Gene Expression Related to Physiological Quality of Soybean Seeds

Production of soybeans with high physiological quality is important to ensure the productivity of improved plant populations. The objective of this work was to select soybean genotypes for high physiological quality of seeds, through germination and vigor tests, and to study gene expression by transcripts and proteins. In a first trial, seeds of 12 soybean cultivars were evaluated and selected six cultivars with contrasting physiological quality levels. At the protein level, the isoenzyme systems alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), sorbitol dehydrogenase (SDH), superoxide dismutase (SOD), catalase (CAT), isocitrate lyase (ICL), esterase (EST), glutamate oxaloacetate transferase (GOT), and heat-resistant proteins were evaluated. For the transcript levels, the real-time PCR technique (qRT-PCR) was used, and the genes coding for the SOD, CAT, MDH, PGI, ICL and PRX enzymes were studied. The germination and vigor tests classified the seeds of cultivars CD201, CA115 and MS8400 as high quality, while the cultivars Syn1263, Syn1279 and CD202 were classified as of low quality. The enzymes involved in the process such as dehydrogenase and phosphoglucose isomerase, are promising markers for assessing the physiological quality of soybean seeds. Higher expression of peroxiredoxin enzyme is related to the low physiological quality of soybean seeds.


Introduction
The development of soybean cultivars with good agronomic characteristics in the sowing and grain industry has increased significantly in recent years in Brazil.Through research, genetic diversity for physiological quality has been observed among soybean cultivars.Improving studies in this line of research is important due to climatic conditions in Brazil during the seed production process.Mainly after the physiological maturity of the seeds, the high temperature and the relative humidity that can accelerate the deterioration process, which leads to the reduction of the physiological quality of seeds.
In the process of seed deterioration a few enzymes are involved that act in respiratory processes, such as alcohol dehydrogenase (ADH), malate dehydrogenase (MDH) and phosphoglucose isomerase (PGI), in addition to those related to the removal of free radicals such as superoxide dismutase (SOD) and catalase (CAT) and also those related to the degradation of reserve materials such as isocitrate lyase (ICL), among others.
In breeding programs, it has increasingly sought to improve technologies for the selection of soybean cultivars with high physiological quality of the seeds.In general, germination and vigor tests have been used in this selection process.However, knowledge at the molecular level, mainly through transcriptomic and proteomic techniques, allows advances in obtaining high-quality seed cultivars.The qualitative and quantitative determination of transcript levels related to the physiological quality of seeds allows differentiated genes to be identified and their metabolic function investigated (Kuhn et al., 2001).Studying genes related to the physiological quality of seeds has been a challenge, mainly due to its genetic control.

Material and Methods
The experiments were conducted in the Central Seed Laboratory and the experimental area of the Department of Agriculture of the Lavras Federal University (UFLA), in Lavras, MG, Brazil.
Initially, for the selection of contrasting genotypes for physiological quality, seeds of 12 conventional soybean cultivars were used: CD201, SYN1263, SYN1279, BMX Potência, UFLA1, CA115, MSoy-8400, CD215, CD202, Conquista, Savana and BRS 820, from the germplasm bank of the Lavras Federal University.For seed multiplication, a randomized complete block design with three replications was used.The experimental units consisted of 4 lines of 5m each, considering only the 2 central lines as useful area.The thinning was carried out maintaining a population of 16 plants per linear meter.
The experiment was sowed using the conventional system with previous desiccation of the weeds, before sowing the soybean, which was performed between 15th and 25th of November, 2015.The fertilization and the management were carried out according to the recommendations for the culture.
The seeds were harvested at the R8 stage (95% maturity of the seeds, with a water content of 18%).Subsequently, they were dried naturally to 12% water content.In the processing, circular sieve was used, between 5.55 mm and 6.35 mm, to standardize the seed size.They were then weighed and stored in paper bags under controlled cold chamber conditions at 10 °C until the next stage of the experiment.
Physiological tests were carried out to identify six contrasting cultivars for physiological quality: CD201, CA115, MS8400, CD202, Syn 1263 and Syn1279.These were again sowed in the field for seed maintenance and multiplication.In addition, they were evaluated for physiological quality, and proteins and transcripts expression.
For the germination and accelerated aging tests, the seeds were treated with Vitavax Thiram 200SC fungicide at 250 ml/kg of seeds dosage.200 seeds per cultivar were used in both tests.
In the germination test, the seeds were sowed on Germitest paper moistened with distilled water in the proportion of 2.5 times the weight of the paper, aiming an adequate moistening and uniformity of the test.The seeds remained in the germinator at 25 ºC.Evaluations were performed at five days (first count) and eight days (final count), computing the percentage of normal seedlings, according to the criteria established in the Brazilian Rules for Seed Analysis (Brasil, 2009) For the accelerated aging test, gerbox minicameras were used, in which 42g of seeds were distributed on a screen suspended inside the box containing 40 mL of water and submitted to 42 ºC for 72 and 82 hours.Afterward the germination test was carried out.A single reading was performed on the fifth day after sowing, and the percentage of normal seedlings was computed (Brasil, 2009).
Controlled deterioration was evaluated using samples of 42 grams of seeds, for each cultivar.These were artificially moistened until reaching a water content of 15%, according to Rossetto and Marcos Filho (1995), and placed in hermetically sealed plastic coated aluminum sachets.These were kept in a water bath at 40 °C for 48 hours.Then, a germination test was performed, and evaluated on the fifth day after sowing, according to the previous item (ISTA, 1995;Rosseto & Marcos Filho, 1995).
For the electrical conductivity test, four replicates of 50 seeds were used for each cultivar, apparently intact and then weighted.They were then placed in plastic cups with a capacity of 200 ml containing 75 ml of deionized water for 24 hours at 25 °C.The conductivity of the seed soak solution of each cultivar was measured by means on a CD 21A (DIGIMED) conductivity equipment, and the results were expressed in μS/cm/g of seeds (Vieira & Carvalho, 1994).
Statistical analysis was performed using the Sisvar software (Ferreira, 2011) and the data were interpreted through the analysis of variance.The Scott-Knott test was used at 5% probability, for comparison of means.
After the harvest, part of the seeds was immediately conditioned in liquid nitrogen and stored at -80 °C for further analysis of gene expression related to the physiological quality of seeds.The real-time PCR (qPCR) technique was used for the transcripts analysis and the technique of electrophoresis for protein analysis.
In the gene expression analysis, an amount of 50 seeds per treatment/cultivar, was removed and conditioned in the deep freezer for later analysis of the activities of the main enzymes of the seed metabolism.
The crude enzymatic extracts were obtained by the maceration of the seeds in the presence of PVP (polyvinylpyrrolidone) and liquid nitrogen, and later stored at -86 ºC.
The electrophoretic run was performed in a discontinuous system of 7.5% polyacrylamide gels (separator gel) and 4.5% (concentrator gel).The gel/electrode system used was Tris-glycine pH 8.9.50 μl of the sample supernatant was applied to the gel well and the run performed at 150 V for 5 hours.
The extraction of RNA was performed by maceration of five seeds of each cultivar, in liquid nitrogen.Afterward, all the materials used were previously submitted H 2 O treated with 0.1% DEPC (diethylpyrocarbonate) to inhibit the action of the RNAs enzymes.The total RNA was extracted in two biological replicates for each sample using PureLink Plant RNA reagent (Invitrogen) according to the manufacturer's recommendations and, for 0.1 g of the seeds, 500 μL of PureLink Plant RNA, vortexed for 20 seconds and maintained in the horizontal position for 5 min, for better homogenization of the material.The material was then centrifuged at 14,000 rpm, 4 ºC for 15 min.After centrifugation, the supernatant was transferred to a new microtube, adding 300 μL of chloroform, 100 μL of potassium chloride (5M), followed by stirring.
Centrifugation was performed again at 14,000 rpm, 4 °C for 10 min.The supernatant was transferred to a new microtube with the same volume of isopropyl alcohol and the microtube held for 10 min.Subsequently, the material was centrifuged at 14,000 rpm at 4 °C for 10 min.Thereafter, the supernatant was discarded and 1 ml of 70% ethanol was added and centrifuged at 14,000 rpm at room temperature for 1 min.The supernatant was removed and the pellet dried for 10 min, which was resuspended in 20 μl of water with 0.1% DEPC and stored at -80 °C.The quantification of total RNA extracted was done in a spectrophotometer, measuring the absorbance at 260 and 280 nm, observing the wavelength ratio 260/280, whose values were in the range 1.8 to 2.0, which indicates high-quality extraction.The integrity of the RNA was also analyzed by agarose gel electrophoresis (1.5%), containing 1× TBE buffer and stained with ethidium bromide solution.The race took place at 90 V for 30 min.
The extracted RNA samples were treated with DNase (Ambion) according to the manufacturer's instructions.To obtain the cDNA, the Hight-Capacity cDNA Reverse Transcription kit (Applied Biosystems) was used.
For the primers design, the NCBI genebank was used.Sequences of the different genes encoding the enzymes evaluated were selected.Subsequently, the primers were designed using the Primer Express 3 program (Applied Biosystems) (Table 1).GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and CYP2 (peptidyl-prolylcis-transisomerase2) genes were chosen as reference for the qRT-PCR analysis.
Table 1.Primers used in the qRT-PCR analysis to selected cultivars of high and low physiological quality Note. (F) forward primer sequence.(R) reverse primer sequence.
Reaction conditions were 50 ºC for 2 min, 95 ºC for 10 min, 45 cycles of 95 ºC for 2 min, 62 ºC for 30 sec and 72 ºC for 30 sec.The amplification reactions were conducted in a final reaction volume of 20 μL containing: 10 μL of SYBR Green PCR Master Mix (Applied Biosystems), 2 μL cDNA (250 ng), 0.4 μL of each of the forward and reverse primers and 7.2 μL of autoclaved ultrapure water.
After amplification by real-time PCR, each amplification product was analyzed by a dissociation curve, certifying that for each gene and treatment, the amplified product had no non-specific bands and/or formation of primer dimers.Data analysis was performed using the 7500 Software SDS (Version 2.0.1)(Applied Biosystems).

Results and Discussion
There was a significant difference in the physiological quality of soybean seeds between cultivars by the majority of germination and vigor tests (P < 0.05) in the first experiment in which seeds of 12 cultivars were evaluated (Table 2).
Table 2. Means obtained from the physiological tests: accelerated aging (E.A), at 72 hours; accelerated aging at 82 hours; first germination count (PC%), final count of germination (GER%); controlled deterioration (D.C) at 5 and 7 days of 12 cultivars of soybean seeds of produced in the first experiment Note.Means followed by the same lowercase letter in the column do not differ by Scott-Knott's test, at 5% significance.
The accelerated aging test for 82 hours allowed greater separation between cultivars.Under these conditions, higher values of vigor were observed in seeds of cultivar MS8400, followed by cultivar CD 201 and lower values were observed for cultivars Syn1263 and CD202.For the other cultivars, intermediate values were observed.When the seeds were aged for 72 hours and evaluated on the fifth day, there was less distinction among the cultivars regarding vigor.
At the first germination count, lower values were observed for the cultivars Conquista and Savana.In the final count, in addition to the cultivars Conquista and Savana classified with lower seed vigor, also the seeds of cultivars SYN 1263, UFLA 1 and BMX Potência presented low physiological quality.
In the controlled deterioration test, there was no statistical difference between the vigor values of the seeds of the twelve cultivars evaluated.
With the result analysis obtained in this first trial, and also based on the previously results in the researches of Baldoni (2013) and Menezes (2008), it was decided to select cultivars and to divide them into two groups: those that presented seeds with high physiological quality (CD201, CA115 and MS8400) and with low physiological quality (YN 1263, SYN 1279 andCD 202).
Table 3 shows the results of germination and vigor tests of the second trial, with the results of the six soybean cultivars, selected as the most contrasting.
In both the first count and the final germination count, the lowest value was verified in seeds of cultivar CD202.
For the other cultivars, there was no statistically significant difference between the values.When seeds were

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