Light Control of Salt-Induced Proline Accumulation Is Mediated by ELONGATED HYPOCOTYL 5 in Arabidopsis

Plants have to adapt their metabolism to constantly changing environmental conditions, among which the availability of light and water is crucial in determining growth and development. Proline accumulation is one of the sensitive metabolic responses to extreme conditions; it is triggered by salinity or drought and is regulated by light. Here we show that red and blue but not far-red light is essential for salt-induced proline accumulation, upregulation of Δ1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1) and downregulation of PROLINE DEHYDROGENASE 1 (PDH1) genes, which control proline biosynthetic and catabolic pathways, respectively. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the transcription factor ELONGATED HYPOCOTYL 5 (HY5) binds to G-box and C-box elements of P5CS1 and a C-box motif of PDH1. Salt-induced proline accumulation and P5CS1 expression were reduced in the hy5hyh double mutant, suggesting that HY5 promotes proline biosynthesis through connecting light and stress signals. Our results improve our understanding on interactions between stress and light signals, confirming HY5 as a key regulator in proline metabolism.

A) Experimental design. Fourteen-days-old wild type Arabidopsis plants were transferred to dark and after 5 days conditioning they were transferred to media containing 0.5 % sucrose (standard growth medium), 3 % of sucrose, 3 % glucose or 3 % mannitol, and treated with 150 mM NaCl under white light or kept in darkness. Concentrations are in W/V %. B) Proline accumulation in plants treated for three days. Note, that salt treatment enhanced proline levels in illuminated plants, but not or only minimally enhanced it in dark-treated plants. None of the sugars (sucrose, glucose) or sugar alcohol (mannitol) could promote proline accumulation in darkness. Error bars indicate standard deviation. Significant differences between means are shown by different letters (p<0.05, Two-way ANOVA, Duncan's test, N=5, fixed parameters were stress and sugar treatments ).
Effect of externally added sucrose, glucose and mannitol on proline accumulation in dark-adapted plants.  Figure S3.
A) Experimental design. Dark-adapted Arabidopsis plants were transferred to white (W), red (R), blue (Bl), far red (Fr) lights or kept in dark (D) and treated with (NaCl) or without (Ctr) 150 mM NaCl. Free proline contents were determined in plants three days later. B) Proline levels in Col-0 and WS ecotypes. Note, that change in proline levels is similar in both Col-0 and WS ecotypes. Significant differences between Col-0 and WS are shown: * p<0.05, (Two-way ANOVA, Tukey test, N=5).

Proline accumulation
Effect of different light regimes on proline accumulation in Arabidopsis Columbia (Col-0) and Wassilewskija (WS) ecotypes.  Figure S4.
Results of a representative experiment. A) Experimental design. Dark-adapted Arabidopsis plants were transferred to white (W), red (R), blue (B) and far red (Fr) lights or kept in dark (D) and treated with or without 150 mM NaCl (NaCl or Ctr, respectively). RNA samples and cDNA templates were prepared in three repetitions. Gene expression of P5CS1 and PDH1 were determined in whole plants after 6 hours using quantitative RT-PCR (reference: Actin2  Reads from the nonimmunoprecipitated mock control (S13) and from three independent biological samples (S1, S2 and S3) were mapped against the Arabidopsis genome (TAIR10). Blue arrow indicates the 5.2 kb transcribed region of P5CS1. Vertical pink lines indicate the approximate borders of read enhancements. Note that peak of the reads is close to the transcription initiation site of P5CS1. More experimental details and the complete set of raw data can be found at the NCBI GEO repository under the accession number GSE117797 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE117797). Figure S7. ChIP-seq result of HY5 binding around the PDH1 locus.

PDH1
Data were compiled from ChIP-seq experiment published in Hajdu et al., (2018) Plant J 96: 1242-1254. Reads from the nonimmunoprecipitated mock control (S13) and from three independent biological samples (S1, S2 and S3) were mapped against the Arabidopsis genome (TAIR10). Blue arrow indicates the 2.9 kb transcribed region of PDH1. Vertical red lines indicate the approximate borders of read enhancements. Note that peak of the reads is approximately in 0.5 kb distance from the transcription initiation site of PDH1. More experimental details and the complete set of raw data can be found at the NCBI GEO repository under the accession number GSE117797 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE117797).

Proline accumulation
Proline accumulation in wild type Arabidopsis (WS) and in the hy5hyh mutant. Results of a representative experiment A) Experimental design. Dark-adapted Arabidopsis plants were transferred to white (W), red (R), blue (B) lights or kept in dark (D) and treated with (NaCl) or without (Ctr) 150 mM NaCl. B,C) Expression of P5CS1 ( B ) and PDH1 ( C ) were determined in whole plants after 6 hours using quantitative RT-PCR (reference: Actin2). Relative transcript levels in WS ecotype and in hy5hyh mutant are shown as 2 -ΔΔCt , where transcript levels in dark samples corresponds to 1. Significant differences between WS and hy5hyh are indicated as: * p<0.05, (Two-way ANOVA, Tukey test, fixed parameters were genotypes and treatments, N=3).