The Arabidopsis splicing regulator SR45 confers salt tolerance in a splice isoform-dependent manner

  • Mohammed Albaqami
  • K. Laluk
  • Anireddy S. N. ReddyEmail author


Key message

Functions of most splice isoforms that are generated by alternative splicing are unknown. We show that two splice variants that encode proteins differing in only eight amino acids have distinct functions in a stress response.


Serine/arginine-rich (SR) and SR-like proteins, a conserved family of RNA binding proteins across eukaryotes, play important roles in pre-mRNA splicing and other post-transcriptional processes. Pre-mRNAs of SR and SR-like proteins undergo extensive alternative splicing in response to diverse stresses and produce multiple splice isoforms. However, the functions of most splice isoforms remain elusive. Alternative splicing of pre-mRNA of Arabidopsis SR45, which encodes an SR-like splicing regulator, generates two isoforms (long—SR45.1 and short—SR45.2). The proteins encoded by these two isoforms differ in eight amino acids. Here, we investigated the role of SR45 and its splice variants in salt stress tolerance. The loss of SR45 resulted in enhanced sensitivity to salt stress and changes in expression and splicing of genes involved in regulating salt stress response. Interestingly, only the long isoform (SR45.1) rescued the salt-sensitive phenotype as well as the altered gene expression and splicing patterns in the mutant. These results suggest that SR45 positively regulates salt tolerance. Furthermore, only the long isoform is required for SR45-mediated salt tolerance.


Pre-mRNA splicing SR45 Salt stress Abiotic stress Stress-responsive genes Arabidopsis Splice isoform 



This research was supported by a Grant from the National Science Foundation (ABI 0743097). We thank Dr. Xiao-Ning Zhang, St. Bonaventure University, St. Bonaventure, NY for providing the seeds of complemented lines; Dr. Salah and Dr. Palusa for their help on this Project; Dr. Prasad for his comments on the manuscript. MA was supported by a Ph.D. fellowship from the government of the Kingdom of Saudi Arabia.

Author contributions

ASNR conceived and supervised the study. MA, KL, and ASNR designed experiments and analyzed data. MA and KL performed experiments. MA and ASNR wrote the manuscript.

Supplementary material

11103_2019_864_MOESM1_ESM.jpg (44 kb)
Supplementary material 1 (JPEG 44 kb) Figure S1: Ion content (mg/g dry weight) of WT,sr45and the complemented lines in plants grown in the presence and absence of NaCl. Three-day-old MS grown seedlings were transferred to MS or MS supplemented with 150 mM NaCl and allowed to grow for two weeks before harvesting. Harvested seedlings were rinsed once with an excess volume of 50 mM EDTA followed by 2 rinses with bi-distilled water. The oven-dried tissues were digested and used for ICP analysis. Data represent the mean ± SE from the average of three independent experiments. Significance was calculated using analysis of variance and Tukey’s test, the significance is indicated by letters associated with bars
11103_2019_864_MOESM2_ESM.pdf (84 kb)
Supplementary material 2 (PDF 83 kb) Figure S2: Sequence alignment ofSOS4isoforms. The amplified products were resolved in a gel and different bands were purified and sequenced. Sequences of cDNAs (A) and predicted proteins (B) were aligned. (C) A schematic diagram showing alternatively spliced transcripts of SOS4. Alternative splicing of the first intron generated three splice isoforms. Primers used in PCR are presented in Suppl. Table 1
11103_2019_864_MOESM3_ESM.pdf (57 kb)
Supplementary material 3 (PDF 56 kb)


  1. Abdel-Ghany SE (2009) Contribution of plastocyanin isoforms to photosynthesis and copper homeostasis in Arabidopsis thaliana grown at different copper regimes. Planta 229:767–779CrossRefGoogle Scholar
  2. Abdel-Ghany SE, Pilon M (2008) MicroRNA-mediated systemic down-regulation of copper protein expression in response to low copper availability in Arabidopsis. J Biol Chem 283:15932–15945CrossRefGoogle Scholar
  3. Ali GS, Reddy AS (2006) ATP, phosphorylation and transcription regulate the mobility of plant splicing factors. J Cell Sci 119:3527–3538CrossRefGoogle Scholar
  4. Ali GS, Reddy AS (2008) Regulation of alternative splicing of pre-mRNAs by stresses. Curr Top Microbiol Immunol 326:257–275Google Scholar
  5. Ali GS, Golovkin M, Reddy AS (2003) Nuclear localization and in vivo dynamics of a plant-specific serine/arginine-rich protein. Plant J 36:883–893CrossRefGoogle Scholar
  6. Ali GS, Palusa SG, Golovkin M, Prasad J, Manley JL, Reddy AS (2007) Regulation of plant developmental processes by a novel splicing factor. PLoS ONE 2:e471CrossRefGoogle Scholar
  7. Ali GS, Prasad KV, Hanumappa M, Reddy AS (2008) Analyses of in vivo interaction and mobility of two spliceosomal proteins using FRAP and BiFC. PLoS ONE 3:e1953CrossRefGoogle Scholar
  8. Alvarez-Aragon R, Haro R, Benito B, Rodriguez-Navarro A (2016) Salt intolerance in Arabidopsis: shoot and root sodium toxicity, and inhibition by sodium-plus-potassium overaccumulation. Planta 243:97–114CrossRefGoogle Scholar
  9. Anschutz U, Becker D, Shabala S (2014) Going beyond nutrition: regulation of potassium homoeostasis as a common denominator of plant adaptive responses to environment. J Plant Physiol 171:670–687CrossRefGoogle Scholar
  10. Assaha DVM, Ueda A, Saneoka H, Al-Yahyai R, Yaish MW (2017) The role of Na+ and K+ transporters in salt stress adaptation in glycophytes. Front Physiol 8:509CrossRefGoogle Scholar
  11. Ausin I, Greenberg MV, Li CF, Jacobsen SE (2012) The splicing factor SR45 affects the RNA-directed DNA methylation pathway in Arabidopsis. Epigenetics 7:29–33CrossRefGoogle Scholar
  12. Barta A, Kalyna M, Reddy AS (2010) Implementing a rational and consistent nomenclature for serine/arginine-rich protein splicing factors (SR proteins) in plants. Plant Cell 22:2926–2929CrossRefGoogle Scholar
  13. Boyer JS (1982) Plant productivity and environment. Science 218:443–448CrossRefGoogle Scholar
  14. Bradley T, Cook ME, Blanchette M (2015) SR proteins control a complex network of RNA-processing events. RNA 21:75–92CrossRefGoogle Scholar
  15. Carvalho RF, Carvalho SD, Duque P (2010) The plant-specific SR45 protein negatively regulates glucose and ABA signaling during early seedling development in Arabidopsis. Plant Physiol 154:772–783CrossRefGoogle Scholar
  16. Carvalho RF, Szakonyi D, Simpson CG, Barbosa IC, Brown JW, Baena-Gonzalez E, Duque P (2016) The Arabidopsis SR45 splicing factor, a negative regulator of sugar signaling, modulates SNF1-related protein kinase 1 stability. Plant Cell 28:1910–1925CrossRefGoogle Scholar
  17. Cheeseman JM (2015) The evolution of halophytes, glycophytes and crops, and its implications for food security under saline conditions. New Phytol 206:557–570CrossRefGoogle Scholar
  18. Chen LH, Zhang B, Xu ZQ (2008) Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na(+)/H (+) antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum). Transgenic Res 17:121–132CrossRefGoogle Scholar
  19. Cramer GR, Urano K, Delrot S, Pezzotti M, Shinozaki K (2011) Effects of abiotic stress on plants: a systems biology perspective. BMC Plant Biol 11:163CrossRefGoogle Scholar
  20. Cui P, Zhang S, Ding F, Ali S, Xiong L (2014) Dynamic regulation of genome-wide pre-mRNA splicing and stress tolerance by the Sm-like protein LSm5 in Arabidopsis. Genome Biol 15:R1CrossRefGoogle Scholar
  21. Day IS, Golovkin M, Palusa SG, Link A, Ali GS, Thomas J, Richardson DN, Reddy AS (2012) Interactions of SR45, an SR-like protein, with spliceosomal proteins and an intronic sequence: insights into regulated splicing. Plant J 71:936–947CrossRefGoogle Scholar
  22. Ding F, Cui P, Wang Z, Zhang S, Ali S, Xiong L (2014) Genome-wide analysis of alternative splicing of pre-mRNA under salt stress in Arabidopsis. BMC Genomics 15:431CrossRefGoogle Scholar
  23. Feng J, Li J, Gao Z, Lu Y, Yu J, Zheng Q, Yan S, Zhang W, He H, Ma L, Zhu Z (2015) SKIP confers osmotic tolerance during salt stress by controlling alternative gene splicing in Arabidopsis. Mol Plant 8:1038–1052CrossRefGoogle Scholar
  24. Filichkin SA, Priest HD, Givan SA, Shen R, Bryant DW, Fox SE, Wong WK, Mockler TC (2010) Genome-wide mapping of alternative splicing in Arabidopsis thaliana. Genome Res 20:45–58CrossRefGoogle Scholar
  25. Floris M, Mahgoub H, Lanet E, Robaglia C, Menand B (2009) Post-transcriptional regulation of gene expression in plants during abiotic stress. Int J Mol Sci 10:3168–3185CrossRefGoogle Scholar
  26. Gassmann W (2008) Alternative splicing in plant defense. In: Golovkin ASNRaM (ed) Nuclear pre-mRNA processing in plants. Springer, Heidelberg, pp 219–233CrossRefGoogle Scholar
  27. Godoy Herz MA, Kubaczka MG, Brzyzek G, Servi L, Krzyszton M, Simpson C, Brown J, Swiezewski S, Petrillo E, Kornblihtt AR (2019) Light regulates plant alternative splicing through the control of transcriptional elongation. Mol Cell 73:1066CrossRefGoogle Scholar
  28. Golovkin M, Reddy ASN (1999) An SC35-like protein and a novel serine/arginine-rich protein interact with Arabidopsis U1-70K protein. J Biol Chem 274:36428–36438CrossRefGoogle Scholar
  29. Gonzalez E, Danehower D, Daub ME (2007) Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5′-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway. Plant Physiol 145:985–996CrossRefGoogle Scholar
  30. Gu J, Xia Z, Luo Y, Jiang X, Qian B, Xie H, Zhu JK, Xiong L, Zhu J, Wang ZY (2018) Spliceosomal protein U1A is involved in alternative splicing and salt stress tolerance in Arabidopsis thaliana. Nucleic Acids Res 46:1777–1792CrossRefGoogle Scholar
  31. Jeong S (2017) SR proteins: binders, regulators, and connectors of RNA. Mol Cell 40:1–9CrossRefGoogle Scholar
  32. Ji H, Pardo JM, Batelli G, Van Oosten MJ, Bressan RA, Li X (2013) The salt overly sensitive (SOS) pathway: established and emerging roles. Mol Plant 6:275–286CrossRefGoogle Scholar
  33. Kawa D, Testerink C (2017) Regulation of mRNA decay in plant responses to salt and osmotic stress. Cell Mol Life Sci 74:1165–1176CrossRefGoogle Scholar
  34. Kosova K, Prail IT, Vitamvas P (2013) Protein contribution to plant salinity response and tolerance acquisition. Int J Mol Sci 14:6757–6789CrossRefGoogle Scholar
  35. Laloum T, Martin G, Duque P (2018) Alternative splicing control of abiotic stress responses. Trends Plant Sci 23:140–150CrossRefGoogle Scholar
  36. Lee SY, Boon NJ, Webb AA, Tanaka RJ (2016) Synergistic activation of RD29A via integration of salinity stress and abscisic acid in Arabidopsis thaliana. Plant Cell Physiol 57:2147–2160CrossRefGoogle Scholar
  37. Liang W, Ma X, Wan P, Liu L (2018) Plant salt-tolerance mechanism: a review. Biochem Biophys Res Commun 495:286–291CrossRefGoogle Scholar
  38. Mandadi KK, Scholthof KB (2015) Genome-wide analysis of alternative splicing landscapes modulated during plant–virus interactions in Brachypodium distachyon. Plant Cell 27:71–85CrossRefGoogle Scholar
  39. Manley JL, Tacke R (1996) SR proteins and splicing control. Genes Dev 10:1569–1579CrossRefGoogle Scholar
  40. Marquez Y, Brown JW, Simpson C, Barta A, Kalyna M (2012) Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis. Genome Res 22:1184–1195CrossRefGoogle Scholar
  41. Mastrangelo AM, Marone D, Laido G, De Leonardis AM, De Vita P (2012) Alternative splicing: enhancing ability to cope with stress via transcriptome plasticity. Plant Sci 185–186:40–49CrossRefGoogle Scholar
  42. Palusa SG, Ali GS, Reddy AS (2007) Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins: regulation by hormones and stresses. Plant J 49:1091–1107CrossRefGoogle Scholar
  43. Petrillo E, Godoy Herz MA, Fuchs A, Reifer D, Fuller J, Yanovsky MJ, Simpson C, Brown JW, Barta A, Kalyna M, Kornblihtt AR (2014) A chloroplast retrograde signal regulates nuclear alternative splicing. Science 344:427–430CrossRefGoogle Scholar
  44. Reddy AS, Shad Ali G (2011) Plant serine/arginine-rich proteins: roles in precursor messenger RNA splicing, plant development, and stress responses. Wiley Interdiscip Rev RNA 2:875–889CrossRefGoogle Scholar
  45. Reddy ASN, Ali GS, Celesnik H, Day IS (2011) Coping with stresses: roles of calcium- and calcium/calmodulin-regulated gene expression. Plant Cell 23:2010–2032CrossRefGoogle Scholar
  46. Reddy AS, Marquez Y, Kalyna M, Barta A (2013) Complexity of the alternative splicing landscape in plants. Plant Cell 25:3657–3683CrossRefGoogle Scholar
  47. Reiland S, Messerli G, Baerenfaller K, Gerrits B, Endler A, Grossmann J, Gruissem W, Baginsky S (2009) Large-scale Arabidopsis phosphoproteome profiling reveals novel chloroplast kinase substrates and phosphorylation networks. Plant Physiol 150:889–903CrossRefGoogle Scholar
  48. Rueschhoff EE, Gillikin JW, Sederoff HW, Daub ME (2013) The SOS4 pyridoxal kinase is required for maintenance of vitamin B6-mediated processes in chloroplasts. Plant Physiol Biochem 63:281–291CrossRefGoogle Scholar
  49. Shi H, Xiong L, Stevenson B, Lu T, Zhu JK (2002) The Arabidopsis salt overly sensitive 4 mutants uncover a critical role for vitamin B6 in plant salt tolerance. Plant Cell 14:575–588CrossRefGoogle Scholar
  50. Tanabe N, Yoshimura K, Kimura A, Yabuta Y, Shigeoka S (2007) Differential expression of alternatively spliced mRNAs of Arabidopsis SR protein homologs, atSR30 and atSR45a, in response to environmental stress. Plant Cell Physiol 48:1036–1049CrossRefGoogle Scholar
  51. Wang X, Wu F, Xie Q, Wang H, Wang Y, Yue Y, Gahura O, Ma S, Liu L, Cao Y, Jiao Y, Puta F, McClung CR, Xu X, Ma L (2012) SKIP is a component of the spliceosome linking alternative splicing and the circadian clock in Arabidopsis. Plant Cell 24:3278–3295CrossRefGoogle Scholar
  52. Wu SJ, Ding L, Zhu JK (1996) SOS1, a genetic locus essential for salt tolerance and potassium acquisition. Plant Cell 8:617–627CrossRefGoogle Scholar
  53. Xing D, Wang Y, Hamilton M, Ben-Hur A, Reddy AS (2015) Transcriptome-wide identification of RNA targets of Arabidopsis SERINE/ARGININE-RICH45 uncovers the unexpected roles of this RNA binding protein in RNA processing. Plant Cell 27:3294–3308CrossRefGoogle Scholar
  54. Yan K, Liu P, Wu CA, Yang GD, Xu R, Guo QH, Huang JG, Zheng CC (2012) Stress-induced alternative splicing provides a mechanism for the regulation of microRNA processing in Arabidopsis thaliana. Mol Cell 48:521–531CrossRefGoogle Scholar
  55. Zhang XN, Mount SM (2009) Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development. Plant Physiol 150:1450–1458CrossRefGoogle Scholar
  56. Zhang XN, Mo C, Garrett WM, Cooper B (2014) Phosphothreonine 218 is required for the function of SR45.1 in regulating flower petal development in Arabidopsis. Plant Signal Behav 9:134Google Scholar
  57. Zhang XN, Shi Y, Powers JJ, Gowda NB, Zhang C, Ibrahim HMM, Ball HB, Chen SL, Lu H, Mount SM (2017) Transcriptome analyses reveal SR45 to be a neutral splicing regulator and a suppressor of innate immunity in Arabidopsis thaliana. BMC Genomics 18:772CrossRefGoogle Scholar
  58. Zhou Z, Fu XD (2013) Regulation of splicing by SR proteins and SR protein-specific kinases. Chromosoma 122:191–207CrossRefGoogle Scholar
  59. Zhu JK (2000) Genetic analysis of plant salt tolerance using Arabidopsis. Plant Physiol 124:941–948CrossRefGoogle Scholar
  60. Zhu JK (2016) Abiotic stress signaling and responses in plants. Cell 167:313–324CrossRefGoogle Scholar
  61. Zhu JK, Liu J, Xiong L (1998) Genetic analysis of salt tolerance in arabidopsis. Evidence for a critical role of potassium nutrition. Plant Cell 10:1181–1191CrossRefGoogle Scholar
  62. Zhu G, Li W, Zhang F, Guo W (2018) RNA-seq analysis reveals alternative splicing under salt stress in cotton, Gossypium davidsonii. BMC Genomics 19:73CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Mohammed Albaqami
    • 1
    • 2
  • K. Laluk
    • 1
  • Anireddy S. N. Reddy
    • 1
    Email author
  1. 1.Department of Biology and Program in Cell and Molecular BiologyColorado State UniversityFort CollinsUSA
  2. 2.Department of Biology, Faculty of Applied ScienceUmm Al-Qura UniversityMeccaKingdom of Saudi Arabia

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