search
Contact Us
HOME

  • Crossref logo
  • Crossref Similarity Check logo
Journal Search Engine

to

Search Advanced Search Adode Reader(link)
Download PDF Export Citaion korean bibliography PMC previewer
ISSN : 2287-5824(Print)
ISSN : 2287-5832(Online)
Journal of The Korean Society of Grassland and Forage Science Vol.42 No.3 pp.137-145
DOI : https://doi.org/10.5333/KGFS.2022.42.3.137

Analysis of Aluminum Stress-induced Differentially Expressed Proteins in Alfalfa Roots Using Proteomic Approach

Dong-Hyun Kim1, Joon-Woo Lee1, Chang-Woo Min1, Md. Atikur Rahman1,2, Yong-Goo Kim1, Byung-Hyun Lee1*
1Division of Applied Life Sciences (BK21), IALS, Gyeongsang National University, Jinju 660-701, Republic of Korea
2Present address: Grassland and Forage Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea
* Corresponding author: Byung-Hyun Lee, Department of Animal Bioscience, Division of Applied Life Sciences (BK21), IALS, Gyeongsang
National University, Jinju 660-701, Republic of Korea. Tel: +82-55-772-1882, Fax: +82-55-772-1889, E-mail: hyun@gnu.ac.kr
September 14, 2022 September 23, 2022 September 24, 2022

Abstract


Aluminum (Al) is one of the major factors adversely affects crop growth and productivity in acidic soils. In this study, the effect of Al on plants in soil was investigated by comparing the protein expression profiles of alfalfa roots exposed to Al stress treatment. Two-week-old alfalfa seedlings were exposed to Al stress treatment at pH 4.0. Total protein was extracted from alfalfa root tissue and analyzed by two-dimensional gel electrophoresis combined with MALDI-TOF/TOF mass spectrometry. A total of 45 proteins differentially expressed in Al stress-treated alfalfa root tissues were identified, of which 28 were up-regulated and 17 were down-regulated. Of the differentially expressed proteins, 7 representative proteins were further confirmed for transcript accumulation by RT-PCR analysis. The identified proteins were involved in several functional categories including disease/defense (24%), energy (22%), protein destination (9%), metabolism (7%), transcription (5%), secondary metabolism (4%), and ambiguous classification (29%). The identification of key candidate genes induced by Al in alfalfa roots will be useful to elucidate the molecular mechanisms of Al stress tolerance in alfalfa plants.


Alfalfa root , Acidic soil , Aluminum stress , Forage , Proteome

초록

    Ⅰ. INTRODUCTION

    Aluminum (Al) is the third most abundant metal in the earth crust, and one of the major factors reducing most plant growth and productivity in acidic soils. Approximately, 30% of the world’s land contains acidic soil, and more than 50% of the arable land is acidic (Uexkull and Mutert, 1995). In acidic soil, Al is solubilized into the toxic form Al3+, which interferes with a number of cellular processes, and inhibits water and nutrient absorption in the roots of plants (Kochian et al., 2015). It is known that the most prominent symptom of Al toxicity is inhibition of root growth. Al inhibits the cell division and elongation of root tissue, and reduces the uptake of inorganic nutrients such as P, K, Mo, Ca, Mg and N (Riaz et al., 2018). Moreover, Al stress increases the production of reactive oxygen species (ROS), which cause oxidative damage to the cellular components (He et al., 2019). Antioxidant enzymes such as SOD, CAT, POD, GST, and GR are involved in the detoxification of the ROS generated by Al stress (Wei et al., 2021).

    Several studies have been reported that various aluminum tolerance mechanisms so far, but they can be grouped into external exclusion mechanisms and internal tolerance mechanisms. (Wei et al., 2021). Plant secretes several organic acids such as citrate, malate, and oxalate from root tips under acidic soils, which help to plants to confer Al stress tolerance (Kochian et al., 2015). For instance, Al-activated malate transporters (ALMT) have been identified from barley (Gruber et al., 2010) and maize (Ligaba et al., 2012). In addition, MATE, multidrug transporters, have been identified from soybean (Glycine max) (Zhou et al., 2019) and maize (Maron et al., 2010). As internal tolerance mechanisms, that is involved in Al uptake, translocation, and detoxification (Li et al., 2014). Studies on the Al tolerance of plants have been actively carried out using molecular mapping with SSR and RFLP markers (Eujayl et al., 2004) and the development of transgenic plants using organic acids (Barone et al., 2008), transporter proteins (Magalhaes et al., 2007), and antioxidant-related genes (Ghanati et al., 2005). In addition, a number of proteomic studies to reveal the mechanisms Al tolerance in plants have been reported from plants such as rice (Yang et al., 2007), soybean (Zhen et al., 2007), and tomato (Zhou et al., 2009). Differentially expressed proteins after exposure to Al stress, of which were associated with oxidative stress, detoxification, organic acid metabolism (Zheng et al., 2014). However, a proteomic analysis of the root protein of alfalfa plants has not yet been reported.

    Alfalfa is one of the most widely cultivated forage legume plants in the world. It is a perennial forage legume that contains high nutritive values, and has excellent nitrogen-fixing ability. In Korea, alfalfa is cultivated in a small scale, and mostly is imported from the USA or other countries. The soil structure and low soil pH in the mountainous grassland or cropland are the major constraints decreasing the productivity of alfalfa in Korea. Therefore, the development of alfalfa varieties tolerant to acidic soil along with the improvement of soil acidity by liming is important for improving the forage productivity and sustainability of mountainous grasslands in Korea. Therefore, understanding of Al-tolerance mechanisms is critical to generate Al-tolerant alfalfa variety in the future. In this study, we identified key proteins involved in Al stress response in alfalfa root tissues, which would be useful for the understanding of the Al stress tolerance mechanisms.

    Ⅱ. MATERIALS AND METHODS

    1. Plant growth and aluminum treatments

    Alfalfa (Medicago sativa. cv. Vernal) seeds were surface sterilized using 70% ethanol following 30% (v/v) sodium hypochlorite solution, and washed three times with distilled water. Seeds were germinated in the dark for 3 days at 25℃ and then transferred to a hydroponic system containing Hoagland solution (pH 7.0) at 25℃ under white florescent light (480 μmol m-1s-1) with 16 h photoperiod. After 15 days, seedlings were treated with 0, 200, 800 and 2000 μM AlCl3 (pH 4.0) for 3 days, and root samples were collected and stored at -80℃ for protein extraction.

    2. Protein extraction, 2DE and gel image analysis

    Proteins were extracted from alfalfa root tissue using a phenol extraction method according to the previous report (Lee et al., 2007). The two-dimensional gel electrophoresis (2-DE) was performed following the method of Lee et al (2007). After SDS-PAGE, The CBB-stained gels were scanned by scanner, the densitometric analysis of 2-DE gel spots were analyzed using PDQuest software (version 7.2; Bio-Rad, Hercules, CA, USA).

    3. In-gel digestion and MALDI-TOF/TOF MS

    The in-gel digestion and MALDI-TOF/TOF MS were performed according to the protocol described previously (Lee et al., 2007). Briefly, differentially expressed protein spots were manually excised from the CBB-stained gels and gel slices were alkylated, digested with trypsin, and the digested peptides were extracted and finally 1 μl of the homogenate was loaded onto the MALDI plate. Peptides were analyzed using a MALDI-TOF/TOF MS (SCIEX-4800 TOF/TOF Mass). The peptide mass fingerprintings (PMFs) obtained from each digested protein were compared with PMFs in the non-redundant National Center for Biotechnology Information (NCBI) database using the ProFound program. The search was performed within green plants (Viridiplantae) (Rahman et al., 2014).

    4. RNA extraction and RT-PCR

    Root samples were collected from alfalfa seedlings that had been treated with Al stress for 0, 6, 12, 24, 48 and 72 h. Total RNA was extracted from the root tissues using a Plant RNeasy mini kit (Qiagen, CA, USA). After isolation, total RNA samples were treated with RNase-free DNase I (Promega, U.K). To determine of quantitative expression of Al-responsive genes, RT-PCR amplification was performed using an RT-PCR kit (TOYOBO, Japan), in accordance with the manufacturer’s instruction. The gene-specific primer sequences were used for RT-PCR are shown in Table 2.

    5. Statistical analyses

    The results of the gel spot intensities were statistically analyzed using on analysis of variance (ANOVA) or Student’s t-test. The values were considered significant at the p≤0.05 level. All data were shown as means ± S.E. of at least three independent experiments.

    Ⅲ. RESULTS AND DISCUSSION

    1. Al stress-induced proteins in alfalfa roots tissues

    To investigate the response of the differentially expressed proteins in alfalfa roots under Al stress, 3 days-old Al stresstreated alfalfa roots were used for proteome analysis. Total proteins were separated one-dimensionally using strips with isoelectric points pI 4 to 7, and separated two-dimensionally according to molecular weight. The 2-DE gel was observed with CBB R-250 staining and spots were analyzed by PDQuest software. About 1,200 proteins spots were found and were compared that the differences in expression of each protein spots between the Al-untreated control and Al-treated samples. It was confirmed that 49 protein spots were differently expressed in response to Al treatment (Fig. 1). These protein spots were excised from the 2-DE gels and digested with trypsin, and peptides were identified by PMF using MALDI-TOF/TOF MS. Finally 45 proteins were identified (Table 1). Among these, 28 were up-regulated, and 17 spots were down-regulated (Fig. 2). These results demonstrate that Al stress treatment under acidic (pH 4.0) condition is toxic to the root cells of alfalfa plants, which would induce expression of Al-responsive proteins in root cells.

    2. Functional categorization of Al stress-induced root protein

    Identified proteins were classified into the following categories including disease/defense (24%), energy (22%), protein destination (9%), metabolism (7%), transcription (5%), secondary metabolism (4%) and miscellaneous (29%) (Fig. 3).

    2.1. Change in disease/defense-related proteins

    Disease/defense-related protein spots, 7, 9, 12, 26, 34 and 41 were increased, and 3, 6, 39 and 45 were decreased. Spots 7 and 12 were identified as alcohol dehydrogenase, which is involved in acetaldehyde detoxification and anaerobic stress resistance (Dolferus et al., 1985). Superoxide dismutase (spot 26) plays a role of defense against ROS generated under stress conditions (Alscher et al., 2002). Isoflavonoids (spot 34) are important secondary metabolites produced primarily in leguminous plants. It plays important roles in plant defense (Wang et al., 2006). The glutathione reductase (spot 3) is an enzyme that catalyzes the conversion of glutathione disulfide (GSSG) to glutathione (GSH) by NADPH-dependent reduction. It is the essential reaction for the keep of glutathione levels. The major role of glutathione is reductant in oxidation-reduction processes, serves in detoxication, and great importance in several cellular functions (Carlberg and Mannervik, 1985). Dehydrin (spot 6) is a protein associated with ABA-related response, low temperature, and salinity stresses (Nylander et al., 2001). Glutathione peroxidase (spot 39) is an enzyme that protects cells from oxidative damage caused by ROS produced by stress (Rodriguez et al., 2003). Thioredoxin (spot 45) and glutathione are major regulators of redox homeostasis, and are essential for postembryonic meristematic activities (Bashandy et al., 2010). Most disease and defense-related genes are thought to play an important role in detoxifying toxins such as ROS.

    2.2. Change of energy-related proteins

    Energy-related protein spots 2, 13, 14, 22, 24, 36, 37, and 47 were increased and spots 1, 4 and 40 were decreased. Aconitate hydratase (spot 1) is an enzyme that catalyzes the resolution of citric acid or isocitric acid into aconitic acid and water in the TCA cycle (Moeder et al., 2007). Plant aconitases are involved in resistance to oxidative stress, regulate gene expression regulating intracellular ROS levels, and regulate cell death (Moeder et al., 2007). Aconitate hydratase appears to protect the plant from ROS due to Al stress (Moeder et al., 2007). Ribulose bisphosphate carboxylase large chain (spot 22) catalyzes the Calvin cycle of carbon dioxide fixation (Andersson et al., 1989). Ribulose bisphosphate carboxylase large chain also catalyzes the initial oxygenation step. Malate dehydrogenase (spot 36 and 47) catalyzes the transform of oxaloacetate and malate. The main organic acids citrate, oxalate, malate are well known for directly deciphering Al toxicity (Tesfaye et al., 2001). Due to Al toxicity, alfalfa seemed to try to protect plants by producing organic acids by malate dehydrogenase to detect or detoxify it. NADP-dependent isocitrate dehydrogenase (spot 37) catalyzes the production of NADPdependent isocitrate dehydrogenase enzyme, an essential component to maintain cell homeostasis. NADPH is a major cofactor for cellular enzymatic reactions, homeostasis of cellular redox essential in the biosynthetic pathway and detoxification processes (Leterrier et al., 2012). Therefore, NADP-dependent isocitrate dehydrogenase is thought to be helpful in detoxifying various stresses with Al stress.

    2.3. Change in miscellaneous proteins

    Several other protein spots also showed differential expression against Al stress treatment; spot 5, 17, 31, 32, 46 and 48, which were increased, and spots 11, 15, 18, 20, 21, 25 and 33, which were decreased. Glutamine synthetase (GS) (spot 32) is the major enzyme of primary nitrogen assimilation, ammonia reassimilation and detoxification (Hossain and Komatsu, 2013). Plant GS requires two magnesium ions for activity so GS can be a potential target of metal stress (Hossain and Komatsu, 2013). The increase in GS expression induces the formation of more glutathione (GSH). The increase in GSH synthesis is not only associated with high metal binding capacity, but also with an improved mechanism of cell defense against oxidative stress (Verbruggen et al., 2009). GSH is the precursor of phytochelatin (Hradilova et al., 2010) which binds to heavy metals including Al. Thus, alfalfa GS would have an important function against Al-induced oxidative stress. OPDA (12-oxophytodienoic acid, spot 5) is known as involved in the stress and defense responses in plants (Matsui et al., 2004). 20S proteasome (spot 11) is a proteolytic complex involved in abnormal cellular proteolysis (Fu et al., 1998).

    2.4. Expression of key genes involved in Al stress response

    Several representative proteins differentially expressed by Al stress treatment in alfalfa roots were selected for further expression analysis with transcriptional level. Among 45 identified proteins, 7 genes (alcohol dehydrogenase, PR protein, SOD, isoflavone reductase protein, general regulatory factor 2, proteasome, and GS) were chosen for mRNA expression analysis by RT-PCR. Transcripts of alcohol dehydrogenase, isoflavone reductase protein, general regulatory factor 2 and glutamine synthetase were increased in response to Al stress treatment and showed similar expression pattern with protein expression (Fig. 4). On the other hand, the expression of proteasome, PR protein and SOD genes showed a different pattern from protein expression. These results suggest that the expression of each gene is differently regulated, such as transcriptional regulation or translational regulation, by Al stress treatment.

    Ⅳ. CONCLUSION

    In this research, data provided an understanding of proteomic responses of alfalfa root under Al stress conditions. Proteomic analysis of Al stress-responsive protein revealed that total 45 proteins were differentially expressed. Proteins were classified as disease/defense, energy, protein destination, metabolism, transcription, secondary metabolism, and unclear classification. These data indicate that most of altered proteins in the Al-stressed alfalfa root involved in different cellular functions under Al stress. Results provide better insights on adaptive mechanisms of alfalfa to Al stress under acidic soil. Further research is needed to define molecular function of each gene under Al stress.

    Ⅴ. ACKNOWLEDGEMENT

    This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D 1A1A01060495).

    Figure

    KGFS-42-3-137_F1.gif

    Two-DE gel profiles of differentially expressed proteins in alfalfa roots after Al stress treatment. A, no Al with pH4.0 ; B, 200 uM Al with pH4.0; C, 800 mM Al with pH4.0; D, 2,000 mM Al with pH4.0.

    KGFS-42-3-137_F2.gif

    Expression levels of the Al stress-induced identified proteins in alfalfa roots under Al stress. Spot intensities were measured densitometer (PDQuest, Bio-Rad) and compared.

    KGFS-42-3-137_F3.gif

    Functional catalogue of Al stress-induced differentially expressed proteins in alfalfa root.

    KGFS-42-3-137_F4.gif

    Expression of Al-responsive genes by aluminum stress treatment at pH 4 with 200 μM Al.

    Table

    Aluminum stress-responsive proteins in alfalfa roots identified by MALDI-TOF/TOF MS analysis

    Gene specific primers were used for RT-PCR amplification

    Reference

    1. Alscher, R.G. , Erturk, N. and Heath, L.S. 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. Journal of Experimental Botany. 372(53):1331-1341.
    2. Andersson, I. , Knight, S. , Schneider, G. , Lindqvist, T. , Lundqvist, Y. , Branden, C.I. and Lorimer, G.H. 1989. Crystal structure of the active site of ribulose-bisphosphate carboxylase. Nature. 337:229-234.
    3. Barone, P. , Rosellini, D. , LaFayette, P. , Bouton, J. , Veronesi, F. and Parrott, W. 2008. Bacterial citrate synthase expression and soil aluminum tolerance in transgenic alfalfa. The Plant Cell. 27:893-901.
    4. Bashandy, T. , Guilleminot, J. , Vernoux, T. , Caparros-Ruiz, D. , Ljung, K. , Meyer, Y. and Reichhelda, J.P. 2010. Interplay between the NADP-linked thioredoxin and glutathione systems in Arabidopsis auxin signaling. The Plant Cell. 22:376-391.
    5. Carlberg, I. and Mannervik, B. 1985. Glutathione reductase. Methods in Enzymology. 113:484-490.
    6. Clarkson, D.T. 1965. The effect of aluminium and some other trivalent metal cations on cell division in the root apices of Allium cepa. Annals of Botany. 29:309-315.
    7. Dolferus, R. , Marbaix, G. and Jaeobs, M. 1985. Alcohol dehydrogenase in Arabidopsis: Analysis of the induction phenomenon in plantlets and tissue cultures. Molecular General Genetics. 199:256-264.
    8. Eujayl, I. , Sledge, M.K. , Wang, L. , May, G.D. , Chekhovskiy, K. , Zwonitzer, J.C. and Mian, M.A.R. 2004. Medicago truncatula EST-SSRs reveal cross-species genetic markers for Medicago spp. Theoretical Applied Genetics. 108:414-422.
    9. Fu, H. , Doelling, J.H. , Arendt, C.S. , Hochstrasser, M. and Vierstra, R.D. 1998. Molecular organization of the 20S proteasome gene family from Arabidopsis thaliana. Genetics. 149:677-692.
    10. Ghanati, F. , Morita, A. and Yokota, H. 2005. Effects of aluminum on the growth of tea plant and activation of antioxidant system. Plant and Soil. 276:133-141.
    11. Gruber, B.D. , Ryan, P.R. , Richardson, A.E. , Tyerman, S.D. , Ramesh, S. , Hebb, D.M. , Howitt, S.M. and Delhaize, E. 2010. HvALMT1 from barley is involved in the transport of organic anions. Journal of Experimental Botany. 61:1455-1467.
    12. He, H. , Li, Y. and He, L.F. 2019. Aluminum toxicity and tolerance in Solanaceae plants. South African Journal of Botany. 123:23-29.
    13. Hossain, Z. and Komatsu, S. 2013. Contribution of proteomic studies towards understanding plant heavy metal stress response. Frontiers in Plant Science. 3:1-12.
    14. Hradilova, J. , Ehulka P.R. , Ehulkova, H.R. , Vrbova, M. , Griga, M. and Brzobohaty, B. 2010. Comparative analysis of proteomic changes in contrasting flax cultivars upon cadmium exposure. Electrophoresis. 31:421-431.
    15. Kochian, L.V. , Hoekenga, O.A. and Pineros, M.A. 2004. How do crop plants tolerate acid soils? Mechanisms of aluminum tolerance and phosphorus efficiency. Annual Review of Plant Biology. 55:459-493.
    16. Kochian, L.V. , Piñeros, M.A. , Liu, J. and Magalhaes, J.V. 2015. Plant adaptation to acid soils: The molecular basis for crop aluminum resistance. Annual Review of Plant Biology. 66:571-598.
    17. Lee, D.K. , Ahsan, N. , Lee, S.H. , Kang, K.Y. , Bahk, J.D. , Lee, I.J. and Lee, B.H. 2007. A proteomic approach in analyzing heat-responsive proteins in rice leaves. Proteomics. 7:3369-3383.
    18. Leterrier, M. , Barroso, J.B. , Palma J.M. and Corpas, F.J. 2012. Cytosolic NADP-isocitrate dehydrogenase in Arabidopsis leaves and roots. Biologia Plantarum. 56(4):705-710.
    19. Li, J.Y. , Liu, J. , Dong, D. , Jia, X. , McCouch, S.R. and Kochian, L.V. 2014. Natural variation underlies alterations in Nramp aluminum transporter (NRAT1) expression and function that play a key role in rice aluminum tolerance. Proceedings of National Academic Sciences. 111:6503-6508.
    20. Ligaba, A. , Maron, L. , Shaff, J. , Kochian, L. and Piñeros, M. 2012. Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux. Plant Cell Environment. 35:1185-1200.
    21. Magalhaes, J.V. , Liu, J. , Guimaraes, C.T. , Lana, U.G.P. , Alves, V.M.C. , Wang, Y.H. , Schaffert, R.E. , Hoekenga, O.A. , Pineros, M.A. , Shaff, J.E. , Klein, P.E. , Carneiro, N.P. , Coelho, C.M. , Trick, H.N. and Kochian, L.V. 2007. A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum. Nature. 39(9):1156-1161.
    22. Maron, L.G. , Piñeros, M.A. , Guimarães, C.T. , Magalhaes, J.V. , Pleiman, J.K. , Mao, C. , Shaff, J. , Belicuas, S.N.J. and Kochian, L.V. 2010. Two functionally distinct members of the MATE (multi-drug and toxic compound extrusion) family of transporters potentially underlie two major aluminum tolerance QTLs in maize. Plant Journal. 61:728-740.
    23. Matsui, H. , Nakamura, G. , Ishiga, Y. , Toshima, H. , Inagaki, Y. , Toyoda, K. , Shiraishi, T. and Ichinose, Y. 2004. Structure and expression of 12-oxophytodienoate reductase (subgroup I) genes in pea, and characterization of the oxidoreductase activities of their recombinant products. Molecular Genetics and Genomics. 271:1-10.
    24. Moeder, W. , Pozo, O.D. , Navarre, D.A. , Martin, G.B. and Klessig, D.F. 2007. Aconitase plays a role in regulating resistance to oxidative stress and cell death in Arabidopsis and Nicotiana benthamiana. Plant Molecular Biology. 63:273-287.
    25. Mun, H.T. , Park, B.K. and Kim, J.H. 1997. Response of plants and changes of soil properties to added acid-soil ameliorants. The Korean Journal of Ecology. 20(1):43-49.
    26. Nylander, M. , Svensson, J. , Palva, E.T. and Welin, B.V. 2001. Stress-induced accumulation and tissue-specific localization of dehydrinsin Arabidopsis thaliana. Plant Molecular Biology. 45:263-279.
    27. Panda, S.K. and Patra, H.K. 2000. Does chromium (III) produce oxidative damage in excised wheat leaves? Journal of Plant Biology. 27:105-110.
    28. Rahman, M.A. , Kim, Y.G. and Lee, B.H. 2014. Proteomic response of alfalfa subjected to aluminum (Al) stress at low pH soil. 2014. Journal of the Korean Society of Grassland and Forage Science. 34:262-268.
    29. Riaz, M. , Yan, L. , Wu, X. , Hussain, S. , Aziz, O. and Jiang, C. 2018. Mechanisms of organic acids and boron induced tolerance of aluminum toxicity: A review. Ecotoxicology Environmental Safety. 165:25-35.
    30. Rodriguez, M.M.A. , Maurer, A. , Huete, A.R. and Gustafson, J.P. 2003. Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways. The Plant Journal. 36:602-615.
    31. Tesfaye, M. , Temple, S.J. , Allan, D.L. , Vance, C.P. and Samac, D.A. 2001. Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum. Plant Physiology. 127:1836-1844.
    32. Uexkull. and Mutert.1995. Global extent, development and economic impact of acid soils. Plant and Soil. 171:1-15.
    33. Verbruggen, N. , Hermans, C. and Schat, H. 2009. Mechanisms to cope with arsenic or cadmium excess in plants. Plant Biology. 12:364-372.
    34. Wang, X. , He, X. , Lin, J. , Shao, H. , Chang, Z. and Dixon, R.A. 2006. Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.). Journal of Molecular Biology. 358:1341-1352.
    35. Wei, Y. , Han, R. , Xie, Y. , Jiang, C. and Yu, Y. 2021. Recent advances in understanding mechanisms of plant tolerance and response to aluminum toxicity. Sustainability. 13:1782.
    36. Yang, Q.S. , Wang, Y.Q. , Zhang, J.J. , Shi, W.P. et al.2007. Identification of aluminum-responsive proteins in rice roots by a proteomic approach: cysteine synthase as a key player in Al response. Proteomics. 7:737-749.
    37. Zhen, Y. , Qi, J. L. , Wang, S. S. , Su, J. et al.2007. Comparative proteome analysis of differentially expressed proteins induced by Al toxicity in soybean. Physiolologia Plantarum. 131:542-554.
    38. Zheng, L. , Lan, P. , Shen, R.F. and Li, W.F. 2014. Proteomics of aluminum tolerance in plants. Proteomics. 14:566-578.
    39. Zhou, S. , Sauve, R. and Thannhauser, T.W. 2009. Proteome changes induced by aluminium stress in tomato roots. Journal Experimental Botany. 60:1849-1857.
    40. Zhou, Y. , Wang, Z. , Gong, L. , Chen, A. , Liu, N. , Li, S. , Sun, H. , Yang, Z. and You, J. 2019. Functional characterization of three MATE genes in relation to aluminum-induced citrate efflux from soybean root. Plant and Soil. 443:121-138.
    Copyright © The Korean Society of Grassland and Forage Science. All rights reserved.