Öğrenme Alanları

Como já relatado, na natureza as plantas são constantemente expostas a estresses

bióticos e abióticos que podem ocorrer isoladamente ou de forma combinada. As

respostas das plantas a estresses combinados são muito complexas e muitos estudos vem

sendo realizados na busca de genes e proteínas que sejam comuns ou específicos a

determinados estresses (FUJITA et al., 2006; MANTRIA et al., 2010; XU E HUANG,

2012; ATKINSON et al., 2013; RASMUSSEN et al., 2013; BADOWIEC E WEIDNER,

2014; SERGEANT et al., 2014), a fim de compreender a tolerância das plantas a estresses

combinados.

Uma das principais respostas de adaptação a condição de combinados estresses

é a modulação da expressão gênica, que possui a especificidade de sua resposta controlada

por uma série de vias regulatórias que envolvem fatores de transcrição, EROS, fatores de

choque térmico e pequenos RNAs que podem interagir uns com os outros (ATKINSON;

URWIN, 2012). Essas respostas podem ser muito diferentes de quando a planta é

desafiada pelos mesmos estresses impostos isoladamente. Às vezes, as exposições das

plantas à combinação dos estresses podem requerer respostas agonísticas ou

antagonísticas ou, até mesmo, respostas não relacionadas com nenhum dos estresses

simples (RASMUSSEM et al., 2013).

Plantas toleram dois ou mais estresses quando ocorrem independentes, mas, não

necessariamente, toleram esses estresses quando ocorrem simultaneamente (NOSTAR et

al., 2013; RAMEGOWDA E SENTHIL-KUMAR, 2015). Tamareira quando exposta à

seca apresentou mais sintomas quando infectado, simultaneamente, com os fungos

Chalara paradoxa e Chalara radicícola (SULEMAN et al., 2001). Além disso, em tabaco

(Nicotiana tabacum) e pimenta (Capsicum annuum) quando expostos a altas temperaturas

suprimiram a resistência ao Tobacco mosaic vírus (TMV) e Tomato spotted wilt vírus

(TSWV), respectivamente (KIRÁLY et al., 2008; MOURY et al., 1998). Contrariamente,

estresses abióticos podem conferir resistência a certos patógenos. Wiese et al. (2004)

mostraram que o estresse salino aumentou a resistência de cevada (Hordeum vulgare) ao

fungo Blumeria graminis em concentrações dose dependente.

Apesar de recentes estudos estarem focando na resposta de plantas a estresses

combinados, a maior parte da pesquisa, em plantas, ainda se baseia na resposta individual

aos estresses, mostrando a importância de trabalhos com esse enfoque.

40

2

HIPÓTESE

O feijão-de-corda (Vigna unguiculata) genótipo BRS-Marataoã, tolerante ao estresse

salino e resistente ao CPSMV, responde ativamente a esses estresses (individualmente ou

simultaneamente expostos) por meio de reprogramação gênica que leva a alterações no

perfil proteômico. Entretanto, as respostas da planta aos estresses individuais são

diferentes de quando os estresses são simultaneamente impostos e, esta segunda condição,

afeta a tolerância da planta ao CPSMV.

3

OBJETIVOS

3.1 Objetivo geral

Estudar as respostas fisiológicas e bioquímicas/moleculares do feijão-de-corda (Vigna

unguiculata, genótipo BRS-Marataoã) quando individualmente ou simultaneamente

submetido ao estresse salino e infecção pelo CPSMV.

3.2 Objetivos específicos

· Avaliar parâmetros fisiológicos referentes à fotossíntese, conteúdo de clorofila e

carotenoides nas folhas de feijão-de-corda, genótipo BRS-Marataoã, submetidas ao

estresse salino e/ou infectadas com o CPSMV (inoculado simultaneamente ou 24 horas

após o estresse salino);

· Detectar a presença do CPSMV nas folhas de plantas de feijão-de-corda, genótipo BRS-

Marataoã, submetidas ao estresse salino e/ou infectadas com o CPSMV (inoculado

simultaneamente ou 24 horas após o estresse salino), via RT-PCR;

· Determinar a cinética enzimática de proteínas antioxidantes (superóxido dismutase,

catalase, ascorbato peroxidase e guaiacol peroxidase) e PR-proteínas (glucanase e

quitinase) em folhas de feijão-de-corda, genótipo BRS-Marataoã, submetidas ao estresse

salino e/ou infectadas com o CPSMV (inoculado simultaneamente ou 24 horas após o

estresse salino);

· Verificar o acúmulo de peróxido de hidrogênio em folhas de feijão-de-corda, genótipo

BRS-Marataoã, submetidas ao estresse salino e/ou infectadas com o CPSMV (inoculado

simultaneamente ou 24 horas após o estresse salino);

· Extrair proteínas de folhas de feijão-de-corda, genótipo BRS-Marataoã, submetidas ao

estresse salino e/ou infectadas com o CPSMV, e obter, por espectrometria de massas

(nano LC-MS/MS), perfis com boa reprodutibilidade entre as repetições técnicas e

biológicas;

· Analisar os perfis espectrométricos obtidos de proteínas de folhas de feijão-de-corda,

genótipo BRS-Marataoã;

· Identificar, classificar e categorizar as proteínas diferencialmente acumuladas em folhas

de feijão-de-corda, genótipo BRS-Marataoã.

42

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CAPÍTULO II

Gel-free/label-free proteomic, biochemical and

photosynthetic analysis of cowpea (Vigna

unguiculata [L.] Walp.) resistance against Cowpea

severe mosaic virus (CPSMV)

Gel-free/label-free proteomic, biochemical and photosynthetic analysis of cowpea

(Vigna unguiculata [L.] Walp.) resistance against Cowpea severe mosaic virus

(CPSMV)

Anna Lídia Nunes Varela,

a

_{Setsuko Komatsu,}

b

_{Xin Wang}

b

_{,Rodolpho Glauber Guedes}

Silva,

a

_{Pedro Filho Noronha de Souza,}

a

_{Ana Karla Moreira Lobo,}

a

_{Ilka Maria}

Vasconcelos,

a

_{Joaquim Albenísio Gomes Silveira,}

a

_{Jose Tadeu Abreu Oliveira,}

a

a

_{Department of Biochemistry and Molecular Biology, Federal University of Ceara, CE,}

Brazil

b

_{National Institute of Crop Science, National Agriculture and Food Research}

Organization, Tsukuba 305-8518, Japan

Corresponding author. Tel.: +55 85 33669823; fax: +55 85 33669789. E-mail address:

52

ABSTRACT

Significant losses in cowpea (Vigna unguiculata) production are caused by Cowpea

severe mosaic virus (CPSMV). In this present study biochemical, physiological, and

proteomic analysis were done to identify pathways and defense proteins that are altered

during the incompatible interaction between the cowpea genotype BRS-Marataoã and

CPSMV. The leaf protein extracts from mock- (MI) and CPSMV-inoculated plantlets (V)

were evaluated at 2 and 6 days post-inoculation (DPI). Data support the assumptions that

the increased of biochemical (high hydrogen peroxide, antioxidant enzymes, and

secondary compounds) and physiological responses (high photosynthesis index and

chlorophyll content), confirmed by the Gel-free/label-free comparative proteomic

approach, in which quantitative changes in proteasome proteins, proteins related to

photosynthesis, redox homeostasis, regulation factors/RNA processing proteins were

observed may be implicated in the resistance of BRS-Marataoã to CPSMV. This

pioneering study provides information for the selection of specific pathways and proteins,

altered in this incompatible relationship, which could be chosen as targets for detailed

studies to advance our understanding of the molecular, physiological, and biochemistry

basis of the resistance mechanism of cowpea and design approach to engineer more

productive plants.

1. Introduction

Cowpea [Vigna unguiculata (L.) Walp.] is an important crop that belongs to the

family Fabaceae, order Leguminosae, mainly grown in tropical and sub-tropical regions

in the world for vegetable and grains [1]. Usually cowpea grains contain high protein (23-

25%) and starch (50-67%) contents, but low lipid level in dry bases [2] and represent a

very important source of other several essential nutrients like vitamins and minerals,

which is related to the prevention of diverse metabolic and cardiovascular disease [3].

Cowpea is a robust crop, but its production is impaired due to various environmental

stresses, including diseases caused by viruses, bacteria, fungi, nematodes, and insects [1].

Cowpea severe mosaic virus (CPSMV) belongs to the genus Comovirus, family

Secoviridae, and has a bipartite linear ssRNA(+) genome composed of RNA-1 and RNA-

2, [4]. It is considered a major problem of cowpea in the world, responsible for important

losses in the crop production and yield [5]. Cowpea plants infected with CPSMV suffer

drastic growth reduction and development, which depend on the intrinsic susceptibility

of each genotype. Typical symptoms in susceptible cowpeas are mosaic, chlorose, yellow

patches, foliar distortion, and leaf morphology alterations that lead to reduction of some

physiological parameters, primarily photosynthesis [6,7]. Therefore, CPSMV infection

provokes strong decrease in cowpea productivity and yield due to reduced photosynthesis

Belgede T.C. BARTIN ÜNİVERSİTESİ SOSYAL BİLİMLER ENSTİTÜSÜ TEMEL İSLAM BİLİMLERİ ANA BİLİM DALI (sayfa 52-0)