Jahn Davik

Research Scientist

(+47) 979 54 473
jahn.davik@nibio.no

Place
Ås H8

Visiting address
Høgskoleveien 8, 1433 Ås

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Abstract

To assess underlying metabolic processes and regulatory mechanisms during cold exposure of strawberry, integrative “omic” approaches were applied to F. ananassa ‘Korona’. Analysis of leaf tissue was emphasized because of its immediate and early responses in the cold acclimation processes. Levels of metabolites, proteins, and transcripts in tissues from plants grown at 20°C were compared to those following 1 to 10 days of cold exposure. When leaves and roots were subjected to GC/TOF-MS-based metabolite profiling, about 160 compounds comprising mostly structurally annotated primary metabolites and secondary metabolites, were found. Overall, ‘Korona’ showed a modest increase of protective metabolites such as amino acids (aspartic acid, leucine, isoleucine, and valine), pentoses, phosphorylated and non-phosphorylated hexoses, and distinct compounds of the raffinose pathway (galactinol and raffinose). Distinctive responses were observed in roots and leaves. By 2DE proteomics a total of 845 spots were observed in leaves; 4.6% changed significantly in response to cold. Twenty-one proteins were identified, many of which were associated with general metabolism or photosynthesis. Transcript levels in leaves were determined by microarray, where dozens of cold associated transcripts were quantitatively characterized, and levels of several potential key contributors (e.g., the dehydrin COR47 and GADb) to cold tolerance were confirmed by qRT-PCR. Cold responses are placed within the existing knowledge base of low temperature stress change in plants, allowing an evaluation of the uniqueness or generality of Fragaria responses in photosynthetic tissues. Overall, the cold response characteristics of ‘Korona’ are consistent with a moderately cold tolerant plant.

Abstract

The woodland strawberry (Fragaria vesca) has become the model plant for the economically important, but genetically complex, octoploid F. × ananassa. Crown rot caused by the oomycete Phytophthora cactorum is a major problem for the strawberry industry and the identification and incorporation of efficient resistance genes into superior cultivars are important for breeding. In the present study, two experimental populations were used in inoculation experiments under controlled greenhouse condition. Studies of a sparse diallel cross between resistant and susceptible F. vesca genotypes concluded that resistance to crown rot is inherited as a dominant trait under nuclear control. Subsequently, an F2 population derived from the grandparents Bukammen (resistant) and Haugastøl 3 (susceptible) collected in Norway, were phenotyped in infection experiments and genotyped using genotyping-by-sequencing. A 416.2-cM linkage map was constructed, and a single major gene locus was identified on linkage group 6 that we attributed to resistance to Phytopthora infection. We propose to name the resistance locus RPc-1 (Resistance to Phytophthora cactorum 1). Gene prediction of the 3.3 Mb QTL recovered 801 genes of which 69 had a potential role in plant disease resistance.

Abstract

The winter hardiness of strawberry varieties used in perennial production systems varies greatly. Still, little information is available on how plant metabolism adapts to cold and freezing temperatures under natural temperature and light conditions. In order to examine the hardening process of overwintering meristematic tissue in Fragaria  ananassa, crown samples of field-grown var. ‘Polka’ and ‘Honeoye’ were consecutively collected over a period of 15 weeks, i.e. from the end of the season (week 35/ end August) until midwinter (week 50/ December). Samples were subjected to qGC MS metabolite profiling to assess the reconfiguration of central metabolism, and characterize the regulation of selected compatible solutes. Besides changes in amino acid patterns (glutamic acid, aspartic acid, and asparagine), monosaccharide levels (fructose) increased strongly in ‘Honeoye’ (180 fold compared to start control) towards the end of the acclimation period. In contrast, ‘Polka’ showed a concentration peak (36-fold) in week 47 and a decline towards week 50. Also sucrose levels were steadily increased throughout the cold hardening period with averagely 6-fold higher levels in ‘Honeoye’ compared to ‘Polka’, thus underscoring cultivar-dependent differences. However, both varieties showed a decline in sucrose levels after week 47. Particularly, the raffinose pathway was affected leading to strongly and transiently increased levels of the precursor galactinol (week 42/ mid-October) and the trisaccharide raffinose (weeks 43 to 47/ end October to mid-November). While galactinol biosynthesis was earlier induced in ‘Polka’ (week 38) compared to ‘Honeoye’ (week 39), subsequent raffinose production was delayed in ‘Polka’ (week 47) compared to ‘Honeoye’ (week 45). Major metabolic changes in both varieties coincided with a decrease in day length below 14 h in mid-September, and a consistent drop below 10°C average day temperature by the end of September.

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Abstract

The use of artificial freezing tests, identification of biomarkers linked to or directly involved in the low-temperature tolerance processes, could prove useful in applied strawberry breeding. This study was conducted to identify genotypes of diploid strawberry that differ in their tolerance to low-temperature stress and to investigate whether a set of candidate proteins and metabolites correlate with the level of tolerance. 17 Fragaria vesca, 2 F. nilgerrensis, 2 F. nubicola, and 1 F. pentaphylla genotypes were evaluated for low-temperature tolerance. Estimates of temperatures where 50 % of the plants survived (LT50) ranged from −4.7 to −12.0 °C between the genotypes. Among the F. vesca genotypes, the LT50 varied from −7.7 °C to −12.0 °C. Among the most tolerant were three F. vesca ssp. bracteata genotypes (FDP821, NCGR424, and NCGR502), while a F. vesca ssp. californica genotype (FDP817) was the least tolerant (LT50 −7.7 °C). Alcohol dehydrogenase (ADH), total dehydrin expression, and content of central metabolism constituents were assayed in select plants acclimated at 2 °C. The LT50 estimates and the expression of ADH and total dehydrins were highly correlated (r adh = −0.87, r dehyd = −0.82). Compounds related to the citric acid cycle were quantified in the leaves during acclimation. While several sugars and acids were significantly correlated to the LT50 estimates early in the acclimation period, only galactinol proved to be a good LT50 predictor after 28 days of acclimation (r galact = 0.79). It is concluded that ADH, dehydrins, and galactinol show great potential to serve as biomarkers for cold tolerance in diploid strawberry.

Abstract

A crucial consideration for strawberry producers in Norway and other northern countries is winter freezing damage. A long-term goal of the Norwegian strawberry breeding is to increase winter hardiness and to improve fruit quality. Due to the complexity involved in regulating and enhancing freezing tolerance, the progress in the improvement of cultivars using traditional screening methods have had limited success. Thus, the development of molecular markers for freezing hardiness would facilitate the selection work for this trait. In this effort, we have developed and adopted state-of-art molecular tools to investigate cold response in strawberry plants during the acclimation phase resulting in the identification of a large number of genes, proteins, and distinct metabolites that correspond to cold/freezing tolerance in strawberry. To identify proteins responsible for freezing tolerance in strawberry we have examined alterations in protein levels in strawberry varieties that differ in cold tolerance using either 2-DE gel analysis followed by LC-MS/MS analysis or a shotgun MS/MS approach. Proteomic analysis suggested 30 potential biomarkers that showed significant changes in the cultivated strawberry in response to cold. In addition, GC-MS-based metabolite profiling revealed the up-regulation of carbohydrates, polyols, amino acids, TCA intermediates, and other distinct secondary metabolites after cold treatment. Transcriptional analysis of the cold acclimated samples also confirmed the regulation upon cold-treatment with varietal differences in strawberry. Moreover, several F2-populations from the model F. vesca parents diverging in cold tolerance have been developed in order to facilitate mapping of QTLs by performing GBS analyses. The knowledge attained from these endeavors is expected to expedite breeding of strawberries to achieve freezing tolerant lines and provide an integrative understanding of the molecular pathways that underlie this characteristic. * Rohloff et al. (2012) Metabolite profiling reveals novel multi-level cold responses in the model Fragaria vesca. Phytochemistry 79:99-109. * Koehler et al. (2012) Proteomic study of low temperature responses in strawberry cultivars (Fragaria x ananassa) that differ in cold tolerance. Plant Physiology 159:1787–1805 * Davik et al., (2012) Low temperature tolerance in diploid strawberry species (Fragaria ssp.) and its correlation to alcohol dehydrogenase levels, dehydrin levels, and central metabolism constituents. Planta (in press; DOI: 10.1007/s00425-012-1771-2).

Abstract

The winter hardiness of strawberry cultivars used in perennial production systems varies greatly, although a strong linkage exists between transcriptional and metabolic changes during cold acclimation. Still, little information is available on how plant metabolism adapts to cold and freezing temperatures under natural temperature and light conditions. In order to examine the hardening process of overwintering meristematic tissue in Fragaria x ananassa, crown samples of field-grown cvs. ‘Polka’ and ‘Honeoye’ were consecutively collected over a period of 15 weeks, i.e. from the end of the season (week 35/ end August) until midwinter (week 50/ December). Samples were subjected to qGC MS metabolite profiling to assess the reconfiguration of central metabolism, and characterize the regulation of selected compatible solutes (amino acids, Krebs metabolites, sugars, polyols). Besides changes in amino acid patterns (glutamic acid, aspartic acid, and asparagine), monosaccharide levels (fructose) were strongly enhanced until the end of the acclimation period in cv. ‘Honeoye’ (180-fold compared to start control). In contrast, ‘Polka’ showed a concentration peak (36-fold) in week 47 and a decline towards week 50. Also sucrose levels were steadily enhanced throughout the cold hardening period with averagely 6-fold higher levels in ‘Honeoye’ compared to ‘Polka’, thus underscoring cultivar differences. However, both cultivars showed a clear decline in sucrose levels after week 47. Particularly, the raffinose pathway was affected leading to strongly and transiently increased levels of the precursor galactinol (week 42/ mid October) and the trisaccharide raffinose (weeks 43 to 47/ end October to mid November). While galactinol biosynthesis was obviously earlier induced in cv. ‘Polka’ (week 38) compared to ‘Honeoye’ (week 39), subsequent raffinose production and concentration peaks were clearly delayed in ‘Polka’ (week 47) in contrast to ‘Honeoye’ (week 45). Major metabolic changes in both cultivars coincided with a decrease in daylength below 14 h after week 37 (mid September), and a consistent drop below 10°C average day temperature in week 39 (end September). The effect of temperature and light conditions on metabolic cold acclimation in field-grown strawberry is discussed. Keywords: Winter hardiness, metabolite profiling, quadrupole gas chromatography-mass spectrometry (qGC-MS), temperature, light

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Abstract

Winter freezing damage is a crucial factor in overwintering crops such as the octoploid strawberry (Fragaria × ananassa Duch.) when grown in a perennial cultivation system. Our study aimed at assessing metabolic processes and regulatory mechanisms in the close-related diploid model woodland strawberry (Fragaria vesca L.) during a 10-days cold acclimation experiment. Based on gas chromatography/ time-of-flight-mass spectrometry (GC/TOF-MS) metabolite profiling of three F. vesca genotypes, clear distinctions could be made between leaves and non-photosynthesizing roots, underscoring the evolvement of organ-dependent cold acclimation strategies. Carbohydrate and amino acid metabolism, photosynthetic acclimation, and antioxidant and detoxification systems (ascorbate pathway) were strongly affected. Metabolic changes in F. vesca included the strong modulation of central metabolism, and induction of osmotically-active sugars (fructose, glucose), amino acids (aspartatic acid), and amines (putrescine). In contrast, a distinct impact on the amino acid proline, known to be cold-induced in other plant systems, was conspicuously absent. Levels of galactinol and raffinose, key metabolites of the cold-inducible raffinose pathway, were drastically enhanced in both leaves and roots throughout the cold acclimation period of 10 days. Furthermore, initial freezing tests and multifaceted GC/TOF-MS data processing (Venn diagrams, Independent Component Analysis, Hierarchical Clustering) showed that changes in metabolite pools of cold-acclimated F. vesca were clearly influenced by genotype.

Abstract

Freezing damage is a crucial factor in the cultivation of perennial crops. Overwintering plants acclimate to decreasing temperatures in their environment and thus, prevent freezing damage of plant tissue. To assess transcriptional and metabolic changes in meristematic tissue (crowns) of octoploid strawberry (Fragaria × ananassa Duch.), acclimation experiments were carried out at above-zero temperature (2 °C) using three cultivars with contrasting cold tolerance: ‘Elsanta’ < ‘Frida’ < ‘Jonsok’. Crowns were sampled after 1 day (d), 2d, 2 weeks (w) and 6w in order to detect short- and long-term metabolic shifts. GC/MS-based metabolite profiling revealed more than 140 metabolites (identified structures, not-annotated mass spectral tags, and unidentified metabolites). Transcriptional changes were assessed at two time points (2d and 6w) using a customized Fragaria microarray chip developed as a joint collaboration between Graminor Breeding Ltd. and NTNU. A total of 4061 differentially regulated transcripts (unique 60-mer probes) with a p-value≤0.05 were detected in all hybridizations. Microarray analysis revealed the up-regulation of ~100 cold-responsive transcripts (TFs and dehydrins), also including enzymes involved in starch breakdown and raffinose biosynthesis. Gene-metabolite correlation analysis revealed strong connectivity in components of Krebs-cycle (citric and succinic acid), amino acids (isoleucine, aspartic acid, glutamic acid, valine and phenylalanine) and the raffinose pathway. Metabolite levels of hexoses (fructose and glucose), trisaccharides (raffinose), amino acids (aspartic acid, alanine and serine), phenols (gallic acid) and several polyphenols still increased during long-term acclimation phase. Varietal differences could be clearly explained by Venn diagrams: frost-tolerant ‘Jonsok’ showed least individual up- or down-regulated transcripts (2 d), and least commonly shared transcripts with frost-sensitive ‘Elsanta’ (2d and 6w). Further multivariate statistics and network analyses underscored genotype-dependent cold responses, and might further guide in the identification of frost-tolerant vs. sensitive plants in diverse Fragaria accessions or cross-breeding populations .

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Abstract

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