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Allantoin accumulation mediated by allantoinase downregulation and transport by Ureide Permease 5 confers salt stress tolerance to Arabidopsis plants

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Abstract

Allantoin, a metabolite generated in the purine degradation pathway, was primarily considered an intermediate for recycling of the abundant nitrogen assimilated in plant purines. More specifically, tropical legumes utilize allantoin and allantoic acid as major nodule-to-shoot nitrogen transport compounds. In other species, an increase in allantoin content was observed under different stress conditions, but the underlying molecular mechanisms remain poorly understood. In this work, Arabidopsis thaliana was used as a model system to investigate the effects of salt stress on allantoin metabolism and to know whether its accumulation results in plant protection. Plant seedlings treated with NaCl at different concentrations showed higher allantoin and lower allantoic acid contents. Treatments with NaCl favored the expression of genes involved in allantoin synthesis, but strongly repressed the unique gene encoding allantoinase (AtALN). Due to the potential regulatory role of this gene for allantoin accumulation, AtALN promoter activity was studied using a reporter system. GUS mediated coloration was found in specific plant tissues and was diminished with increasing salt concentrations. Phenotypic analysis of knockout, knockdown and stress-inducible mutants for AtALN revealed that allantoin accumulation is essential for salt stress tolerance. In addition, the possible role of allantoin transport was investigated. The Ureide Permease 5 (UPS5) is expressed in the cortex and endodermis of roots and its transcription is enhanced by salt treatment. Ups5 knockout plants under salt stress presented a susceptible phenotype and altered allantoin root-to-shoot content ratios. Possible roles of allantoin as a protectant compound in oxidative events or signaling are discussed.

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Acknowledgments

This work was supported by Grants (PICT-2009-0114) of the National Fund of Science and Technology (FONCyT, Argentina) and of the Secretary of Science and Technology of the National University of Córdoba (SECyT-UNC, Argentina). C.I.L. is grateful for a scholarship at the Multidisciplinary Institute of Plant Biology (IMBIV-CONICET). We thank Dr. Alejandra Trenchi for microscopy assistance.

Author contributions

C.I.L. did the cloning work, generation of transgenic plants and expression studies. Plant phenotyping was carried out by C.I.L., C.M. and C.A.G. The writing of the manuscript was performed by C.I.L., C.A.G. and M.D.

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Authors and Affiliations

  1. Multidisciplinary Institute of Plant Biology, National University of Córdoba, CONICET, FCEFyN, Vélez Sarsfield Av. 299, 5000, Córdoba, Argentina

    Carlos Ignacio Lescano, Carolina Martini, Claudio Alejandro González & Marcelo Desimone

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  1. Carlos Ignacio Lescano
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  2. Carolina Martini
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  3. Claudio Alejandro González
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  4. Marcelo Desimone
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Correspondence to Marcelo Desimone.

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Fig. S1

Characterization of two aln T-DNA insertion lines and AtALN stress-inducible lines used in this study. (a) Two independent T-DNA insertion lines in the AtALN gene, Salk_146783 (designated aln-1) and Salk_142607 (designated aln-2) were analyzed. The insertion in aln-1 (aln-2) was localized in the twelfth (ninth) exon (intron). (b) AtALN T-DNA lines verification by PCR. Primers were designed using SALK’s T-DNA Primer tool. LP = Left genomic Primer, RP = Right genomic primer, LB = Left border primer of the T-DNA insertion. (c) Expression of AtALN mRNA in WT and aln T-DNA insertion lines. RNA was extracted from 14 day-old plants. A 641 bp AtACT2 (control) and a 682 bp AtALN fragments of wt and aln T-DNA insertion lines were amplified by PCR (28 and 32 cycles) or adding two extra PCR cycles (30 and 34 cycles). (d) Map of the construct pCRD29A::ALN used for the generation of transgenic plants with a stress-inducible-AtALN. (e) Expression of AtALN mRNA in WT, aln T-DNA insertion lines and RD29A::ALN/aln-2 under salt stress. 14 day-old seedlings were transferred to 0.5× MS vertical plates supplemented with 0 and 150 mM NaCl. Material harvested after 24 h salt treatment was used for RT-PCR. AtALN fragment was amplified by PCR (32 cycles) and AtACT2 fragment was used as control (28 cycles) (TIFF 16821 kb)

Fig. S2

Characterization of lines with T-DNA insertions in the AtUPS5 gene used in this study. (a) Two AtUPS5 T-DNA insertion lines Salk_044810 (designated ups5-1) and Salk_123120 (designated ups5-2) were analyzed. The insertion in ups5-1 and ups5-2 were localized in the UTR 5′ and in the promoter region of AtUPS5, respectively. (b) AtUPS5 T-DNA lines verification by PCR. Primers were designed using SALK’s T-DNA Primer tool. LP = Left genomic Primer, RP = Right genomic primer, LB = Left border primer of the T-DNA insertion. (c) Expression of AtUPS5 mRNA in WT and ups5 mutants. RNA was extracted from 14 day-old plants. A 641 bp AtACT2 fragment (control) and a 510 bp AtUPS5 fragment were amplified by RT-PCR (28 and 30 cycles), or adding two extra PCR cycles (30 and 32 cycles) (TIFF 14852 kb)

Fig. S3

Phenotype of ups5 and aln mutants grown with allantoin as a sole nitrogen source. WT, the two ups5 and the two aln T-DNA insertion lines previously described were grown in either solid 0.5× MS standard medium containing 30 mM total inorganic nitrogen (control) or 0.5× MS medium without nitrogen supplemented with 7.5 mM allantoin as sole nitrogen source for 7 days. (a) Representative seedlings grown on vertical plates (bar = 1 cm), (b) Root length and (c) Fresh weight per seedling are shown. Values of root length and fresh weight are relative to control plants. Bars represent the means and standard errors of at least 15 (b) or 3 (c) independent measurements. Asterisks indicates significant differences between genotypes and treatments (N = 221, *P < 0.05, Kruskal–Wallis for b; N = 31, *P < 0.05, DGC Test for c) (TIFF 88510 kb)

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Lescano, C.I., Martini, C., González, C.A. et al. Allantoin accumulation mediated by allantoinase downregulation and transport by Ureide Permease 5 confers salt stress tolerance to Arabidopsis plants. Plant Mol Biol 91, 581–595 (2016). https://doi.org/10.1007/s11103-016-0490-7

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