The rice Osmyb4 gene enhances tolerance to frost and improves germination under unfavourable conditions in transgenic barley plants

The Osmyb4 rice gene, coding for a transcription factor, proved to be efficient against different abiotic stresses as a trans(cis)gene in several plant species, although the effectiveness was dependent on the host genomic background. Eight barley transgenic lines carrying the rice Osmyb4 gene under the control of the Arabidopsis cold inducible promoter cor15a were produced to test the efficiency of this gene in barley. After a preliminary test, the best performing lines were subjected to freezing at −11°C and −12°C. Frost tolerance was assessed measured the Fv/Fm parameter widely used to indicate the maximum quantum yield of photosystem II photochemistry in the dark adapted state. Three transgenic lines showed significantly increased tolerance. These selected lines were further studied under a complex stress applying cold and hypoxia at germinating stage. In these conditions the three selected transgenic lines outperformed the wild type barley in terms of germination vigour. The transgenic plants also showed a significant modification of their metabolism under cold/hypoxia conditions as demonstrated through the assessment of the activity of key enzymes involved in anoxic stress response. None of the transgenic lines showed dwarfism, just a slight retarded growth. These results provide evidence that the cold dependent expression of Osmyb4 can efficiently improved frost tolerance and germination vigour at low temperature without deleterious effect on plant growth.

[1]  V. Bajic,et al.  Supra-optimal expression of the cold-regulated OsMyb4 transcription factor in transgenic rice changes the complexity of transcriptional network with major effects on stress tolerance and panicle development. , 2010, Plant, cell & environment.

[2]  Chungui Lu,et al.  Plant responses to cold: Transcriptome analysis of wheat. , 2010, Plant biotechnology journal.

[3]  E. Mazzucotelli,et al.  Abiotic stress response in plants : when post-transcriptional and post-translational regulations control transcription , 2008 .

[4]  R. Henry,et al.  Microarray analysis of gene expression in germinating barley embryos (Hordeum vulgare L.) , 2005, Functional & Integrative Genomics.

[5]  Chin-Shang Li,et al.  Regulatory genes involved in the determination of frost tolerance in temperate cereals , 2009 .

[6]  M. Thomashow,et al.  The 5′-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression , 1994, Plant Molecular Biology.

[7]  E. Segura,et al.  Glutamate dehydrogenase and aspartate aminotransferase in Trypanosoma cruzi. , 1977, Comparative biochemistry and physiology. B, Comparative biochemistry.

[8]  K. Bradford,et al.  Seed development, dormancy and germination , 2007 .

[9]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[10]  M. Iriti,et al.  The ectopic expression of the rice Osmyb4 gene in Arabidopsis increases tolerance to abiotic, environmental and biotic stresses , 2006 .

[11]  P. Waterhouse,et al.  Marker gene elimination from transgenic barley, using co-transformation with adjacent `twin T-DNAs' on a standard Agrobacterium transformation vector , 2001, Molecular Breeding.

[12]  A. Hanson,et al.  Induction of lactate dehydrogenase isozymes by oxygen deficit in barley root tissue. , 1986, Plant physiology.

[13]  N. Saibo,et al.  Transcription Factors and Regulation of Photosynthetic and Related Metabolism under Environmental Stresses , 2022 .

[14]  D. Weigel,et al.  The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene , 2005, Planta.

[15]  L. Sodek,et al.  Alanine metabolism and alanine aminotransferase activity in soybean (Glycine max) during hypoxia of the root system and subsequent return to normoxia , 2003 .

[16]  C. Vannini,et al.  Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants. , 2004, The Plant journal : for cell and molecular biology.

[17]  A. Hanson,et al.  Control of Lactate Dehydrogenase, Lactate Glycolysis, and α-Amylase by O2 Deficit in Barley Aleurone Layers , 1984 .

[18]  K. Thompson,et al.  Seeds: Physiology of Development and Germination , 1986 .

[19]  K. Yamaguchi-Shinozaki,et al.  Transcriptional Regulatory Networks in Response to Abiotic Stresses in Arabidopsis and Grasses1 , 2009, Plant Physiology.

[20]  W. L. Butler,et al.  Fluorescence quenching in photosystem II of chloroplasts. , 1975, Biochimica et biophysica acta.

[21]  A. Mustroph,et al.  Tolerance of crop plants to oxygen deficiency stress: fermentative activity and photosynthetic capacity of entire seedlings under hypoxia and anoxia. , 2003, Physiologia plantarum.

[22]  U. Grossniklaus,et al.  A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta[w] , 2003, Plant Physiology.

[23]  R. Kalla,et al.  Agrobacterium tumefaciens‐mediated barley transformation , 1997 .

[24]  M. Zhou,et al.  CBF-dependent signaling pathway: A key responder to low temperature stress in plants , 2011, Critical reviews in biotechnology.

[25]  J. Doyle,et al.  Isolation of plant DNA from fresh tissue , 1990 .

[26]  A. Macgregor,et al.  Barley : chemistry and technology , 1993 .

[27]  L. Cattivelli,et al.  Use of chlorophyll fluorescence to evaluate the cold acclimation and freezing tolerance of winter and spring oats , 2001 .

[28]  Manuela Campa,et al.  Evaluation of transgenic tomato plants ectopically expressing the rice Osmyb4 gene , 2007 .

[29]  R. Kalla,et al.  Gibberellin-regulated expression of a myb gene in barley aleurone cells: evidence for Myb transactivation of a high-pI alpha-amylase gene promoter. , 1995, The Plant cell.

[30]  Jianhua Zhu,et al.  Cold stress regulation of gene expression in plants. , 2007, Trends in plant science.

[31]  P. Perata,et al.  Repression of α-Amylase Activity by Anoxia in Grains of Barley is Independent of Ethanol Toxicity or Action of Abscisic Acid , 2002 .

[32]  Elena Baldoni,et al.  Osmyb4 expression improves adaptive responses to drought and cold stress in transgenic apples , 2008, Plant Cell Reports.

[33]  B. Menand,et al.  Post-transcriptional Regulation of Gene Expression in Plants during Abiotic Stress , 2009, International journal of molecular sciences.

[34]  C. Vannini,et al.  Overexpression of Osmyb4 enhances compatible solute accumulation and increases stress tolerance of Arabidopsis thaliana , 2005 .

[35]  Patrick Schweizer,et al.  Large-scale analysis of the barley transcriptome based on expressed sequence tags. , 2004, The Plant journal : for cell and molecular biology.

[36]  F. Locatelli,et al.  Metabolic response to cold and freezing of Osteospermum ecklonis overexpressing Osmyb4. , 2010, Plant physiology and biochemistry : PPB.

[37]  P. Perata,et al.  Effect of anoxia on the induction of α-amylase in cereal seeds , 1993, Planta.

[38]  W. Peacock,et al.  Evidence for a role for AtMYB2 in the induction of the Arabidopsis alcohol dehydrogenase gene (ADH1) by low oxygen. , 1998, Genetics.

[39]  James Z Zhang,et al.  Overexpression analysis of plant transcription factors. , 2003, Current opinion in plant biology.

[40]  L. Voesenek,et al.  Flooding stress: acclimations and genetic diversity. , 2008, Annual review of plant biology.

[41]  B. Dolinka,et al.  Complex stressing vigour test: a new method for wheat and maize seeds , 1988 .

[42]  L. Cattivelli,et al.  Effects of growth stage and hardening conditions on the association between frost resistance and the expression of the cold-induced protein COR14b in barley , 2008 .