Plant Molecular Biology Reporter https://doi.org/10.1007/s11105-017-1059-6
ORIGINAL PAPER
Comparative Transcriptional Profiling of Soybean Orthologs of Arabidopsis Trichome Developmental Genes under Salt Stress Özge Çelik 1 & Çimen Atak 1 & Zekiye Suludere 2
# Springer Science+Business Media, LLC, part of Springer Nature 2017
Abstract The aim of this study was to determine the ultrastructural changes and regulation of trichome-metabolism-related genes against salt stress in soybean (Glycine max L. Merr.) plants. The 14-day-old Ataem-7 and S04-05 soybean seedlings were subjected to 0, 50, 100, and 150 mM NaCl stress. While the chlorophyll quantities were reduced, the activities of guaiacol peroxidase were increased in both varieties due to increasing NaCl concentrations. In S04-05 soybean variety, trichome densities were increased on both surfaces of the leaves whereas decreases were recorded in Ataem-7 variety at 150 mM NaCl treatment. Stomatal densities were increased on both surfaces of the leaves of both soybean varieties after salinity stress. We also performed a qRT-PCR analysis to evaluate the relative transcription levels of the soybean orthologs of Arabidopsis trichome developmental genes. qRTPCR analysis demonstrated an induction of the soybean orthologs of GL2 and GL3 genes in soybean plants after 50, 100, and 150 mM NaCl treatments in both varieties. While the expression level of TTG1 ortholog gene was negatively affected in both soybean varieties under different concentrations of salinity, GL1 ortholog gene expression profile differed as a result of changing salt concentrations in both varieties with respect to control plants. It is observed that the regulation of trichome formation differs between two soybean varieties. Keywords Salt stress . Trichome density . Stomatal density . Trichome initiation . Glabrous ortholog genes . Soybean
Introduction The climate changes limit the agricultural production worldwide. Beside the biotic factors, abiotic stress factors have the major limitation efficiency in product (Munns 2002). Water is an important constituent of a plant life. Plants have more than 70–90% water of their fresh weights. It plays critic roles in biochemical reactions like as an electron donor in photosynthesis. Water availability is the major factor to affect the survival Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11105-017-1059-6) contains supplementary material, which is available to authorized users. * Özge Çelik
[email protected] 1
Faculty of Science and Letters, Department of Molecular Biology and Genetics, İstanbul Kültür University, Ataköy, 34156 İstanbul, Turkey
2
Faculty of Science, Department of Biology, Gazi University, Teknik Okullar, 06500 Ankara, Turkey
under environmental stresses. Salinity is one of the hazardous abiotic stress factors limiting the productivity of the plants. NaCl is the main salt causing salinity in soil because of the Na+ toxicity. The studies are focused to understand which mechanisms affected from salinity stress and to develop the tolerant species (Silva et al. 2008). When a plant subjected to abiotic stress, the homeostasis between production of ROS and detoxifying activity of the antioxidants is disturbed (Cheong and Yun 2007). Higher concentrations of salt cause primary and secondary effects that negatively affect the plant development (Hossain et al. 2006). Also salinity effects germination, growth, photosynthesis, protein synthesis, chlorosis, and senesence (Hassan et al. 2004; Manchanda and Garg 2008; Munns 2002). The effects of soil salinity depend on stress duration, plant species, and the developmental stage of the plant when subjected to salinity. Under salinity stress, Na + and Cl − ions interact with aminoacids non-covalently thus it leads to functional losses and conformational changes in proteins (Zushi et al. 2009). The reasons of decreases in plant growth are physiological drought caused by osmotic adjustments by decreased water
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potential, decreased turgor, and increased ion concentrations. Decreases in chlorophyll and carotenoid concentrations, photosynthesis and closure of stomata, and less transpiration are the important physiological changes observed under higher salinity stresses (Azevedo Neto et al. 2004; Manchanda and Garg 2008; Yan and Guizhu 2007). Plants have species-specific salt tolerance capacities. Induction of antioxidant defenses, accumulation of osmolytes, and vacuolar localization of Na+ are the components of salt tolerance mechanisms (Ashraf 2004; Hernández et al. 2000; Koca et al. 2007). It is known that, leaf area modulations can be seen as responses to decreases of leaf water potentials (Steduto et al. 2000). Structural adaptations like changes in leaf size, stomatal opening/closure, and modifications of leaf anatomy against salinity stress also play important role in salt tolerance (Abbruzzese et al. 2009; Hameed et al. 2011). Trichomes are specialized epidermal cells on leaf or stem surfaces. These hairs are produced as a result of the outward growth of one or more epidermal cells. Epidermal tissues give rise to appendages (trichomes) such as spines, hairs, or glands that give plant leaves or stems distinctive textures. Trichomes have potential to accumulate bioactive compounds which have role in defense mechanisms against different stress factors (Ekanayaka et al. 2014; Harada et al. 2010; Roy et al. 1999; Szymanski and Marks 1998). The trichome-metabolism-related genes and their molecular mechanisms were identified in Arabidopsis. The molecular mechanism of trichome formation in the epidermis is under control of GLABROUS 1 (GL1), GLABROUS 3 (GL3), T R A N S PA R E N T T E S TA G L A B R A 1 ( T T G 1 ) , a n d GLABROUS 2 (GL2) genes. These genes are the major genes which have role in trichome initiation and spacing of leaf trichomes. TTG1 codes WD-40 repeat containing protein. GL1 gene encodes R2R3 MYB-transcription factor is expressed in developing leaves of the plants (Gonzalez et al. 2008; Hauser et al. 2001; Lillo et al. 2008). GL3 plays as a positive regulator of trichome initiation and growth (Gao et al. 2008). The formation of GL1-GL3 and TTG1 complex is essential for the cell to determine the formation of trichome (Gao et al. 2008). GL2 (HD-ZIP IV family) is an environmental change-sensitive gene-encoding leucine zipper transcription factor and is regulated by the TTG1 and GL1 and WEREWOLF (WER) genes (Marks 1997; Masucci et al. 1996; Rerie et al. 1994; Szymanski and Marks 1998; Feng et al. 2016; Xiao et al. 2017). In this study, we subjected 14-day-old soybean plantlets to three different concentrations of NaCl. We aimed to evaluate the biochemical and ultrastructural changes on the leaves under salt stress in Ataem-7 and S04-05 soybean varieties. We also compared the phenotypic data with the results of expression analysis of the genes which are responsible of trichome formation.
Materials and Methods Plant Material Glycine max L. Merrill seeds (Ataem-7 and S04-05) were taken from Black Sea Agricultural Research Institute, Samsun, Turkey. These varieties were improved and selected for breeding studies by the institute due to their highly adaptive features to agricultural conditions of Turkey. Soybean seeds were planted onto perlite and irragated with ½ Hoagland solution for 14 days.
Salinity Experiment Fourteen-day-old soybean seedlings were subjected to salinity stress at 0, 50, 100, and 150 mM NaCl. The doses were determined according to the results of previous studies (Çelik and Ünsal 2013). Ninety-millimolar NaCl was determined as the tolerance limit for these cultivars. Therefore, we used 50mM NaCl as the lowest stress dose and 150-mM salt concentration was used as the higher dose than the tolerance limit. The salinity studies were performed in growth chamber conditions with 25 °C and 16 h/8 h light/dark regime. Salinity treatments were continued for 1 week. The leaves were harvested for biochemical, molecular analyses, and electron microscopy studies.
Chlorophyll Content Determination The extraction of leaf pigments was done with 80% acetone, and the absorbances were measured at 663 and 645 nm with Amersham Spectrophotometer. Chlorophyll a, chlorophyll b, and total chlorophyll quantities were calculated in accordance with Arnon method (Arnon 1949).
Guaiacol Peroxidase Enzyme Activity Assay 0.5 g of leaf samples were homogenized with an ice-cold 50-mM sodium phosphate buffer (pH 7.8) containing 1mM EDTANa 2 , 2% (w/v) polyvinylpolypyrrolidone (PVPP). After centrifugation of the homogenates at 13.000 g for 40 min, supernatants were used to determine the guaiacol peroxidase activity according to Scebba et al. (2006) at 25 °C. Guaiacol is using as a hydrogen donor, and the principle of the method is to calculate the H2O2 decomposition rate by peroxidase. The reaction mixture contained 11-mM H2O2 in 65-mM phosphate buffer (pH 6.0), 2.25-mM guaiacol and 50 μl of the enzyme extract in a final assay volume of 2 ml. GPX activity was determined due to increase in absorbance of oxiguaiacol at 470 nm and was expressed as μmol min−1 g−1 FW.
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Scanning Electron Microscopy Study Fresh leaves were fixed for 24 h in 2.5% gluteraldehyde (pH 7.2, phosphat buffered). Then, specimens were rinsed two or more times with distilled water. The leaves were subjected to graded series of ethanol (from 70 to 100%) for dehydration. Leaves were then put into amyl acetate. The samples were next dried at the critical point with CO2 (Polaron, CPD 7501). They were mounted with double-sided tape on SEM stubs, coated with gold in Polaron SC 502 Sputter coater, and examined with Jeol JSM 6060 Scanning Electron Microscope at 0.5–20 kV. Measurements and counting of trichomes, glandular trichomes, and stomata on the adaxial and abaxial surface of leaves were made × 100 and × 500 magnification on SEM. Micrographs were taken digitally. Trichomes were removed by tweezers in the buffer solution after fixation step. After removing them, the base of trichomes remains intact and easy to count of them on the leaves. The adaxial and abaxial surfaces of the leaves of control and salt-stressed soybean leaves were examined by SEM. The total leaf area and the area of stomata on both surfaces of the leaves were determined by Scion Image programme. Leaves were photographed with Panasonic DMC FZ30. The images were converted to grayscale. The final version was saved as a TIFF file without LZW compression. We used public domain software (Image 4.0.2 for Windows, National Institute of Health, Bethesda, MD) downloaded from https://imagej.nih.gov/ij/ to measure the areas of the samples (O’neal et al. 2002). We opened TIFF file to be analyzed within the Image Software. We selected the set scale option from the analyze menu. We selected a unit (cm) to convert pixels to a unit of measurement. A ruler within the image was used for calibrating the pixel convertion. When the object is outlined, the surface area was calculated by selecting the measure option from the analyze menu (O’neal et al. 2002). These measurements were replicated for five times.
RNA Isolation Total RNA was extracted from the leaves of control and saltstressed soybean plants with MoBio UltraClean Plant RNA Isolation Kit (USA). First-strand cDNA synthesis was performed in a total volume of 20 μl with iScript cDNA synthesis kit for real-time polymerase chain reaction (RT-PCR) (BioRAD, USA). Each reaction mixture contained 1 μg of total RNA, 4 μl iScript reaction mix. The reaction mixtures were incubated at 25 °C for 5 min, 42 °C for 30 min, and 85 °C for 5 min, then stored at 4 °C.
Quantitative RT-PCR Analyses Soybean orthologs of Arabidopsis trichome-metabolismrelated genes were given by Hunt et al. (2011). The primers
were designed by using Prtimer3Plus (http://www. bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) due to the ortholog gene sequences and given in Table 1. The synthesized cDNA was subjected to quantitative RT-PCR analysis using SYBR Green Supermix (Bio-Rad, USA) in Mini Opticon™ Real-Time PCR System (Bio-Rad, USA). The PCR conditions were as follows: a denaturation step at 94 °C for 3 min was followed by 35 cycles of 54 °C for 15 s, 72 °C 1.5 min and 94 °C for 15 s. Soybean EF-1αA4 gene was found the most stable reference gene under different abiotic stress treatments (Ma et al. 2013; Miranda et al. 2013; Bansal et al. 2015); therefore, the elongation factor EF1αA4 (GenBank accession no. XM_003553244.3) (Ma et al. 2013; Celik et al. 2014) was used as internal control to estimate the relative transcript level of the genes tested. Each PCR reaction was carried out in triplicate. A melting curve analysis was performed at the end of the PCR run over the range 30–85 °C, increasing the temperature stepwise by 0. 5 °C every 10 s.
Relative Quantification Baseline and quantification cycles were determined. The raw Ct values obtained fom the CFX ManagerTM Software were converted into relative quantities via the delta-delta Ct method (Pfaffl et al. 2003), and the different expression levels between the salt-stressed and control soybean samples were determined.
Statistical Analysis Each data was the mean of three independent experiments and respresented as the mean ± SD. All the statistical analyses were performed by using ANOVA, and we applied Duncan’s multiple range test to compare the results of the experiments (P < 0.05) (Duncan 1955). Relationships between trichomemetabolism-related gene expression levels and trichome densities of control and salt-stressed soybean plants were determined by Pearson’s correlation analysis. For each of variables, the interactions among all trichome-metabolism-related genes were also evaluated using Kuskal-Wallis test and post-test Dunn’s multiple comparison test (Celik et al. 2014).
Results Chlorophyll concentrations were determined and given in Fig. 1. Chlorophyll a, chlorophyll b, and total chlorophyll concentrations showed decrease in contrast to increasing NaCl concentrations. While chorophyll b parameters were decreased 50% in Ataem-7 variety, 62.50% decrease was observed in S04-05 variety at 150-mM NaCl treatment. When we evaluate the total chlorophyll results, Ataem-7 soybean variety showed higher decreases than S04-05 variety.
Plant Mol Biol Rep Table 1 Primer sequences of trichome-metabolism-related genes used in qRT-PCR analysis
Gene
Soybean orthologs
Primer sequence
Amplicon length (bp)
GL1
Glyma07g05960
Forward:5’-GGCATCTTTAGGTGCTCTGC-3’
210
Glyma07g08340
Reverse:5’-TGATTTCCATTGGCCTTCTC-3’ Forward:5’-GTTTCTTGAGCTTGCCTTGG-3’
174
Glyma08g01810
Reverse:5’-GCCTCAGACACAAACCCATT-3’ Forward:5’-GAGGCCATTGTGATGTGTTG-3’
203
Glyma06g14180
Reverse:5’-TTGGTTTGAGGGGTGACTTC-3’ Forward:5’-GCGTCGACATCCTCTCTTTC-3’
183
XM_003553244.3
Reverse:5’-GCGTCGACATCCTCTCTTTC-3’ Forward:5’-GACCTTCTTCGTTTCTCGCA -3’
195
GL2 GL3 TTG1 EF1α4A
Reverse: 5’-CGAACCTCTCAATCACACGC-3’
We indicated the level of stress which the soybean plants subjected via activity of guaiacol peroxidase. In both varieties, GPX enzyme activities were given in Fig. 2. GPX activities were increased due to increasing NaCl concentrations. In control groups, the enzyme activities were 0.535 and 0.425 μmol H2O2/min mg FW for S04-05 and Ataem-7 varieties, respectively. After salt treatments, while the determined GPX acitivities of Ataem-7 variety were 1.384, 2.674, and 3.454 μmol H 2 O 2 /min mg FW, in S04-05 variety, the
Fig. 1 Effects of salt stress on chlorophyll a, b, and total chlorophyll concentrations of a Ataem-7 and b S04-05 soybean varieties. Results are given as the means of three replicative experiments with standard error. Mean values in columns with different letters are significant at 5% level according to Duncan’s multiple range test
activities were found as 1.028, 2.197, and 2.967 μmol H2O2/ min mg FW at 50, 100, and 150-mM NaCl, respectively. The reduction of growth induced by salinity was observed in decreased total leaf size. The leaf areas of the salt-stressed plants were 12.93, 47.69, and 54.85% inhibited relative to control plants after 50, 100, and 150-mM NaCl treatments, respectively, in Ataem-7 cultivar and given in Fig. 3. At the same salt concentrations, the relative decrement rates for S0405 variety were 34.12, 47.32, and 72.68%, respectively.
Plant Mol Biol Rep Fig. 2 Effects of salt stress on guaiacol peroxidase activities of the leaves of Ataem-7 and S04-05 soybean variety. Results are given as the means of three replicative experiments with standard error. Mean values in columns with different letters are significant at 5% level according to Duncan’s multiple range test
On the seventh day of salinity experiment, the areas of stomata on the leaves of soybean plantlets were determined and given in Fig. 4. Stomata areas showed decrease in contrast to increasing NaCl concentrations on both surfaces of the leaves in comparison to control in Ataem-7 variety. The stomatal area on the abaxial surface was found higher than adaxial surface in Ataem-7 control group. The stomatal areas were highly affected at 50-mM NaCl treatment in Ataem-7 variety. The decreases of stomatal area were also observed higher on the abaxial surface than adaxial surface in response to increasing salt concentrations. For S04-05 variety, at 50-mM NaCl treatment, stomata areas were 7.29 and 18.32% decreased on abaxial and adaxial surfaces of the leaves, respectively. Higher concentrations of NaCl caused significant decrease in stomata areas on both surfaces of the leaves of S04-05 variety, while 86.05 and 76.10% decreases were observed on the abaxial and Fig. 3 Total leaf areas of differentially salt-stressed soybean plants belonging to Ataem-7 and S04-05 varieties. Results are given as the means of three replicative experiments with standard error. Mean values in columns with different letters are significant at 5% level according to Duncan’s multiple range test
adaxial surfaces of the leaves after 100-mM NaCl treatment and 94.31 and 96.09% decreases were observed after 150-mM NaCl treatments with respect to control, respectively. The number of trichomes, glandular trichomes, and stomata on adaxial and abaxial surface of the control and salt stressed plantlets was determined by SEM per square millimeter for Ataem-7 and S04-05 soybean varieties, as provided in Table 2, respectively, and defined as densities. After 7 days of treatment, in Ataem-7 variety, on the abaxial surface, densities of the trichomes were 89.13%, 116.67% increased at 50 and 100-mM NaCl treatments according to the control, respectively. At 150-mM NaCl concentrations, trichome densities were 14.49% decreased on abaxial surface with respect to Ataem-7 control plant. In S04-05 variety, 27.20 and 31.20% were observed on the abaxial surfaces of the leaves at 50 and 100-mM NaCl treatments. Representative photographs of
Plant Mol Biol Rep Fig. 4 Effects of salt stress on the stomata areas of the leaves of Ataem-7 and S04-05 soybean varieties. Results are given as the means of three replicative experiments with standard error. Mean values in columns with different letters are significant at 5% level according to Duncan’s multiple range test
trichome densities at 0, 50, 100, and 150-mM NaCl treatments of Ataem-7 and S04-05 soybean plants were given in Fig. 5. The glandular trichome densities on the abaxial surface of the Ataem-7 and S04-05 varieties showed increase due to increasing NaCl concentrations. The increases in Ataem-7 variety were found higher than S04-05 variety (Table 2). While the glandular trichome densities were decreased on adaxial surfaces of both varieties, 2.98 and 1.99-fold increases were recorded at 150-mM NaCl treatment in Ataem-7 and S04-05 soybean varieties with respect to control plants. While the stomatal densities on the leaves of NaCl-treated plants showed 28.22, 64.89, and 16.21% increases with respect to control leaves on the abaxial surfaces of Ataem-7 variety, whereas 177.56, 477.66, and 56.26% increment rates were observed in S04-05 variety at 50, 100, and 150-mM NaCl concentrations, respectively. On adaxial surfaces, stomatal densities were increased as a response to increasing salt concentrations. The increment rates were found higher in S0405 variety than Ataem-7 variety. 1.15, 1.24, and 1.56-fold increases in stomatal densities were determined on adaxial surfaces of the leaves of Ataem-7 soybean variety at 50, 100, and 150-mM NaCl treatments while 1.47, 2.61, and 2.62-fold increases were recorded in adaxial surfaces of the leaves of S04-05 variety, respectively. GL1, GL2, GL3, and TTG1 gene expression levels were determined in control and salt-stressed plants in both soybean cultivars by qRT-PCR. The changes in the transcript levels of the trichome-metabolism-related genes are shown in Fig. 6. The data given in Fig. 6 represent the changes in expression levels of GL1, GL2, GL3, and TTG1 genes in the leaves of soybean plants in comparison to control plants. The differences between the expression levels of trichome development ortholog genes between the soybean varieties at different salt concentrations were statistically evaluated (P < 0.05). According to the results of qRT-PCR, we found that the expression level of GL1 gene was 0.3-fold increased at 50-mM NaCl and 0.5 and 0.65-fold decreased after 100 and 150-mM NaCl treatments in Ataem-7 variety in comparison to control
plants, respectively. In S04-05 soybean variety, GL1 gene expression was 0.25-fold increased at 50-mM NaCl treatment, whereas 0.4 and 0.5-fold decreases were observed at 100 and 150-mM NaCl treatments, respectively. The expression levels of GL2 in all salt treatment groups were upregulated. At 50 mM, 0.24-fold increment was observed in Ataem-7 variety, whereas 0.12-fold increment was observed in S04-05 variety. 0.2 and 0.38-fold increases were recorded in Ataem-7 variety after 100 and 150-mM NaCl treatments, respectively. In S04-05 soybean variety, upregulation fold rates of GL2 gene were found as 0.3 and 0.42 after 100 and 150 mM salt treatments with respect to control, respectively. GL3 gene showed increasing expression profile in relation to increasing salt concentration in both soybean varieties. While this increase was gradually in Ataem-7 variety, the increment was found lower at 150 mM than 100 mM in S04-05 variety. For Ataem-7 variety, 0.1, 0.2, and 0.24-fold increases were found at 50, 100, and 150-mM NaCl treatment with respect to control, respectively. 0.2, 0.3, and 0.28-fold increases were also recorded at the same salt concentrations in S04-05 variety, respectively. TTG1 gene expression analyses demonstrated 0.2, 0.4, and 0.53-fold decreases in TTG1 gene expression levels under 50, 100, and 150-mM salt stress for Ataem-7 plants, in comparison with control plants, respectively. TTG1 gene levels were gradually decreased due to increasing salt concentrations. In S04-05 variety, the transcipt level of TTG1 gene was decreased as a response to increasing salt concentration. 0.17, 0.3, and 0.42fold decrease was recorded with respect to conrol group at 50, 100, and 150-mM NaCl treatments, respectively. The relations between the expression levels of GL1, GL2, GL3, and TTG1 genes and trichome densities on both surfaces of Ataem-7 and S04-05 soybean leaves were evaluated by Pearson’s correlation analysis (Supplementary Fig. 7). In Ataem-7 soybean variety, the relation between trichome densities on both surfaces of leaves and GL1 gene expression was found statistically significant (P < 0.05) as distinct from the finding of the lowest NaCl treatment on abaxial surface. At
S04-05
12.9 ± 5.4a 12.5 ± 3.8a 7.6 ± 1.9a Adaxial Abaxial Adaxial
372 ± 42.5a 56.7 ± 9.1a 106.1 ± 11.5a 79.4 ± 8.3a
26.1 ± 5.4b 14.9 ± 4.3a 15.9 ± 5.1b 9.7 ± 3.4a 12.8 ± 6.5a 13.4 ± 5.7a 33.9 ± 12.7a 33.4 ± 0.1a 13.8 ± 3.7a Abaxial
24.8 ± 9.1b 7.3 ± 1.4b 31.5 ± 6.2b 12.1 ± 3.1b
613.4 ± 18.1c 70.3 ± 10.1c 612.9 ± 17.3c 207.2 ± 9.8c Ataem-7
Trichome Glandular trichome Trichome
Stomata
50 mM NaCl Control
Glandular trichome
Stomata Leaf surface Variety
* The data are represented as the means ± SD. Means that per type of parameters are statistically different between the salt treatments are indicated with different letters after Duncan’s multiple range test at a 5% level of probability
432.3 ± 51.5b 88.8 ± 15.7d 165.8 ± 15.3d 208.1 ± 9.9c 37.0 ± 16.6c 6.96 ± 0.9b 39.3 ± 9.5c 16 ± 7.1c 29.9 ± 14.3b 16.5 ± 4.23b 16.4 ± 4.9b 10.9 ± 3.4b 477 ± 70.2b 65.3 ± 14.9b 294.5 ± 43.3b 116.7 ± 26.5b
39.2 ± 13.3d 6.2 ± 1.2b 67.6 ± 14.6d 22.4 ± 7.1d
Trichome Glandular trichome Trichome Stomata
Trichome, glandular trichome, and stomata densities (number per mm2) of control and salt-stressed soybean plants belonging to Ataem-7 and S04-05 Table 2
11.8 ± 4.9a 12.4 ± 4.8a 48.9 ± 12.7c 35.1 ± 6.5c
150 mM NaCl 100 mM NaCl
Glandular trichome
Stomata
Plant Mol Biol Rep
50 mM NaCl treatment, the only statistically significance was determined between GL2 gene expression level and trichome density on abaxial surface of the leaves. In S04-05 variety, trichome densities were found in relation with the transcript levels of GL1 and GL2 as a response to salt stress (P < 0.05). The divergent significances were determined between GL3 and trichome density at 50 mM NaCl treatment on abaxial surface of the leaves. At 100-mM NaCl treatment, trichome densities on the adaxial surfaces of the leaves were found in relation with GL1 and GL2 transcript levels while only GL2 expression level is seem to be statistically important in regulation of trichome density after 150-mM NaCl treatment (P < 0.05). We also determined the relation among transcript levels of trichome-metabolism-related genes in both soybean varieties against salt-stress treatments. The regulations between TTG1 and GL2 and TTG1 and GL3 were found inversely correlated in Ataem-7 and S04-05 varieties, respectively, (P < 0.05) as well as the regulation of other genes was not correlated.
Discussion Soil salinity is an important abiotic stress factor that affects plant productivity. The analysis of leaf ultrastructural changes under abiotic stress conditions is effective to clarify the different strategies to overcome the detrimental effects of salinity. Inhibition of cell division and enlargement, reduced shoot growth, and changes in morphological and physiological traits are the results of osmotic and ionic stresses to cope with the stress (Babaeian et al. 2011; Wang and Frei 2011). Photosynthetic parameters are the first physiological parameters affected by salt stress. Therefore, we determined the contents of chlorophyll a, b, and total chlorophyll. In both soybean varieties, photosynthetic pigments were gradually decreased in accordance with increasing salt concentrations. Production of reactive oxygen species (ROS) is responsible from damages in thylakoid membranes, cell membranes, organelles, changes in protein synthesis, and photosynthetic capacity. Decreases in plant growth are the visible changes under abiotic stress conditions. Adaptation to salinity stress is under a complex-coordinated system (Azevedo Neto et al. 2006, 2004; Tuteja 2007). Peroxidases catalyze the reduction of H2O2 to H2O and O2. Hydrogen peroxide which is a byproduct of metabolic processes is toxic radical that may interact with various biomolecules due to its unpaired electron configuration. Guaiacol peroxidase as a non-specific peroxidase has been defined as the most important hydrogen peroxide scavenging enzyme beside catalase leading to salt tolerance. In order to support the results of physiologic parameters, we investigated the activity of guaiacol peroxidase enzyme. In both soybean varieties, the enzyme activity was increased
Plant Mol Biol Rep Fig. 5 Representative photographs of trichome densities of abaxial surface of the leaves of Ataem-7 and S04-05 soybean plants at 0, 50, 100, and 150 mM NaCl treatments. The photographs were screened by scanning electron microscopy analysis (× 100). a Ataem-7 Control. b Ataem-7 50 mM NaCl. c Ataem7100 mM NaCl. d Ataem7150 mM NaCl. e S04-05 Control. f S04-05 50 mM NaCl. g S04-05100 mM NaCl. h S0405150 mM NaCl
statistically significant in comparison to control groups (P < 0.05). Leaf area was the most critical growth feature in answer to salinity conditions (Abbruzzese et al. 2009; Silva et al. 2008). Munns reported that leaf growth is more negatively affected by salinity. In our experiment, we observed decreases in leaf
area in accordance with increasing salinity concentrations in both soybean varieties. Forty-seven and 92% reduction of leaf areas were reported in mangabeira and young guava plants at 125 and 150-mM NaCl concentrations, respectively (Silva et al. 2008). Azevedo Neto et al. (2004) observed no significant affect of increasing
Plant Mol Biol Rep
Fig. 6 The relative changes in expression profiles of soybean orthologs of GL1, GL2, GL3, and TTG1 genes after salt stress teratments (Mean ± SD). Gene-specific fragments were amplified by RT-PCR with 35 cycles. EF1αA4 was used as a housekeeping gene in response to salt stress. Results
are given as the means of three replicative experiments with standard error. Mean values in columns with different letters are significant at 5% level according to Duncan’s multiple range test
salinity stress on leaf area in maize and Silva et al. (2008) reported the similar results in young umbu plants. In contrast Abbruzzese et al. (2009) reported that 14P11 genotype Populus alba showed decrease in leaf area due to the level of salinity stress. Stomata are important to regulate the CO2 exchange that is related to transpirational cooling. Stomata also respond to photosynthetic metabolites, hormones, or regulators (especially ABA as a stress signal) and hydraulic linkages. Changes in stomata size (stomatal area) are based on the stomatal closure, and it seems as another adaptive mechanism of plants improved against salinity conditions. Therefore, we evaluated effects of different concentrations of NaCl on the stomatal areas. In this study, at 50mM NaCl treatment, in Ataem-7 variety represented a sharp decrease in stomatal area than S04-05 variety. However, on both sufaces of the leaves belonging to both varieties showed decreases in stomatal areas in contrast to increasing NaCl concentrations. Abbruzzese et al. observed decreases of stomatal area after salinity treatments at 50–250-mM NaCl concentrations in Populus alba L. genotypes. The osmotic stress caused by salinity stress helps stomata open (Bañon et al. 2004). Silva et al. (2008) realized that there is no correlation between salinity and stomatal closure; the contrary was showed by Abbruzzese et al. (2009), and they observed that the variations were time dependent. Trichomes are important surface caharacteristics to keep water droplets off the leaf surface and the ion balance. It is known that salinity increases trichome density (Botti et al. 1998; Huttunen
et al. 2010). The main task of the trichome under excessive salinity is to excrete the excess salt. This adaptive mechanism plays role under salt stress treatments which indicates changes in water imbalances in relation to oxidative metabolism (Rossi et al. 2016). Huttunen et al. (2010) reported that increased trichome density is an inducible defense mechanism against herbivors and water deficiencies for plants. Salinity has shown to have an effect on density and structure of the trichomes (Xiao et al. 2017). Halophytes are also observed to have specialized trichomes functioning in accumulation of salt and/or secretion to gain tolerance capacity (Adebooye et al. 2012). Even though according to our results, trichome densities were increased on both surfaces of the leaves at 50, 100, and 150-mM NaCl treatments in S04-05 variety. In Ataem-7 variety, at 150-mM trichome densities were decreased below control groups on abaxial and adaxial surfaces of the leaves. These data were determined as trichome numbers per square millimeter (trichome density). Increase of leaf pubescence caused by reduced water availability is reported by many researchers (Gonzalez et al. 2008). Glandular trichomes are also important to secrete different types of chemical compounds that are toxic to several insects (Gonzalez et al. 2008). While glandular trichome densities of Ataem-7 variety were increasing due to increasing NaCl concentrations on abaxial surfaces of the leaves, on adaxial surfaces, decreases were determined in comparison with control. In S04-05 variety, in spite of the decrease at 50 mM NaCl, glandular trichome densities on abaxial surface were increased after 100 and 150 mM NaCl treatments, with respect to control.
Plant Mol Biol Rep
It has been known that stomatal density can differ on the upper and lower surfaces of the leaf as physiological responsiveness (Gutschick 1999). The salt treatments reduced stomatal area on both surfaces of the leaves of Ataem-7 soybean variety compared with the control. In S04-05 variety, unlike Ataem-7, 50 mM NaCl treatment caused decreases in stomatal area on both surfaces of the leaves with respect to control plants. By increasing NaCl concentrations, stomatal areas on abaxial and adaxial surfaces showed decreases in comparison with control. GL1, which encodes R2R3 MYB transcription factor, has role in signal transduction pathways of stress hormones such as absisic acid (ABA), jasmonic acid, and salicylic acid. ABA is known to cause stomatal closure (Ambawat et al. 2013). In this study, the transcriptional regulation of GL1 in accordance with increasing severity of the salt treatment confirms the observed decreases in stomatal area caused by the closure as a response to salinity. The genes that have roles in trichome initiation and elongation have recognized in Arabidopsis (Gao et al. 2008). Studies have shown that formation of GL1/GL3/TTG1 complex is important in initiation of trichome development. GL2 gene is expressed in leaf trichomes, and it is thought to be involved in regulation of cell differentiation (Digiuni et al. 2008). Kirik et al. (2005) reported that GL1 expression is not dependent to the expression levels of GL3 and TTG1, and they also added that there is no regulation between TTG1 expression and GL3-GL1 genes in Arabidopsis. Payne et al. (2000) declared that GL3 protein can synergetically interact with GL1 to start tichome initiation independent from TTG1. However, TTG1 is essenial for function of the trichome initiation complex. Zhang et al. (2003) observed increased GL3 expression in developing leaves. In this study, the gene expression patterns of soybean orthologs of Arabidopsis trichome-metabolism-related genes were evaluated. This is the first study done in soybean plants under salt stress. Our qRT-PCR analysis that we perfomed by using primer pairs designed due to conserved regions of these genes, demonstrated an induction of the soybean orthologs of GL2 and GL3 in soybean plants after 50, 100, and 150 mM NaCl treatments in both varieties. While the expression level of TTG1 ortholog gene was negatively affected in both soybean varieties under different concentrations of salinity, GL1 ortholog gene expression profile differed as a result of changing salt concentrations in both varieties with respect to control plants. In Ataem-7 variety, GL2 and GL3 ortholog gene expressions were upregulated at all salt treatments, whereas GL1 ortholog gene expression level was downregulated at higher salt concentrations in comparison to control. At 50 mM NaCl treatment, the trichome development-related ortholog genes except TTG1 ortholog gene were upregulated and were in correlation with increased trichome numbers on both surfaces of the leaves in accordance with control group. GL1 and TTG1 ortholog genes are downregulated in Ataem-7 soybean variety
with respect to control. In Ataem-7 variety, trichome densities on both surfaces of the leaves were positively correlated with GL1 expression level (P < 0.05). In S04-05 soybean variety, the transcript levels of the trichome development related genes were showed the same pattern with Ataem-7 variety after 50 and 100 mM NaCl treatments. Electron microscopy results were showed that the trichome densities were increased after 50, 100, and 150 mM NaCl treatments on both surfaces of the S04-05 leaves with respect to control. According to the results of our experiments, this complex formation is important in trichome formation for soybean orthologs as described for Arabidopsis, but their transcript levels were differently affected by salt stress. We observed upregulation of GL1 ortholog gene in Ataem-7 variety, while the transcript levels of GL2 and GL3 ortholog genes were increased in S04-05 soybean variety at higher concentrations of NaCl stress. The soybean varieties presented different expressional profile through different arrays of gene responses to overcome the adverse effects of salt stress. GL2 (homodomain-leucine zipper transcription factor) has been reported to be involved in mediating plant responses to salt stress and known to be negatively regulated by GL1 which encodes a R2R3 MYB transcription factor. GL2 has a regulatory gene role in phenylpropanoid pathway that is involved in plant defense (Fraser and Chapple 2011; Reinprecht et al. 2013; Chen et al. 2014). Accumulation of phenylpropanoids and their derivatives which can be termed as secondary metabolytes has been defined in relation to plant survival under several stresses. Positive regulatory relationship between phenylpropanoid compounds and glandular trichome density has been reported in several plant species such as soybean, common bean (Reinprecht et al. 2013), and Oleo europaea cultivars (Rossi et al. 2016); therefore link between glandular trichome density and plant salt sensitivity can be correlated (Rossi et al. 2016). In relation to these studies, glandular trichome density on abaxial surface of the leaves increased 1.54-fold in Ataem-7 variety compared to S04-05 variety at 150 mM NaCl concentration, in this study. In this novel study, transcriptional differences of the trichomemetabolism-related genes between two soybean varieties were evaluated. In respect to the findings of the study, we suggest further investigations of regulatory genes and compounds of phenylpropanoid pathway under salinity stress for better understanding the tolerance capacities of the soybean varieties.
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