000 rpm for 8 min, the supernatant was collected and the precipitate was subjected to two new extractions with 1 mL of 80% ethanol (v/v), under the same conditions. The supernatants were collected at the end of the procedures, totaling 4 mL of extract per sample, while the residues were used for the extraction and determination of starch. TSS was determined by the anthrone method, which was established by Yemm and Willis (1954). RS was quantified by the colorimetric method of dinitrosalicylic acid - DNS (MILLER, 1959). TFAA was estimated by the method of Peoples et al. (1989), using the ninhydrin reagent, with glycine as the standard amino acid. The results for TSS, RS and TFAA were expressed in μmol g-1 DM.

4.5.4. Starch - starch extraction was performed using the precipitate generated from the extraction of low-molecular-weight soluble compounds (TFAA, TSS and RS). The precipitate was macerated with 2 mL of 30% perchloric acid (v/v) for its resuspension. The extract obtained was centrifuged at 3000 rpm for 5 min, collecting the supernatant and precipitates re-extracted with 1 mL of 30% perchloric acid (v/v) for two more times. At the end of the procedures, the supernatants were gathered, generating 4 mL of extract per sample. Starch content was quantified following the anthrone reagent method (YEMM and WILLIS, 1954; MORRIS, 1948).

4.6. Statistical analysis and experimental design

The treatments were distributed in a completely randomized experimental design, in a factorial scheme (2 x 2 x 7), with two storage conditions (natural environment at 26 ± 3 °C / 55 ± 12% relative humidity and air-conditioned chamber at 10 ± 1 °C / 53 ± 7% relative humidity) x two forms of processing (naked seeds and seeds with diaspores) x seven storage periods (0; 60; 120; 180; 240; 300 and 360 days) and four replicates per treatment. Quantitative factor data for the four treatments were subjected to analysis variance and the means were compared by Tukey test at 5% probability level. For storage periods, regression analysis was performed, selecting the significant models (F > 0.05) with best biological expression. The data were analyzed using Assistat 7.7 beta software.

17

Results and Discussion

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18

5.RESULTSANDDISCUSSION

__________________________________________________________________________

C. leprosum seeds initially had a moisture content of 10.3% (Table 1). However, it is worth noting that the seeds, due to their hygroscopic characteristics, showed variation in moisture content under all storage conditions and periods. Thus, it was possible to verify that the naked seeds and diaspores stored in an air-conditioned environment were the ones that varied the most in terms of moisture content throughout the storage period. However, it is valid to consider that the difference is not significant when compared to seeds stored in natural environment.

TABLE 1. Moisture content in Combretum leprosum Mart. seeds as a function of forms of

seed processing, environment conditions and storage periods.

The performance in relation to the moisture content of C. leprosum seeds is explained by the variation in the environment at the storage site and the type of permeable paper package used. According to Marcos-Filho (2015), when seeds are stored in packages that allow exchange of water vapor with the atmospheric air, there may be absorption of moisture in places with high relative humidity, consequently leading to faster seed deterioration. This can be verified in the experiment of Gentil et al. (2018) with seeds of Psidium friedrichsthalianum O. Berg. In this study, the authors found reduction in physiological quality for seeds with moisture content of 15.4% at three months of storage and death of seeds at six months of storage.

Based on the summary of the analysis of variance for physiological variables, there was an interaction between the environment and the storage period for emergence and emergence speed index. Total seedling length was influenced only by the storage periods. Total seedling dry mass was significantly affected by the single factors environment and storage periods (Table 2).

Forms of processing Environment Moisture content (%)

0 60 120 180 240 300 360 Naked seeds Natural environment 10.3 12.3 11.0 11.0 9.9 10.1 11.0 Air-conditioned chamber 10.3 12.3 11.4 12.3 12.1 11.7 11.4

Seeds with diaspores

Natural

environment 10.3 12.7 11.5 11.0 10.0 10.0 10.9 Air-conditioned

19

TABLE 2. Summary of analysis of variance for emergence (E), emergence speed index

(ESI), total seedling length (TSL), total seedling dry mass (TSDM) of Combretum leprosum Mart. as a function of the forms of seed processing, environment conditions and storage periods.

It was found in the present study that the reduction in the viability of C. leprosum seeds was evidenced by the decrease in emergence percentage along the storage period, being more pronounced from 180 days for air-conditioned environment, with a 28% reduction in emergence. The natural environment was more beneficial than the cold chamber, promoting a 22% reduction in emergence (Figure 1).

Probably the response of the natural environment which resulted in the smallest reduction in emergence may be related to the lower oscillation in the moisture content of the seeds during the storage period. It is pertinent to consider that the increase in moisture content during storage can trigger seed deterioration and promote reactivation of metabolism, as well as the development of microorganisms (BLACK et al., 2006).

FIGURE 1. Emergence of Combretum leprosum Mart. seeds as a function of environment

conditions and storage periods.

Mean squares

Source of variation DF E ESI TSL TSDM

Forms of processing of seeds (FPS) 1 24.14 ns _0.002 ns _0.36ns _0.00003 ns

Environment (E) 1 315.57* 1.21** 0.29ns _0.01* Storage periods (SP) 6 3512.61** 3.49 ** 12.49** 0.06 * FPS x E 1 11.57ns _0.021ns _0.027ns _0.00001ns FPS x SP 6 48.80ns _0.016ns _1.62ns _0.0006ns E x SP 6 166.90* 0.28** 0.77ns _0.0047ns FPS x E x SP 6 89.57ns _0.079ns _1.85ns _0.0023ns Erro 84 62.33 0.066 1.39 0.002 CV (%) 10.94 14.14 9.83 16.70 DF = degrees of freedom; CV = coefficient of variation; ns _{not significant and **significant at 1%}

20 The germination performance observed in the present study is similar to that of other forest seeds, when storage environments are evaluated under controlled and uncontrolled conditions in the different studies. Abbade and Takaki (2014), working with Tabebuia roseoalba seeds, found that the storage period significantly compromised germination percentage, which decreased from 99 to 52% at the end of the twenty-four months of storage. Similarly, Felix et al. (2020) worked with Pityrocarpa moniliformis seeds and also found a progressive reduction in germination throughout storage. The authors reported that the initial germination was 80% and this viability was maintained for 12 months when seeds were stored in a domestic refrigerator (6±3 ºC); however, for seeds that were stored in a growth chamber environment (27±4 ºC), the germination percentage was reduced to 54% at the end of the same period.

As expected, the reduction in the viability of C. leprosum seeds was accompanied by a decline in vigor, verified by the decrease in emergence speed index (ESI), seedling length and dry mass accumulation observed in seeds stored for 360 days. For ESI, the condition of natural environment was fundamental to maintain greater vigor of the seeds along the 180 days of storage compared to those stored in an air-conditioned chamber (Figure 2). This probably occurred because the storage in laboratory environment led to a slower and more gradual process of deterioration compared to that of the cold chamber (CISNEIROS et al., 2003).

Similar responses were reported by Guedes et al. (2010), who found the highest emergence speed indices of Amburana cearensis seedlings in seeds kept in the laboratory environment. On the other hand, Oliveira et al. (2018), in a study conducted with Schinus terebinthifolius Raddi seeds, pointed out the condition of domestic refrigerator as more efficient, for maintaining this index and percentage of normal seedlings with better results, compared to the ambient condition.

FIGURE 2. Emergence speed index of Combretum leprosum Mart. seeds as a function of the

21 According to the results obtained for seedling length, there was no interaction between the studied factors. However, it is possible to note the simple effect with linear tendency, which resulted in a slight reduction in performance for the variable during the storage period (Figure 3).

FIGURE 3. Length of Combretum leprosum Mart. seedlings as a function of storage periods.

As for the dry mass content of seedlings, there was a reduction along the storage period (Figure 4). This reduction may be associated with higher consumption of seed reserves during storage, mainly for maintaining viability for a longer period, which consequently led to a reduction in seedling vigor (ALEGRETTI et al., 2015). Similar results were found by Pádua et al. (2019), who observed that Acacia mangium seeds that were stored in semi-permeable polyethylene bags and subjected to three environment conditions (domestic refrigerator, freezer and air-conditioned room) originated seedlings with reduction in dry mass over 15 months of storage.

FIGURE 4. Total dry mass of seedlings of Combretum leprosum Mart. as a function of

storage periods.

Also with regard to dry mass of seedlings, the results show significance for the environment factor, with better average for the natural environment (Table 3). In agreement with the results presented, the controlled environment led to a rapid reduction in the dry mass

22 content of seedlings obtained from seeds of A. cearensis (GUEDES et al., 2010). Thus, it was found that C. leprosum seeds stored in natural environment maintain greater viability and vigor for a period of up to 180 days.

TABLE 3. Total dry mass of seedlings of Combretum leprosum Mart. as a function of the

environment conditions.

Environment Average

Natural environment 0.30 a Air-conditioned chamber 0.28 b

*significant at 5% probability, by test F.

During the storage period of C. leprosum seeds, biochemical analyses were also carried out. In these analyses, the results of the analysis of variance showed a triple interaction (form of seed processing x environment x storage periods) for neutral lipids and lipid peroxidation. The variables total soluble sugar, reducing sugar and starch were influenced only by the single factor storage periods. For total free amino acids, there were significant effects of the single factors seed processing, environment and storage periods (Table 4).

TABLE 4. Summary of analysis of variance for neutral lipid (NL), lipid peroxidation (LP), total

soluble sugar (TSS), reducing sugar (RS), starch content (SC) and total free amino acids (TFAA) of Combretum leprosum Mart. seeds as a function of forms of seed processing, environment conditions and storage periods.

Parallel to the reductions in viability and vigor, the loss of viability was also accompanied by the biochemical responses, based on the degradation of lipid reserve,

Mean squares

Source of variation DF NL LP TSS RS SC TFAA

Forms of processing of seeds (FPS) 1 3.59 ns 1.26ns 1.04ns 0.026ns 3584.69ns 8.79** Environment (E) 1 10.33* 24.41ns _52.60ns _0.0007ns _4901.48ns _44.19** Storage periods (SP) 12 19.24 * 41.52** 2967.82** 1.21* 55321.91** 0.63* FPS x E 1 8.13ns _0.22ns _2.627ns _0.016ns _20151.02ns _2.62ns FPS x SP 12 1.32ns 2.53* 112.01ns _0.028ns _11880.18ns _0.59ns E x SP 12 4.55ns _5.58ns _104.06ns _0.025ns _11625.18ns _1.27ns FPS x E x SP 12 5.60* 2.59* 228.86ns _0.097ns _5484.45ns _0.20ns Erro 156 2.12 14.41 127.93 0.04 6563.50 0.68 CV (%) 7.02 109.43 10.69 13.22 17.37 10.32 DF = degrees of freedom; CV = coefficient of variation; ns _{not significant and **significant at 1%}

23 accumulation of soluble metabolites, such as sugars and amino acids, and hydrolysis of reserve polysaccharides, such as starch.

For neutral lipid, naked seeds showed a greater reduction in lipid content when stored in an air-conditioned chamber environment. On the other hand, diaspores had better results when stored in a natural environment (Figure 5). Possibly, the lipids stored in C. leprosum seeds are degraded more significantly when the seeds are kept in the cold chamber, which contributes to the reductions in viability (Figure 1) and seed vigor (Figure 2).

FIGURE 5. Neutral lipids of Combretum leprosum Mart. seeds as a function of forms of seed

processing, environment conditions and storage periods.

Degradation of reserve lipids was also verified in Moringa oleifera Lam. seeds, in a more intense way, both in a chamber environment and in a refrigerator, from twelve months of storage (OLIVEIRA et al., 2017). In T. roseoalba seeds (ABBADE and TAKAKI, 2014), there was also a progressive and marked reduction in lipid content after twelve months of storage. Based on these previously mentioned studies and taking into account the seed deterioration process, it can be inferred that these are closely related to non-enzymatic reactions, in which storage lipids are subject to cleavage through peroxidation and de-esterification reactions (BLACK et al., 2006).

Also with regard to lipid degradation, the analysis of peroxidation showed triple interaction (forms of seed processing x environment x storage period). In terms of thiobarbituric acid reactive substances, all treatments showed an increase in malonaldehyde concentration until 240 days of storage and, after this period, the values began to decrease

24 (Figure 6). Probably, the decrease in malonaldehyde level was due to the ease of oxidation, or due to the reduction in the content of unsaturated fatty acid (linolenic acid), which is the precursor of this reaction (BEUGE and AUST, 1978).

FIGURE 6. Lipid peroxidation in Combretum leprosum Mart. seeds as a function of forms of

seed processing, environment conditions and storage periods.

In general, the natural environment promoted higher values of malonaldehyde concentration for both forms of seed processing, but the concentration was more significant in naked seeds. When lipid peroxidation occurs at the cell membrane level, it affects the permeability, promoting the loss of solutes to the medium, thus reducing seed viability (ATAÍDE et al., 2016).

Although lipid peroxidation is considered one of the main indicators of seed deterioration in many studies, the present experiment did not show this relationship, considering that one of the main consequences of peroxidation is the destruction of amino acids (ARAÚJO, 1994). This fact is not observed when comparing Figures 6 and 9 because, as the malonaldehyde concentrations increase, the level of amino acid increases.

Regarding the concentrations of total soluble sugars (Figure 7A) and reducing sugars (Figure 7B) in C. leprosum seeds stored under different conditions, the treatments did not cause significant differences. However, there was an accumulation of total soluble sugars until 180 days of storage and, soon after this period, the behavior was marked by a reduction until the end of storage. Similar results were found in Jatropha curcas L. seeds, for which the content of total soluble sugars increased along the storage, followed by a reduction until 12 months (MONCALEANO-ESCANDON et al., 2013).

25 The decrease in the concentration of soluble sugars is probably related to the respiratory process of the seed, which involves the oxidation of this compound for energy production (BEWLEY et al., 2013). Biochemical changes in forest seeds still lack scientific elucidation, which can be reinforced by the results obtained with A. mangium seeds (Pádua et al., 2019), in which it was not possible to detect a pattern of alteration in the degradation of total soluble sugars and non-reducing sugars as a response to deterioration over 15 months of storage of seeds subjected to three thermal conditions (-20 ± 3 ºC; 6 ± 3 ºC; and 27 ± 4 ºC).

During the seed deterioration process, when the content of total soluble sugars decreases, there is an increase in the levels of reducing sugars (MARCOS-FILHO, 2015). According to the same author, as a consequence, it causes losses in the ability to use carbohydrates, affecting the mobilization of reserve tissues to the embryonic axis, with reductions in germination and vigor.

FIGURE 7. Total soluble sugars (A) and reducing sugars (B) of Combretum leprosum Mart.

seeds as a function of the storage periods.

For starch content, there was significant effect of the single factor storage period. There was a linear decrease in the content of these polysaccharide over the storage period (Figure 8). Differing from this result, in seeds of M. oleifera (OLIVEIRA et al., 2017) and P. moniliformis (Félix et al., 2020), it was found that starch content did not show significant changes over the storage periods.

Considering that the metabolism of C. leprosum seeds is active, it certainly promoted greater activity of amylases. With this, there was cleavage of carbohydrates while the starch was broken to maintain the stock of soluble sugars and to be used as a respiratory substrate (MARCOS-FILHO, 2015).

26

FIGURE 8. Starch content in Combretum leprosum Mart. seeds as a function of the storage

periods.

Regarding the concentrations of amino acids in C. leprosum seeds, there were significant effects of the three single factors, with better results for naked seeds and natural environment (Table 5).

Table 5. Total free amino acids of Combretum leprosum Mart. seeds as a function of the

forms of seed processing and environment conditions.

Variation source Average

Forms of processing of seeds Naked seeds 8.36a Seeds with diaspores 7.72 b Environment conditions Natural environment 8.77a Air-conditioned chamber 7.31 b **significant at 1% probability level by F test.

Also in relation to total free amino acids, there was an increase in their contents until 180 days, decreasing from this period until the end of storage (Figure 9). Such increase in amino acid content over the seed storage period may be related to the increase in proteolytic activity (SMITH and BERJAK, 1995). However, the reduction is possibly linked to the occurrence of the Maillard reaction, as it is characterized by the non-enzymatic attack on amine groups by reducing sugars (VESELOVA et al., 2015).By comparing the results presented for reducing sugars (Figure 7B) and amino acids (Figure 9), it was possible to note an association between the data, which consequently culminated in the reduction of seed viability and vigor.

27

FIGURE 9. Total free amino acids of Combretum leprosum Mart. seeds as a function of

storage periods.

In Jatropha curcas L. seeds kept at ambient temperature or under refrigeration for 12 months, accumulation of amino acids was also observed (MONCALEANO-ESCANDON et al., 2013). Conversely, in M. oleifera Lam. seeds, the amino acid content remained unchanged throughout the storage period, regardless of the environment condition (OLIVEIRA et al., 2017).

In general, based on the results of the present study, C. leprosum seeds should be stored for a period of 180 days, regardless of the form of seed processing and storage condition. Until this period, it was found that there are no significant variations in the contents of lipids, sugars, starch and amino acids that could compromise the viability and vigor of the seeds.

28

Conclusions

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29

6. CONCLUSIONS

__________________________________________________________________________ 1. C. leprosum seeds, when stored under condition of natural environment, maintain their viability and vigor for a period of 180 days and lower lipid degradation, regardless of the form of processing.

2. Seed deterioration after 180 days of storage is associated with the attack of reducing sugars against amino acids, with reductions in viability and vigor.

3. During the 360-day period of storage of C. leprosum seeds, no influence of seed processing was observed on viability and vigor.

30

Acknowledgements

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31

7. ACKNOWLEDGEMENTS

__________________________________________________________________________ This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Cited Literature

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__________________________________________________________________________ Abbade, L.C.; Takaki, M. Biochemical and physiological changes of Tabebuia roseoalba (ridl.) sandwith (bignoniaceae) seeds under storage. Journal of Seed Science, v.36, n.1, p.100-107, 2014.

Alegretti, A.L.; Wagner Júnior, A.; Bortolini, A.; Hossel, C.; Zanela, J.;Citadin, I. Armazenamento de sementes de cerejas-do-mato (Eugenia involucrata) DC. submetidas ao recobrimento com biofilmes e embalagem a vácuo. Revista Ceres, v.62, n.1, p.124-127, 2015.

Alves Filho, F.; Cavalcanti, P.M.; Passaglia, R. C.; Ballejo, G. Long-lasting endotheliumdependent relaxation of isolated arteries caused by an extract from the bark of Combretum leprosum. Einstein (Sao Paulo) [s.l.], v.13, n.3, p.395-403, 2015.

Amorim, I.L.; Sampaio, E.V.S.B.; Araújo, E.L. Fenologia de espécies lenhosas da caatinga do Seridó, RN. Revista Árvore, Viçosa, v. 33, n.3, p.491-499, 2009.

Araújo, J.M.A. Oxidação de lipídios. Viçosa: Imprensa Universitária/UFV, 1994. 22 p.

Ataíde, G.M; Borges, E.E.L.; Leite Filho, A.T. Alterações fisiológicas e biométricas em sementes de Melanoxylon brauna Schott durante germinação em diferentes temperaturas. Revista Árvore, v.40, n.1, p.61-70, 2016.

Baudet, L.; Villela, F.A. Armazenamento de Sementes. In: Peske ST, Lucca Filho, O.A., Barros, A.C.S.A. (Eds.). Sementes: fundamentos científicos e tecnológicos. 2.ed. Pelotas: Gráfica Universitária-UFPel. 2006. pp. 427-472.

Bailly, C.; El-Maarouf-Bouteau, H.; Corbineau, F. From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology. Comptes Rendus Biologies , v.331, p.806–814, 2008.

Barbosa, D.C.A.; Barbosa, M.C.A.; Lima, L.C.M. Fenologia de espécies lenhosas da caatinga. In: LEAL, I.R.; TABARELLI, M.; SILVA, J.M.C. (Eds.). Ecologia e conservação da caatinga. Recife: Universitária UFPE. 2003. p. 657-693.