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Loss of all three calreticulins, CRT1, CRT2 and CRT3, causes enhanced sensitivity to water stress in Arabidopsis

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Abstract

Key message

The calreticulin triple knockout mutant shows growth defects in response to abiotic stress.

Abstract

The endoplasmic reticulum (ER) is an essential organelle that is responsible for the folding and maturation of proteins. During ER stress, unfolded protein aggregates accumulate in the cell, leading to the unfolded protein response (UPR). The UPR up-regulates the expression of ER-stress-responsive genes encoding calreticulin (CRT), an ER-localized Ca2+-binding protein. To understand the function of plant CRTs, we generated a triple knockout mutant, t123, which lacks CRT1, CRT2 and CRT3 and examined the roles of calreticulins in abiotic stress tolerance. A triple knockout mutant increased sensitivity to water stress which implies that calreticulins are involved in the Arabidopsis response to water stress. We identified that the cyclophilin AtCYP21-2, which is located in the ER, was specifically enhanced in the t123 mutants. Seed germination of the atcyp21-1 mutant was retarded by water stress. Taken together, these results suggest that regulatory proteins that serve to protect plants from water stress are folded properly in part with the help of calreticulins. The AtCYP21-2 may also participate in this protein-folding process in association with calreticulins.

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Abbreviations

2-DE:

2-Dimensional electrophoresis

ABA:

Abscisic acid

CNX:

Calnexins

CRT:

Calreticulin

DTT:

Dithiothreitol

ER:

Endoplasmic reticulum

IVT:

In vitro transcription

MS:

Murashige and Skoog

PPiase:

Peptidyl-prolyl isomerase

TM:

Tunicamycin

UPR:

Unfolded protein response

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Acknowledgments

This work was supported by a grant from the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (to Hojoung Lee, 2012; grant #2012-112068-3) and by a grant from the National Research Foundation (to Suk-Whan Hong; #2012R1A1A4A01006448).

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Authors and Affiliations

  1. Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, 1, 5-ka Anam-dong, Seongbuk-ku, Seoul, 136-713, Republic of Korea

    Jun Hyeok Kim, Nguyen Hoai Nguyen & Hojoung Lee

  2. Institute of Life Science and Natural Resources, Korea University, Seoul, 136-713, Republic of Korea

    Jun Hyeok Kim & Nguyen Hoai Nguyen

  3. Department of Molecular Biotechnology, College of Agriculture and Life Sciences, Bioenergy Research Center, Chonnam National University, Gwangju, Republic of Korea

    Ngoc Trinh Nguyen & Suk-Whan Hong

Authors
  1. Jun Hyeok Kim
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  2. Nguyen Hoai Nguyen
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  3. Ngoc Trinh Nguyen
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  4. Suk-Whan Hong
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  5. Hojoung Lee
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Corresponding authors

Correspondence to Suk-Whan Hong or Hojoung Lee.

Additional information

Communicated by K. Chong.

Electronic supplementary material

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299_2013_1497_Fig9_ESM.jpg

Figure S1. Multiple alignments of amino acid sequences of CRT1, CRT2 and CRT3 proteins. Identical residues are shaded in black (*), and two identical residues are shaded in gray (two dots). CRT1A is CRT1, and CRT1B is CRT2. The CRT1 is At1g56340, CRT2 is At1g09210 and CRT3 is At1g08450. (JPEG 2512 kb)

299_2013_1497_Fig10_ESM.jpg

Figure S2. Growth phenotypes of Arabidopsis wild-type, t12, t13, t23 and t123 mutant seedlings Seven-day-old Arabidopsis wild-type (Col-0), t12, t13, t23 and t123 seedlings grown on normal MS1/2 medium (2 % sucrose) were transferred to soil and allowed to grow for 3 (a) or 7 (b) more weeks before being photographed. The seedlings shown were typical of three separate experiments performed at 23 °C (n = 5-8 seedlings per experiment). (JPEG 1977 kb)

299_2013_1497_Fig11_ESM.jpg

Figure S3. Complementation analysis. A plasmid expressing the CRT1 or CRT3 cDNA under the control of the CRT1 or CRT3 promoter was introduced into the t123 mutant, and 10 independent homozygous transgenic lines were recovered for the complementation analysis shown in the picture. Following sterilization and 3 days of stratification at 4 °C, seeds were germinated in standard MS agar medium supplemented with 2 % sucrose and allowed to grow for 10 days before picture was taken. (JPEG 668 kb)

299_2013_1497_Fig12_ESM.jpg

Figure S4. The expression of CNX1 in Col-0, cyc20-1, cyc21-1, cyc21-2 and t123 seedlings in response to DTT, MG132 or TM Col-0, cyc20-1, cyc21-1, cyc21-2 and t123 mutant seedlings were grown on normal growth medium for two weeks and then transferred to medium containing DTT (10 μM), MG132 (10 μM) or TM (10 μM) for 6 h prior to RNA extraction. The transcript level of CNX1 is shown, and rRNA was used as a loading control. (JPEG 530 kb)

299_2013_1497_Fig13_ESM.jpg

Figure S5. Expression of ATCYP21-2 in Arabidopsis Col-0, t1, t2, t3 and t123 seedlings. (a) Transcription of the Cyclophilin, ATCYP21-2, in 3-week-old Col-1, t1, t2, t3 and t123 plants was determined using qRT-PCR. ACTIN was used as the internal loading control. Ten-day-old seedlings were grown on normal MS1/2 medium before total RNA extraction for RT-PCR. (b) Col-0, t12 and t123 mutant seedlings were grown on normal growth medium for 2 weeks and then transferred to medium containing 300 mM mannitol for 0, 3, 6 or 24 hrs prior to RNA extraction using the ATA reagent. Transcript level of CYP21-2 is shown. rRNA levels were used as loading control. (JPEG 696 kb)

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Kim, J.H., Nguyen, N.H., Nguyen, N.T. et al. Loss of all three calreticulins, CRT1, CRT2 and CRT3, causes enhanced sensitivity to water stress in Arabidopsis . Plant Cell Rep 32, 1843–1853 (2013). https://doi.org/10.1007/s00299-013-1497-z

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