Genetics and Molecular Biology, 35, 1 (suppl), 304-314 (2012) Copyright © 2012, Sociedade Brasileira de Genética. Printed in Brazil www.sbg.org.br Research Article Subtractive libraries for prospecting differentially expressed genes in the soybean under water deficit Fabiana Aparecida Rodrigues1, Juliana Marcolino-Gomes1, Josirlei de Fátima Corrêa Carvalho1, Leandro Costa do Nascimento2, Norman Neumaier1, José Renato Bouças Farias1, Marcelo Falsarella Carazzolle2, Francismar Corrêa Marcelino1 and Alexandre Lima Nepomuceno1 1Embrapa Soja, Laboratório de Biotecnologia Vegetal, Londrina, PR, Brazil. 2Departamento de Genética, Evolução e Bioagentes, Universidade Estadual de Campinas, Campinas, SP, Brazil. Abstract Soybean has a wide range of applications in the industry and, due to its crop potential, its improvement is widely de- sirable. During drought conditions, soybean crops suffer significant losses in productivity. Therefore, understanding the responses of the soybean under this stress is an effective way of targeting crop improvement techniques. In this study, we employed the Suppressive Subtractive Hybridization (SSH) technique to investigate differentially ex- pressed genes under water deficit conditions. Embrapa 48 and BR 16 soybean lines, known as drought-tolerant and -sensitive, respectively, were grown hydroponically and subjected to different short-term periods of stress by with- holding the nutrient solution. Using this approach, we have identified genes expressed during the early response to water deficit in roots and leaves. These genes were compared among the lines to assess probable differences in the plant transcriptomes. In general, similar biochemical processes were predominant in both cultivars; however, there were more considerable differences between roots and leaves of Embrapa 48. Moreover, we present here a fast, clean and straightforward method to obtain drought-stressed root tissues and a large enriched collection of tran- scripts expressed by soybean plants under water deficit that can be useful for further studies towards the under- standing of plant responses to stress. Key words: gene expression, drought, transcriptome. Introduction that plant responses were more uniform in the V4 stage Soybean is one of the most important crops in the (Fehr et al., 1971). The genetic background of the cultivars world, and Brazil is its second largest producer. Due to its BR 16 (drought sensitive) and Embrapa 48 (drought toler- use in animal feed, vegetable oil and biodiesel, there is a ant) under drought conditions was studied by Oya et al. sustained demand for soybeans. Nevertheless, abiotic stres- (2004), and they found that at the vegetative stage, tolerant ses, such as water deficit, cold, wind, heat or waterlogging, cultivars present higher growth rate and a larger leaf area reduce productivity and present a major challenge in the compared to sensitive plants. However, to better under- quest for sustainable soybean production. In order to meet stand plant physiology under stress, productivity analyses the ever-increasing demand, studies to improve productiv- are hardly enough to distinguish significant tolerance dif- ity are very important. Crop yield is highly affected by wa- ferences among cultivars. The data indicate how cultivars ter deficit stress, particularly when it occurs during flower- behave under stress and, therefore, suggest which ones are ing and early pod expansion (Pedersen et al., 2005). suitable for further biochemical and molecular studies. However, soybean plants can acclimate to such conditions A stress response is initiated when plants recognize by triggering protective mechanisms that enable these the stress at the cellular level, it triggers a complex signal plants to survive subsequent events of drought (Bray, transduction network which is modulated by molecular 2004). While studying soybean plants exposed to stress at events. Gene products involved in water-deficit responses different developmental stages, Kron et al. (2008) observed can be classified into two groups: functional or regulatory genes. Functional genes include the following: proteins, osmolytes, chaperones, heat-shock proteins (HSPs), wa- Send correspondence to Alexandre Lima Nepomuceno. Embrapa Soja, Laboratório de Biotecnologia Vegetal, Rodovia Carlos João ter-channel proteins (multifunctional proteins involved in Strass s/n, 86001-970 Londrina, PR, Brazil. E-mail: transport facilitation of solutes and water in leaves and [email protected]. roots), and other compounds probably conferring direct tol- Rodrigues et al. 305 erance to abiotic stresses. Under water stress conditions, a Material and Methods chemical imbalance in the cell leads to production of reac- tive oxygen species (ROS), which are damaging to lipids Plant growth and water stress treatment and proteins (Carvalho, 2008; Molina et al., 2008). En- Seeds of the soybean cultivars Embrapa 48 and BR zymes involved in detoxification metabolism are found to 16, classified as tolerant and sensitive to water deficit, re- protect against the stress in the tolerant common bean spectively, were germinated on filter paper during four cultivar (Phaseolus acutifolius) (Türkan et al., 2005). In days in a growth chamber at 25 ± 1 °C of temperature and drought-stressed tissues, the water movement within and 100% relative humidity. Seedlings were placed in 36 L between cells can be facilitated by aquaporins (Cocozza et boxes containing 50% Hoagland’s solution (Hoagland and al., 2010). The second group of genes involved in the re- Arnon, 1950), which was continuously aerated and re- sponse to water deficit includes molecules involved in fur- placed on a weekly basis. These boxes were then trans- ther regulation of signal transduction and stress-responsive ferred to a greenhouse under a natural photoperiod of ap- gene expression. These molecules are regulatory proteins, proximately 12/12 h light/dark cycle, temperature of represented by various transcription factors such as 30 ± 5 °C and 60 ± 10% relative humidity (RH), where the DREB1, ABA-responsive element, nitrogen assimilation plants were allowed to grow until the V4 stage (Fehr et al., control protein, ethylene-response factors, kinases, phos- 1971). The experimental plan was a randomized complete phatases, enzymes involved in phospholipid metabolism, block 2x7 factorial design with three repetitions. The fac- and other signaling molecules, such as calmodulin-binding tors were two cultivars (BR 16 and Embrapa 48) and seven protein (Shinozaki and Yamaguchi-Shinozaki, 2007). In times of water deficit (0, 25, 50, 75, 100, 125 and 150 min). the peanut, a predicted drought tolerant legume, a set of The stress was applied by removing the plants out of the hy- functional and regulatory genes were detected in enriched droponic solution and leaving them in boxes without nutri- subtractive libraries probably acting in a gradual process of ent solution for up to 150 min under ambient-air exposure. drought acclimation (Govind et al., 2009). For each time of water deficit, roots from 10 plants were As discussed, the ability to tolerate water deficit is a collected, pooled and frozen in liquid nitrogen before stor- complex trait controlled by many genes (Molina et al., age at - 80 °C. The same procedure was performed for the leaf samples. 2008; Ergen and Budak, 2009). Collections of cDNAs ex- pressed under stressed conditions have been a useful re- Physiological parameters measurements source to study gene expression and regulation. The cDNA libraries were associated to normalization steps and PCR Photosynthetic rate (A), stomatal conductance (gs), amplification to obtain just the transcripts presented in a intercellular CO2 concentration (ci), transpiration rate (E) target situation (Diatchenko et al., 1996). A suppressive and leaf temperature (Tl) were evaluated using a LI-6400 subtractive library approach makes it possible to search for Portable Photosynthesis System (LiCor, Inc.). Measure- genes expressed in treated samples but not in the control ments were taken on the middle leaflet of the youngest soy- samples, if the forward subtraction is done. It can be used to bean leaf, fully expanded, under photon flux density of m -2 -1 assess changes in metabolism and in quantitative gene ex- 1.000 mol m s . The water use efficiency (WUE) of pression studies, such as those based on arrays. The method plants was determined through the ratio between photo- has been employed successfully in different research areas, synthetic (A) and transpiration (E) rate. Data was submitted such as the monitoring of citrus responses to fungus infec- to ANOVA analysis through the programs Sanest and SAS tion (González-Candelas et al., 2010) and in fish infected (Statistical Analysis System version 8.0), and the treatment by iridovirus (Xu et al., 2010). Using the subtracted library means were compared by the Tukey test (p < 0.05). of pigeonpea, Priyanka et al. (2010) isolated a gene encod- cDNA subtracted libraries synthesis ing a hybrid-rich-proline protein, which affords tolerance to water deficit and other abiotic stresses such as salt, heat, Total RNA was extracted from roots and leaves using cold and abscisic acid treatment. In addition, cDNA Trizol reagent (Invitrogen) according to the manufacturer’s subtractive libraries have also allowed the identification of instructions. Samples from each biological replicate were genes involved in the flower development of orange plants extracted separately, and equimolar amounts of total RNA (Zhang