Abstract

Phenylpropanoid metabolism is known to increase in canola upon exposure of the plants to subzero temperatures. Normally, low temperatures result in an increase in the level of oxygen radical formation in plant tissues causing damage to DNA and proteins, which adversely effects the ability of the plant to survive winter/spring conditions. Phenolics produced by the phenylpropanoid pathway are powerful antioxidants in plant tissues. These compounds also serve as precursors to suberins and lignins which act as physical protectants for plants against cold temperatures and freeze/thaw conditions associated with spring conditions in Canada and Europe. We have successfully utilized the doubled haploid /UV mutagenesis system coupled with various chemical selection methods to isolate plants with superior phenylpropanoid metabolism. Plants that have been identified with this trait will be subjected to simulated winter/spring conditions to determine survival rates. Plants with superior winter/spring tolerance will be subjected to field testing in various locations in Canada. Increased cold tolerance should allow for the reintroduction of winter canola to Canadian growers as well as improved varieties for the world market.

Introduction

Subzero winter temperatures and waterlogged spring soils are the primary factors that negatively influence survival of winter canola (Brassica napus) in Canada. Superior yield over spring cultivars makes winter canola the preferred commodity for producers; however limited survivability severely reduces the geographic regions where it can be grown economically. In order to develop high yielding winter hardy cultivars for Canadian producers, it is essential that we understand the biochemical responses to these environmental conditions at the cellular level during osmotic stress and the extreme temperature fluctuations of late winter and early spring.

It is widely accepted that cold acclimation and freezing tolerance is a three step process in herbaceous winter annual plants (Kacperska-Palacz 1978; Kulesza et al., 1986; Kacperska, 1989; Solecka and Kacperska 1995). Plants are subjected to near freezing temperatures (>0^oC), then subzero temperatures and finally the freeze thaw cycles that occur with the approach of spring. Frost tolerance in many species of plants has been attributed to an increase in the cell wall lignification or suberization (Chalker-Scott and Fuchigami 1989). Suberins are large molecules associated with the cell wall composed of phenolics and sugars providing a protective layer which normally is associated with wounding or infection. Several groups have discovered that increased suberization and lignification of cell walls provides an increase in frost tolerance commonly observed with conditions in late winter and early spring. Solecka and Kacperska have proposed that increased levels of specific phenolics may be involved in the response of the plant to cold temperature stress. This group has also reported that levels of Phenylalanine ammonia-lyase (PAL) increase rapidly after a laboratory freeze/thaw treatment.

Phenolic levels have been altered in tissue culture using inhibitors of PAL and or proline analogues such as hydroxyproline (Yang and Shetty, 1998). Proline metabolism is linked to phenolic metabolism by providing the precursors for the production of phenylalanine, the first substrate in phenylpropanoid biosynthesis (Hare and Cress, 1997). We have proposed to increase the ability of the plant to tolerate not only cold stress, but also freezing tolerance and osmotic stress, conditions normally associated with winter and spring survival. Using the doubled haploid system and UV mutagenesis coupled with in vitro chemical selection with inhibitors of PAL and proline biosynthesis, we have successfully selected plants with increased phenolic levels specifically in leaf and stem tissues. These plants and the role of phenolics, proline and winter survival are described in this paper.

Experimental

Plant Material

As a starting point, we prescreened 47 winter genotypes from harsh climatic zones at the Elora, Ontario Research Station for winter survival in 1996-1997. Winter hardiness was scored by a scale of 1-5 on root injury (i.e. 1= firm, undamaged, with new roots; 5= rotted dead root) and 1-5 on cold injury (i.e. 1= no injury; 5= dead plant) (Schwab et al., 1996). Individuals that survived the field test were grown indoors at the University of Guelph and donor plants were used for doubled haploid extraction and mutagenesis (Fletcher et al., 1998).

Doubled Haploid Extraction, Mutagenesis and Chemical Selection

Donor plants were grown indoors for the first 3 weeks in flats of 72 under 16 hour days at 20°C days and 18°C nights. After this initial period plants were vernalized for 8 weeks at 4°C under 8 hour days and 16 hour nights. Plants were removed from vernalization to under 16 hour days at 20°C days and 18^oC night conditions and were not fertilized for the first 7 days. Once plants began to elongate, they were transferred to 15 cm pots. After the first flowers opened, racemes were collected for microspore isolation (Fletcher et al., 1998). Hydroxyproline was added to liquid media after isolation, and then the petri plates were irradiated with long wave UV light for a period of 90 seconds at a rate of 800 μW/cm². Survivors were processed via the normal doubled haploid procedure.

Chemical selection was also performed on solid media. After UV mutagenesis and normal embryo development, embryos at the 28 day stage are transferred to solid media containing hydroxyproline. Survivors are carried through the normal doubled haploid system.

Phenolic testing

Phenolic levels in leaves and seeds were tested using Folin and Ciocalteu’s reagent. Briefly, a 100 mg sample of leaf or seed is ground in 95% ethanol and allowed to extract for 48-72 hours. To 1 mL of sample, 1.5 mL of 95% ethanol and 5 mL of distilled water are added. To this mixture, 0.5 mL of 50% Folin’s reagent and 1 mL of 5% NaCO₃ are added and vortexed. The mixture was allowed to stand in the dark for 1 hour before reading each sample at 725 nm. Samples were quantitated using various concentrations of gallic acid as a standard curve.

Results

Phenolic levels in winter and spring canola

We tested various winter and spring lines for phenolic levels both prior to cold temperature treatment and after 4 days of exposure to 4°C (Table 1).

Table 1: Response of phenolic levels in leaf tissue to low temperatures

As illustrated in Table 1, phenolic levels in both spring and winters increase when exposed to low temperatures. Phenolic levels in winter varieties are higher than those found in springs, which may be an adaptation to surviving winter conditions. The highest phenolic levels were found in Debut, a Brassica rapa variety known to survive the harsh winter conditions of Eastern Europe. The line 509-02 is the result of in vitro mutagenesis and selection from another winter hardy variety from Eastern Europe selected solely for low glucosinolates. This result encouraged us to carry out mutagenesis and selection studies aimed at increasing the phenolic levels of winter canola for superior winter survival.

In vitro mutagenesis and selection

From our mutagenesis and chemical selection in liquid media with hydroxyproline, we produced a haploid plant that contained significantly higher phenolic levels than the parent. Phenolic levels in the parent were 1.41 mg/g FW without exposure to cold temperatures, whereas the mutagenized and hydroxyproline selected line contained 2.38 mg/g FW, an increase of 170%. This new line was characterized by slow growth, thick waxy leaves and a “woody” stem. This line has been chromosome doubled using colchicine and we are currently in the process of obtaining seed from this individual. Seed will be grown to the 3 week old stage and then plants subjected to a 2 week acclimation period at 4°C and then subjected laboratory freeze/thaw test at -15°C to evaluate freezing tolerance.

Seed Selection

Seeds from an Eastern European winter hardy variety (Otradnenskjii: open pollinated seed) were incubated in a solution of L-α-bromo-phenylalanine for a 24 hour period. This compound is a known analogue of phenylalanine (personal communication, Dr. C. Lanthier). This analogue was used to inhibit PAL activity and served as a selection agent to identify seed that could override this blocking agent. Seeds which germinated (600 of 2000) were allowed to grow for 3 weeks until subjected to an acclimation period for 2 weeks at 4^oC. Plants were then placed at -15°C for 7 hours, brought out to 4°C for 12 hours and then placed in normal conditions (20°C day, 18°C night, 16 hour day). Two plants out of the 600 survived the abrupt temperature conditions (0.33%). These plants have set seed and will be re-evaluated for cold tolerance in future experiments.

CONCLUSIONS

We have found that proline metabolism and phenylpropanoid metabolism are essential in the adaptation of plants to cold temperatures. Our in vitro selection method has proven effective at increasing phenolic levels and seed treatment with an inhibitor of PAL are useful tools in selecting winter canola with superior cold tolerance. Future studies will prove that these techniques will generate new winter and spring canola varieties with superior cold tolerance for the world marketplace.

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