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Cold Response of Dedifferentiated Barley Cells at the Gene Expression, Hormone Composition, and Freezing Tolerance Levels: Studies on Callus Cultures

Abstract

In this study, data is presented how dark-grown, embryogenic barley callus cells respond to cold without any light-dependent, chloroplast-related mechanism, independently of the systemic signals. The expression of HvCBF9, HvCBF14, and HvCOR14b genes, members of one of the most important cold-inducible regulatory system, was measured by real-time PCR. Characteristic of the cold response was similar in the crowns of seedlings and in dark-grown callus cultures, however, gene expression levels were lower in calli. Endogenous concentration of auxins, abscisic acid, and salicylic acid did not change, but phaseic acid and neophaseic acid showed robust accumulation after cold acclimation. Freezing tolerance of the cultures was also higher after 7 days of cold-hardening. The results suggest the presence of a basal, light-independent, cold-responsive activation of the CBF–COR14b pathway in barley cultures. The effects of Dicamba, the exogenous auxin analog used for maintaining tissue cultures were also studied. Dicamba seems to be a general enhancer of the gene expression and physiological responses to cold stress, but has no specific effect on the activation. Our data along with previous findings show that this system might be a suitable model for studying certain basic cellular mechanisms involved in the cold acclimation process in cereals.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid (IUPAC: (2,4-dichlorophenoxy) acetic acid)

ABA:

Abscisic acid (IUPAC: (S)-5-(1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexen-1-yl)-3-methyl-2,4-pentanedienoic acid)

AREB/ABF:

ABA-Responsive Element Binding Protein/Factor

BAP:

6-Benzylaminopurine (IUPAC: N-(phenylmethyl)-7H-purin-6-amine)

CBF:

C-repeat binding factor

COR:

Cold-regulated gene

Dic:

Dicamba (IUPAC: 3,6-dichloro-2-methoxybenzoic acid)

DPA:

Dihydrophaseic acid (IUPAC: (2Z,4E)-5-[(1R,3S,5R,8S)-3,8-dihydroxy-1,5-dimethyl-6-oxabicyclo[3.2.1]oct-8-yl]-3-methylpenta-2,4-dienoic acid)

IAA:

Indole-3-acetic acid (IUPAC: 2-(1H-indol-3-yl)acetic acid)

IAA-Asp:

IAA-aspartate (IUPAC: 2-[[2-(1H-indol-3-yl)acetyl]amino]butanedioate)

IAA-GE:

IAA-glucose ester

ICE:

Inducer of CBF expression

JA:

Jasmonic acid (IUPAC: (1R,2R)-3-oxo-2-(2Z)-2-pentenyl-cyclopentaneacetic acid)

Neo-PA:

Neophaseic acid (IUPAC: (2Z,4E)-5-[(1S,3S,8S)-3,8-dihydroxy-1,5-dimethyl-6-oxabicyclo[3.2.1]octan-8-yl]-3-methylpenta-2,4-dienoate)

PA:

Phaseic acid (IUPAC: (2Z,4E)-5-[(1R,5R,8S)-8-hydroxy-1,5-dimethyl-3-oxo-6-oxabicyclo[3.2.1]octan-8-yl]-3-methylpenta-2,4-dienoic acid)

PGR:

Plant growth regulator

SA:

Salicylic acid (IUPAC: 2-hydroxybenzoic acid)

TTC:

2,3,5-Triphenyltetrazolium chloride (IUPAC: 2,3,5-triphenyl-2H-tetrazolium chloride)

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Acknowledgments

The authors are grateful to Nicola Pecchioni for providing the barley (H. vulgare L. cv. Nure) seeds, to Mónika E. Fehér for her technical assistance and to the Applied Genomics Department (AI CAR HAS) for the use of laboratory equipment. This study was supported by the Hungarian Scientific Research Fund (OTKA K84190), the Hungarian TÁMOP-4.2.2/B-10/1-2010-0025 grant, the Norwegian Financial Mechanism (OTKA NNF78866), the Czech Science Foundation (project no. 522/09/2058), and the European Union (Agrisafe 203288—EU-FP7-426 REGPOT 2007-1). BT is a Bolyai Fellow of the Hungarian Academy of Sciences.

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Supplemental Figure 1

Effect of Dicamba on hormone levels of endogenous auxins after four days of cold treatment. Relative hormone levels of endogenous auxins: indole-3-acetic acid (IAA), IAA-aspartate (IAA-Asp) and IAA-glucose ester (IAA-GE) in Dicamba-treated, Dic(+), and Dicamba-free, Dic(−), calli (mean ± SE). The results show the hormone levels in cold-treated samples after four days of hardening relative to the unhardened control (grown at +24 °C) (%). Three biological repeats are given for each data point. Statistical analysis was performed using unpaired (Student’s) and Welch’s t test (* 0.01 < p<0.05; ** 0.001 < p<0.01). (TIFF 752 kb)

Supplemental Figure 2

Effect of Dicamba on hormone levels of ABA and its catabolites after four days of cold treatment. Relative hormone levels of endogenous ABA and dihydrophaseic acid (DPA) (A), and phaseic acid (PA) and neophaseic acid (Neo-PA) (B) in Dicamba-treated, Dic(+), and Dicamba-free, Dic(−), calli (mean ± SE). The results show the hormone levels in cold-treated samples after four days of hardening relative to the unhardened control (grown at +24 °C) (%). Three biological repeats are given for each data point. Statistical analysis was performed using unpaired (Student’s) and Welch’s t test (* 0.01 < p<0.05; ** 0.001 < p<0.01). (TIFF 1319 kb)

Supplemental Figure 3

Effect of Dicamba on hormone levels of jasmonic acid and salicylic acid after four days of cold treatment. Relative hormone levels of endogenous jasmonic acid (JA) and salicylic acid (SA) in Dicamba-treated, Dic(+), and Dicamba-free, Dic(−), calli (mean ± SE). The results show the hormone levels in cold-treated samples after four days of hardening relative to the unhardened control (grown at +24 °C) (%). Three biological repeats are given for each data point. Statistical analysis was performed using unpaired (Student’s) and Welch’s t test (* 0.01 < p<0.05; ** 0.001 < p<0.01). (TIFF 566 kb)

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Vashegyi, I., Marozsán-Tóth, Z., Galiba, G. et al. Cold Response of Dedifferentiated Barley Cells at the Gene Expression, Hormone Composition, and Freezing Tolerance Levels: Studies on Callus Cultures. Mol Biotechnol 54, 337–349 (2013). https://doi.org/10.1007/s12033-012-9569-9

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Keywords

  • ABA
  • Barley
  • Callus
  • CBF
  • Cold
  • COR14b
  • Dicamba
  • IAA