Frost injury in potato can involve the interaction of both freezing temperatures and high light. Thus frosts can result in accumulation of activated oxygen species that change the redox potential of cells. Activated oxygen species can either act as signals that induce protection mechanisms or accelerate injury. In comparison with Solanum tuberosum L., Solanum commersonii Dun., is better adapted to low temperature. In this study the freezing tolerance was studied by isolating the cold-regulated genes glutathione S-transferase (ScgstF1), heat shock cognate 70 kDa (Schsc70) and dehydrin2 (Scdnh2) from S. commersonii and characterizing gene expression in potato genotypes that differ in freezing tolerance. Fluorescence measurements (Fv/Fm, 1 − qP) showed that photosynthesis of a freezing-tolerant genotype was transiently reduced during frost whereas in S. tuberosum the reduction was higher and irreversible damage occurred. In a freezing-tolerant genotype the transient reduction of photosynthesis occurred coincident with accumulation of ScgstF1 and Scdhn2 transcripts. In cold-acclimated and H2O2-treated plants, ScgstF1 and Scdhn2 accumulated in freezing-tolerant genotypes whereas Schsc70 transcript was more abundant in S. tuberosum. Pre-treatment with H2O2 also improved the freezing tolerance of S. commersonii suggesting that signal pathways associated with low temperature cold acclimation or H2O2 may overlap.
In temperate climates crop productivity is affected by freezing and chilling stresses. Cultivated potato, Solanum tuberosum L., is sensitive to freezing and is severely injured at temperatures below −3 °C (Chen & Li 1980). Solansm tuberosum possesses little genetic variation in freezing tolerance (Chen & Li 1980; Seppänen, Nissinen & Perälä 2001) which limits the improvement of this trait by conventional plant breeding. In contrast the wild potato species such as Solanum commersonii and Solanum acaule, are freezing tolerant and could serve as a source of germplasm for this trait. Research has shown that somatic hybrids between S. commersonii and S. tuberosum and the segregating populations derived from these hybrids exhibit altered freezing tolerance (Cardi et al. 1993; Nyman & Waara 1997; Seppänen et al. 1998). In addition to freezing tolerance, photosynthesis in wild potato species (Steffen & Palta 1986, 1989) and hybrids between S. commersonii and S. tuberosum (Seppänen et al. 2001) are more tolerant to low temperature photo-inhibition.
Weiser (1970) suggested that improved freezing tolerance during cold acclimation is a result of altered gene expression. Genetic variation in freezing tolerance can be due to difference in either the composition and/or the regulation of protective genes. Studies with winter and spring wheat indicate that both genotypes have the same protective genes; however, regulation in the spring genotype varies from the winter genotype during low temperature stress (Sahran & Danyluk 1998). The situation could be the same in potato. When the genetic make-up of S. commersonii is compared with S. tuberosum, one copy of a cold-inducible Scgst1 (S. commersonii glutathione S-transferase 1) was found in both genomes, however, Scgst1 appeared to be expressed at higher levels during low temperature stress in S. commersonii (Seppänen et al. 2000).
In the field, freezing injury starts to develop in the potato canopy when the air temperature approaches the 50% lethality (LT50)-value of the genotype (Seppänen et al. 2001). When plants are exposed to high intensity light after freezing the severity of these freezing injuries increases significantly (Steffen & Palta 1989; Seppänen et al. 2001). Because in field conditions freezing nights are often followed by high light during the morning, light is an important component of frost injuries. The harmful effects of light in the frozen potato canopy are related to an imbalance between energy supply and demand. At suboptimal temperatures the energy flow to the photosystem exceeds demand and leads to excess excitation energy in the chloroplasts. If oxygen is present, the potential exists for the formation of active oxygen species (AOS) and injury from oxidative stress (Krause & Weiss 1991; Krause 1994). H2O2 generated in the chloroplasts can also signal the expression of genes such as ascorbate peroxidase and act as a systemic signal leading to improved tolerance to photo-oxidation (Karpinski et al. 1997, 1999). In addition, scavenging of H2O2 leads to changes in the reduction state of glutathione and ascorbate, which is an important mechanism regulating gene expression during abiotic stresses (Foyer & Noctor 2000). The redox state of photosynthetic electron transport chain can also serve as a stress signal. Thus redox changes not only regulate genes involved in the photosynthesis but are also likely to play a role in regulating stress-responsive genes (Maxwell, Laudenbach & Huner. 1995; Gray et al. 1997; El Bissati & Kirilovsky 2000). Alterations in photosystem II excitation pressure (1 − qP) may serve as a signal in the low-temperature signal transduction pathway. Thus, photosynthetic redox status may act as an environmental sensor and co-ordinate the expression of cold-responsive genes (Huner, Öquist & Sahran 1998). In addition, the redox status of the plastoquinone pool of light-stressed plants has been proposed to be a signal for rapid induction of gene transcription (Karpinski et al. 1997). In these light-stressed plants, the redox signal was actually detected prior to a transient burst of H2O2.
Under field conditions it is possible that induction of stress-related genes may involve both low temperature as well as signals produced by changes in the photosynthetic redox state. Such signal molecules could include H2O2 generated during photo-inhibitory conditions. In this study we investigated whether differences in freezing injury between potato genotypes that vary in freezing tolerance involves signals produced by photo-inhibition.
MATERIALS AND METHODS
Tubers of Solanum tuberosum cv. Pito, were obtained from the Tuber Seed Center (Tyrnävä, Finland). The somatic hybrid, SH9A (S. commersonii PI243503 (+) S. tuberosum SPV11), was kindly provided by T. Cardi, University of Naples, Italy (Cardi et al. 1993) and selfed to produce a S1 population (Seppänen et al. 1998). The S1 genotypes were characterized for freezing tolerance and acclimation capacity (Seppänen et al. 1998) and the most freezing-tolerant (2019, LT50 = −4.3 °C) and -sensitive (2051, LT50 = −3.1 °C) genotypes were selected for experiments. The freezing tolerance (LT50) of S. commersonii, SH9A, S.tuberosum cv. SPV11 and Pito was −4.7, −3.8, −3.0 and −2.5 °C, respectively (Seppänen et al. 1998, 2001). The hybrids and parental plants were propagated in vitro and grown for 4 to 6 weeks before transfer to the greenhouse. Greenhouse plants were grown in pots (7.5 L) containing 10 : 1 (v/v) peat and sand with 20/15 °C day/night temperatures and an 18 h photoperiod supplemented with 220 µmol m−2 s−1 of light supplied by Lucalux, LU-T/250/400/12 GE lamps (General Electrics, Budapest, Hungurg).
Plants were cold acclimated for 1 or 2 d in growth chambers (Weiss Bio 2000; Weiss, Reiskirchen, Germany) at a day/night temperature regime of 2/1 °C and a 12 h photoperiod at a light intensity of 100 µmol m−2 s−1 (Osram L 30 W/13–830; Lumilus Plus, Institut für Mikroteclinik, Mainz, Germany). In a growth chamber the plants were arranged in a random block design and divided into three replicate blocks.
For the H2O2 treatment leaf discs (diameter 1 cm) were incubated at room temperature for 1 or 24 h with either 0.1 or 1.0 mm H2O2. Control leaf discs were treated with water for the same time period. The freezing tolerance (LT50) of the H2O2-treated leaf discs was measured using the ion-leakage method (Seppänen et al. 1998). In each experiment, the LT50-value was calculated as an average of leaf discs from three plants in three individual experiments.
Experiments of freezing (−1 or −3 °C) and light (HL, 700 µmol m−2 s−1) were performed by using leaf discs (diameter 3 cm). At least three leaf discs from three individual plants were used for the experiments. In the freezing experiments, the leaf discs were placed in test tubes containing a piece of wet tissue. The tubes were put into a controlled cooling unit (Lauda RK20 KP; Lauda, Könighofen, Germany) at 1 °C and the temperature was decreased to −1 °C over 2 h. Ice nucleation was initiated by adding small ice crystals to the tubes. The temperature was decreased 1 °C h−1 to the desired minimum temperature. The minimum temperature was maintained for 1 h before the temperature was increased to 4 °C during a time period of 5 h. The leaf discs were then kept in the dark at 4 °C overnight. To determine the influence of light on freezing injury the leaf discs were exposed to high light intensity (700 µmol m−2 s−1) (+ HL) for 4 h after freezing. For the light treatment the leaf discs were transferred to Petri dishes on a piece of wet paper towel. The Petri dishes were kept on ice and the temperature and moisture conditions were monitored throughout the light treatment. Recovery (RE) from freezing and light treatments involved placing the treated leaf discs contained in the Petri dishes into a growth chamber at 15/10 °C (day/night) and an 18 h photoperiod of 100 µmol m−2 s−1. In some experiments, as indicated in the figure captions, the freezing step was omitted and the leaf discs were exposed to high light only (HL) or kept in the dark on ice (DARK) for the same period of time.
Chlorophyll fluorescence measurements
The ratio of variable (Fv) to maximum (Fm) chlorophyll a fluorescence was measured with a fluorometer (Hansatech FMS-2; Hansatech, King Lynn, UK), after 15–30 min of dark adaptation. Coefficients for photochemical (qP) and non-photochemical quenching (qNP) were measured and excitation pressure on PSII (1 − qP) calculated according to van Kooten & Snell 1990) and Bolhàr-Nordenkampf & Öquist (1993). Measurements were performed at room temperature (25 °C) using saturating light flashes (2000 µmol m−2 s−1; OSRAM 64255 8 V, 25 W) and the temperature was monitored using a temperature leaf-clip attached on a standard tripod mount.
Cloning of cold-induced genes from S. commersonii
Total RNA was extracted from 3, 5 and 7 d cold-acclimated (2/1 °C) leaves of S. commersonii and equal amounts of total RNA from each cold acclimation treatment were mixed and used for a cDNA library following instructions provided by the supplier (UniZap-cDNA Synthesis Kit; Stratagene, La Jolla, CA, USA). cDNAs corresponding to cold-induced mRNA were isolated by differential screening and the positive recombinant cDNAs were excised in vitro into pBluescript SK-(Stratagene) following the supplier's specifications. The expression of cold-specific cDNAs was verified by Northern blot analysis. Both strands of the acclimation-specific cDNA clone were sequenced (Center for Gene Research and Biotechnology, Oregon State University, Corvallis, OR, USA and A.I. Virtanen Institute, Kuopio, Finland). The cDNA sequences were then used in a BLAST search to identify potential similarities.
RNA gel blot analysis
Total RNA was isolated using the Plant RNeasy extraction kit (Qiagen GmbH, Hilden, Germany). Ten micrograms were used for agarose formaldehyde gels and blotted onto nylon membranes (MagnaGraph, Microseparations, Westborough, MA, USA) (Sambrook, Fritsch & Maniatis 1989). Membranes were pre-hybridized for 1 h at 65 °C in hybridization solution [5 × SSPE, 0.5% sodium dodecyl sulphate (SDS), 5 × Denhardt’s, 100 µg mL−1 ssDNA). Gel-purified cDNA fragments of S. commersonii glutathione S-transferase (ScgstF1), S. commersonii dehydrin2 (Scdnh2) and S. commersonii heat shock cognate 70 kDa (Schsc70) were labelled with 32P either by nick translation (Pharmacia, Uppsala, Sweden) or by a random primed labelling system (Ladderman labelling kit; Takara Biomedicals, Otsu, Shiga, Japan) and hybridized to the blotted RNA overnight at 65 °C. Membranes were washed once with 2 × SSC, 0.1% SDS at 65 °C for 5 min; once with 1 × SSC at 65 °C for 20 min and finally once with 0.5 × SSC, 0.1% SDS for 10–30 min. After visualization of the corresponding mRNA by autoradiography or by phosphoimaging (Molecular Dynamics Storm System: Molecular Dynamics, Sunnyvale, CA, USA), the probe was removed by stripping the membranes according to the instructions provided by the supplier and rehybridized with a 32P-labelled probe corresponding to a soybean ribosomal gene (rRNA). At least two individual plants of each genotype were used for gene expression analysis.
Identification of cold-inducible cDNAs
The S. commersonii glutathione S-transferase (AF002692) was previously cloned and characterized (Seppänen et al. 2000) and in the present article has been renamed ScgstF1, according to the suggestions for gene and enzyme nomenclature of plant GSTs (Edwards, Dixon & Walbot 2000). Two additional low temperature-induced cDNAs were isolated from cold-acclimating leaves of S. commersonii by differential screening. One cDNA Schsc70 (AF002667) is similar to tomato (Lycopersicon esculentum L) 70 kDa heat shock cognate (HS71_LYCES) (X54029). The Schsc70 (AF002667) cDNA is a partial, 1149 bp fragment of the 3′ end. The second isolated cDNA, Scdnh2 (AF386075), shows 51% identity with cold-inducible dehydrin/RAB homolog (S70185) that was earlier characterized from potato tubers during storage at low temperature by van Berkel, Salami & Gebhardt (1994).
Cold acclimation induces transcript accumulation
Previously it was shown that during the two days of cold acclimation, freezing tolerance improved 1.8, 1.4 and 0.5 °C in S. commersonii, SH9A and SPV11, respectively (Seppänen & Fagerstedt 2000). Both S1 hybrids have inherited S. commersonii's ability to cold acclimate (Seppänen et al. 1998; Seppänen & Fagerstedt 2000) whereas Pito has no acclimation capacity similarly to other members of S. tuberosum family (data not shown). Here we observed transcript accumulation for all three genes in all genotypes studied (Fig. 1). In comparison to the two S. tuberosum genotypes and the somatic hybrid SH9A, ScgstF1 and Scdnh2 accumulated to higher levels in leaves of S. commersonii during cold acclimation. For 2019 and 2051 little difference in ScgstF1 accumulation was observed during cold acclimation, whereas the abundance of Scdnh2 increased similarly in the two S1 hybrids. During cold acclimation the abundance of Schsc70 was similar among the potato genotypes except in freezing-sensitive S. tuberosum cv. Pito where slightly higher transcript accumulation was detected.
Cold-inducible gene transcripts accumulate during simulated night frosts
In the field, frost injury develops after freezing nights when the temperature declines close to the LT50 of S. tuberosum. The severity of freezing injury can increase if plants are exposed to high intensity light the following morning (Seppänen et al. 2001). To determine whether low temperature and light influence the expression of cold-inducible genes in freezing-tolerant and -sensitive potato genotypes, severe (−3 °C) and mild (−1 °C) night frosts were simulated in growth chambers. Somatic hybrid SH9A instead of S. commersonii was used as a freezing-tolerant genotype because the leaves of SH9A are morphologically similar to S. tuberosum cv. Pito. This helps in the selection of leaves of same age for experiments. The freezing was followed either with or without exposure to high light and then the expression of cold-inducible genes was monitored. In the somatic hybrid SH9A, severe night frosts resulted in accumulation of all three cold-inducible genes (Fig. 2a). Moreover, exposure to high intensity light (700 µmol m−2 s−1) after severe freezing increased the amount of ScgstF1 and Scdhn2 transcripts but not Schsc70 (Fig. 2a). In S. tuberosum cv. Pito no changes in transcript levels were observed during severe night frost (Fig. 2a). The ScgstF1 and Scdnh2 transcript accumulation was also followed in SH9A and S. tuberosum cv. Pito during mild night frost (Fig. 2b). In SH9A transcripts of both genes accumulated similarly than during severe night frost. In addition, some transcript accumulation was detected in Pito (Fig. 2b). However, compared to SH9A, the accumulation of ScgstF1 and Scdhn2 transcripts were lower in Pito. During recovery transcript levels decreased in both SH9A and Pito after exposure to mild night frosts (Fig. 2b) and severe night frost for SH9A (Fig. 2a). No transcripts were detected after recovery from severe night frost for Pito because this was lethal.
To determine the individual effects, both freezing and high light were studied separately. As shown in Fig. 3 only Scdhn2 was light inducible. Schsc70 was not light inducible but transcript accumulation increased when leaf discs were held on ice in dark. ScgstF1 transcript was detected only 24 h after the light treatment during recovery (RE) (Fig. 3). In contrast, freezing induced all three genes.
Severe frosts caused significant changes in photosynthetic parameters
Signals generated from alterations in photosynthesis have been proposed to be involved in the regulation of cold-inducible genes (Gray et al. 1997). It was shown that growth conditions, which elevate the excitation pressure of PSII (1 − qP), resulted in accumulation of Wsc19 transcript (Gray et al. 1997). Whether severe (−3 °C) or mild (−1 °C) night frosts generate alterations in the reduction state of photosystem II was studied in freezing-tolerant (SH9A) and -sensitive (S. tuberosum cv. Pito) genotypes. In the somatic hybrid SH9A severe night frosts caused a significant decrease in Fv/Fm (Fig. 4a) as well as an increase in the excitation pressure of PSII evaluated as 1 −qP (Fig. 4b). During recovery (RE) the fluorescence parameters almost fully recovered to the initial level. Thus, the changes in photosynthesis were reversible in SH9A. In contrast in S. tuberosum cv. Pito severe frosts led to dramatic decrease in Fv/Fm (Fig. 4a) and increase in 1 − qP after light stress (Fig. 4b). After recovery Fv/Fm decreased to zero indicating to irreversible photo-oxidative damage to chloroplasts. After mild night frost hardly any change in Fv/Fm or 1 − qP was observed in both genotypes (Fig. 4a & b). The amount of qNP reflected the freezing tolerance of the genotype (Fig. 4c). In Pito qNP decreased dramatically after exposure to severe frost whereas in SH9A only some reduction was detected. However, after exposure to mild night frost there were no changes in qNP in either genotype (Fig. 4c).
Transcript accumulation is correlated with reversible changes in chlorophyll fluorescence
The response to severe night frosts (−3 °C) was studied further in the hybrid parents and selected S1 genotypes. Increased levels of Scdhn2 and Schsc70 transcripts were detected in all genotypes studied during simulated night frosts (Fig. 5). The ScgstF1 transcript accumulated similarly in S. commersonii and in the freezing-tolerant S1 hybrid 2019, whereas hardly any transcript was detected in the freezing-sensitive S1 hybrid 2051. It should be noted that the freezing temperature that was used in frost treatment was close to the LT50 value of 2051 and S. tuberosum cv. SPV11 (Seppänen & Fagerstedt 2000). Fluorescence measurements revealed that transcript accumulation was detected during frost when the changes in Fv/Fm were reversible and irreversible photo-oxidation damage had not occurred (Figs 5 & 6). The reduction in Fv/Fm was reversible in S. commersonii, SH9A, SPV11, 2019 and 2051 and irreversible in Pito (Fig. 6). The irreversible damage to photosynthesis correlated with failure of transcript accumulation of all cold-inducible genes in Pito (Figs 2a & 6). Despite the freezing temperature was close to the LT50 of 2051, the reduction in Fv/Fm was reversible (Fig. 6). Both 2051 and 2019 have inherited the ability to cold acclimate from S. commersonii (Seppänen et al. 1998; Seppänen & Fagerstedt 2000). The fact that S1 hybrid 2051 can acclimate may explain its better tolerance to night frost compared to S. tuberosum.
Excess light energy during frost can lead to over-excitation of photosynthesis and to a formation of AOS such as H2O2. When the capacity of scavenging enzymes to destroy AOS is exceeded, AOS can accumulate. Thus, during night frosts the signal transduction pathway from photosynthesis may involve H2O2. The induction of nuclear-encoded defence enzymes such as ascorbate peroxidase (Karpinski et al. 1999), glutathione S-transferase, and catalase (Polidoros & Scandalios 1999) is mediated by H2O2. As shown here the induction of ScgstF1, Scdhn2 and Schsc70 expression was induced after of H2O2 treatment (Fig. 7). Treatment of S. commersonii and S. tuberosum cv. Pito with 0.1 or 1 mm H2O2 resulted in rapid and concentration-dependent transcript accumulation of cold-inducible genes within 1 h. The induction was transient and after 24 h the transcript levels were similar to control levels. Similarly to cold-acclimated plants (Fig. 1), the level of Schsc70 accumulation was slightly higher in freezing-sensitive genotype S. tuberosum cv. Pito whereas ScgstF1 and Scdhn2 accumulated more in the tolerant genotype S. commersonii. When the freezing tolerance of S. commersonii was measured after 20 h, the treatment with 0.1 mm H2O2 had improved the freezing tolerance by approximately 1 °C (Fig. 8). No similar effect on freezing tolerance could be detected iin SH9A or S. tuberosum SPV11 (data not shown).
Stress gene expression in freezing-tolerant and -sensitive potato genotypes
The cold-inducible genes studied here represent members of three gene families. Glutathione S-transferases (GST) are glutathione-dependent enzymes, involved in detoxification and transport of toxic substances to vacuole (Daniel 1993). Recent results also show that some GSTs transfer the anthocyanin precursor, cyanidin 3-glucoside, to the vacuole (Alfenito et al. 1998). Some GSTs also have glutathione peroxidase (GPX) activity and transgenic plants with elevated GST/GPX activity were more tolerant to low temperature-induced lipid peroxidation (Roxas et al. 1997). Members of 70 kDa HSP family act as molecular chaperons to forestall misfolding of proteins and prevent the formation of biologically incorrect protein structures (for review see Boston, Viitanen & Vierling 1996). Not all Hsp70 genes are induced by low temperature (Li, Haskell & Guy 1999; Sung, Vierling & Guy 2001). In Arabidopsis, 14 Hsp70 family members have been identified and both mitochondrial and most of the nuclear localized Hsp70 genes are induced at low temperature whereas chloroplastic localized genes were not low temperature induced (Sung et al. 2001). The biochemical role of dehydrins is not well understood, but dehydrins are thought to function as structural stabilizers of numerous targets (Close 1996). The accumulation of dehydrins is associated with adaptation to cellular dehydration, which in the case of freezing stress is proposed to be especially important during freeze–thaw cycling (Zhu et al. 2000).
In the present study the expression of three cold-inducible genes coding for GST (ScgstF1), Hsp70 (Schsc70) and dehydrin (Scdhn2) were studied in potato genotypes with varying degrees of freezing tolerance. Increased transcript levels were detected within 2 d of cold acclimation as well as after simulated night frosts. In cold-acclimated S. commersonii, ScgstF1 and Scdhn2 transcript accumulation was higher than in freezing-sensitive S. tuberosum genotypes. In contrast, Schsc70 was most abundant in the S. tuberosum cv. Pito which was the most freezing-sensitive genotype studied. Thus, the accumulation of Schsc70 transcripts at low temperature is not necessarily related to improved tolerance but rather to increased damage to proteins or problems in protein folding. In S1 hybrids, which were able to cold acclimate, the relationship between transcript level and freezing tolerance was not as clear. Freezing tolerance and acclimation capacity are independent traits in S1 hybrids (Seppänen et al. 1998). Although the S1 hybrids used in these experiments differed significantly in freezing tolerance before cold acclimation, their capacity to cold acclimate was almost the same. Their similar response to low temperature stress may explain the lack of correlation between freezing tolerance and transcript accumulation.
After night frosts the accumulation of ScgstF1 and Scdhn2 was the highest after exposure to high light although only Scdhn2 was up-regulated solely by light. Under severe night frost, transcript accumulation coincided with increased excitation pressure of photosystem II (1 −qP) indicating a regulative role of photosynthesis. However, under milder night frost, transcript accumulation was detected in freezing-tolerant potato hybrid without significant alterations in 1 − qP. This observation supports our hypothesis related to gene regulation via photosynthesis during frost. As suggested by Foyer & Noctor 2000) the redox state of H2O2 scavenging molecules such as ascorbate and glutathione is another important signal for stress gene expression. As both ScgstF1 and Scdhn2 transcript accumulation was induced by H2O2, this suggests that H2O2 may serve as a signalling molecule during frost. Whether H2O2 accumulates during night frost treatments needs further investigation.
Photosynthetic responses to night frosts
In these experiments, the reduction state of photosynthesis was modulated by temperature during the frost treatments. In the potato hybrid (S. commersonii (×) S. tuberosum cv. SPV11), freezing at −3 °C and following exposure to high intensity light resulted in a reversible increase in the excitation pressure of PSII, estimated as 1 − qP. If the freezing temperature was increased to −1 °C, 1 − qP remained unchanged. The milder freezing temperature than −3 °C resulted in higher qNP. One mechanism to dissipate excess light energy and prevent over-reduction of plastoquinone (Q) is qNP (Demmig-Adams & Adams 1996; Anderson, Park & Chen 1997; Gilmore 1997). In the potato hybrid, the degree of qNP was affected by freezing temperature indicating that these scavenging mechanisms are temperature sensitive. The data also suggest that failure in qNP can result in a transient increase in the excitation pressure of PSII and accumulation of defence gene transcripts in the freezing-tolerant genotype.
The value of 1 − qP correlated with the freezing tolerance of the potato genotype. In the freezing-sensitive cultivated potato, PSII appeared inactive after freezing to −3 °C. This result is in accordance with observations on rice and barley where 1 − qP was found to be an important parameter reflecting susceptibility to chilling-induced photo-inhibition (Xu, Ah Jeon & Lee 1999). According to Huner et al. (1998) any process which can decrease the over-reduction of Q can influence the redox state of photosynthesis. Furthermore, in winter wheat an increased resistance to photo-inhibition correlated with increased capacity to keep Q oxidized. Similarly in tomato, Q was more oxidized during low-temperature stress in the more chilling-tolerant wild tomato Lycopersicon peruvianum (Mill) (Brüggemann et al. 1995). The freezing-tolerant wild potato species which are able to cold acclimate, are also more tolerant to low temperature photo-inhibition (Steffen & Palta 1986, 1989; Kristjandottir & Merker 1993; Seppänen et al. 2001). The results indicate that improved capacity to keep Q oxidized after night frost is one of the tolerance mechanisms missing in S. tuberosum species which are unable to cold acclimate.
Experiments using all S1 potato populations showed that photosynthesis of all genotypes was able to recover from the severe frost treatment independent on their freezing tolerance. The recovery was accompanied with accumulation of ScgstF1 and Scdhn2 transcripts. In addition, the transcript accumulation correlated with changes in Fv/Fm parameters so that the highest levels were detected simultaneously with the highest reduction of Fv/Fm. Under these stress conditions, the cultivated potato Pito suffered irreversible damage to photosynthesis. Although the freezing temperature was elevated above the calculated LT50 value of the genotype, only some transcript accumulation was observed. This observation indicates that there may be differences in the regulation of cold-inducible genes and photo-inhibition tolerance between S. tuberosum genotypes. The small, but detectable, capacity of S. tuberosum cv. SPV11 to cold acclimate that was measured in earlier studies may explain this observation (Seppänen & Fagerstedt 2000). Thus, even a slight capacity to cold acclimate can protect photosynthesis from irreversible frost damage and affect expression of cold-inducible genes. The characterization of missing elements in the cold acclimation signal transduction pathway in Pito would be one approach to improve low-temperature tolerance in cultivated potato.
Role of hydrogen peroxide
Hydrogen peroxide is involved in increasing number of plant stress responses. Analysis of Arabidopsis transcriptome by cDNA microarray technology revealed expressed sequence tags (ESTs) for GST (GST6), dehydrin (rab28) and numerous heat shock proteins that were regulated by H2O2 (Desikan et al. 2001). Accumulation of H2O2 is also a well-known consequence of exposure to excess light energy (Karpinski et al. 1997, 1999) and could be a potential signal molecule during frost stress in potato. Pre-treatment with H2O2 improved freezing tolerance of S. commersonii which correlated with transcript accumulation of ScgstF1, Schsc70 and Scdhn2. In maize and tomato it has been shown that H2O2 can activate chilling acclimation (Prasad et al. 1994; Kerdnaimongkol et al. 1997) whereas in S. tuberosum microplants H2O2 was implicated in heat acclimation (Lopez-Delgado et al. 1998). Our results expand these observations to freezing stress in potato. Pre-treatment with H2O2 activated cold acclimation processes in S. commersonii resulting in improved freezing tolerance and altered membrane integrity.
Hydrogen peroxide is a signal for several GSTs but to our knowledge only maize GST, Bz2, is also regulated by low temperature (Christie, Alfenito & Walbot 1994). In contrast, dehydrin transcript accumulation is not usually associated with oxidative stress but rather with dehydration and stress signals such as abscisic acid (ABA) (Close 1996). However, evidence in Vicia faba guard cells suggests that both ABA and H2O2 can be a part of same signal transduction pathway and H2O2 may be an intermediate in ABA signalling (Zhang et al. 2001). Similar to freezing and chilling, heat shock causes H2O2 accumulation in plant cells and is therefore a potential signal for heat shock protein family members (Dat et al. 1998; Desikan et al. 2001). Interestingly, in both cold-acclimated and H2O2-treated plants, Schsc70 transcript was more abundant in freezing-sensitive genotype S. tuberosum cv. Pito whereas ScgstF1 and Scdhn2 transcripts were more abundant in the freezing-tolerant genotype S. commersonii. It can be hypothesized that the signalling pathways by low temperature and H2O2 overlap. Thus, S. tuberosum is deficient or less efficient in transducing signals caused by low temperature or H2O2. To some extent this observation can explain the lack of adaptation responses, such as capacity to cold acclimate, in S. tuberosum.
The Finnish Cultural foundation, University of Helsinki and the Finnish Academy are gratefully acknowledged for the funding of this research. Ms Eerika Karli, Lilia Sarelainen and Saija Perälä are acknowledged for their excellent technical assistance.
Received 12 January 2002; received in revised form 1 July 2002; accepted for publication 27 August 2002