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DREB2C acts as a transcriptional activator of the thermo tolerance-related phytocystatin 4 (AtCYS4) gene

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Abstract

Phytocystatins are proteinaceous inhibitors of cysteine proteases. They have been implicated in the regulation of plant protein turnover and in defense against pathogens and insects. Here, we have characterized an Arabidopsis phytocystatin family gene, Arabidopsis thaliana phytocystatin 4 (AtCYS4). AtCYS4 was induced by heat stress. The heat shock tolerance of AtCYS4-overexpressing transgenic plants was greater than that of wild-type and cys4 knock-down plants, as measured by fresh weight and root length. Although no heat shock elements were identified in the 5′-flanking region of the AtCYS4 gene, canonical ABA-responsive elements (ABREs) and dehydration-responsive elements (DREs) were found. Transient promoter activity measurements showed that AtCYS4 expression was up-regulated in unstressed protoplasts by co-expression of DRE-binding factor 2s (DREB2s), especially by DREB2C, but not by bZIP transcription factors that bind to ABREs (ABFs, ABI5 and AREBs). DREB2C bound to and activated transcription from the two DREs on the AtCYS4 promoter although some preference was observed for the GCCGAC DRE element over the ACCGAC element. AtCYS4 transcript and protein levels were elevated in transgenic DREB2C overexpression lines with corresponding decline of endogenous cysteine peptidase activity. We propose that AtCYS4 functions in thermotolerance under the control of the DREB2C cascade.

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Acknowledgments

This work was supported by the Next-Generation BioGreen 21 Program (SSAC, grant no. PJ009514032013, Rural Development Administration, Republic of Korea) and Basic Science Research Program (Grant No. 2010-0009175, through the NRF funded by the Ministry of Education, Science and Technology, Republic of Korea). J. Je was supported by BK21 fellowship from the Ministry of Education, Science and Technology, Republic of Korea.

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  1. Division of Applied Life Science, Systems and Synthetic Agrobiotech Center and PMBBRC, Gyeongsang National University, **ju, 660-701, Korea

    Jihyun Je, Chieun Song, Jung Eun Hwang, Woo Sik Chung & Chae Oh Lim

  2. Department of Biochemistry, Gyeongsang National University, **ju, 660-701, Korea

    Chae Oh Lim

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  1. Jihyun Je
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Correspondence to Chae Oh Lim.

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11248_2013_9735_MOESM3_ESM.tif

Supplementary Fig. S1 Analysis of AtCYS4 overexpression and knock-down lines. A Schematic diagram of the AtCYS4 gene from the putative transcription start site (TSS; -1) to the end of the AtCYS4 ORF (+492). The position of the T-DNA insertion (+74) in 5’-UTR of the knock-down mutant is shown by the inverted triangle. The position and orientation of the P4 to P5 primers that were used for RT-PCR is shown by the arrows. B Comparison of AtCYS4 transcript levels. RT-PCR analysis was performed on total RNA isolated from 10-day-old plants of WT, two independent Prom 35S :AtCYS4 transgenic lines (OX18, OX21) and the AtCYS4 knock-down mutant (cys4-KD) using primers P2 and P5. Actin2 gene (At3g18780) is shown as loading control. C Comparison of AtCYS4 protein levels. Shown is a western blot of resolved total protein extracts of 10-day-old plants that was performed using anti-AtCYS4 polyclonal antibody and visualized using an ECL system. D Comparison of endogenous CP activities. CP activities were measured in total protein extracts of 10-day-old plants and are expressed as a percentage of the mean CP activity of WT extracts. Bars represent mean ±S.E. (n = 3; *P < 0.05, **P < 0.01 by Student’s t-test). (TIFF 147699 kb)

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Je, J., Song, C., Hwang, J.E. et al. DREB2C acts as a transcriptional activator of the thermo tolerance-related phytocystatin 4 (AtCYS4) gene. Transgenic Res 23, 109–123 (2014). https://doi.org/10.1007/s11248-013-9735-2

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