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Published online 27 May 2005
Published in Crop Sci 45:1379-1386 (2005)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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CROP PHYSIOLOGY & METABOLISM

Root Temperature and Aeration Effects on the Protein Profile of Canola Leaves

Jennifer A. Franklina,*, Nat N. V. Kavb, William Yajimab and David M. Reidc

a Dep. of Forestry, Wildlife and Fisheries, 274 Ellington Plant Sciences Bldg., Univ. of Tennessee, Knoxville, TN 37996-4563
b Dep. of Agricultural, Food and Nutritional Science, Univ. of Alberta, Edmonton, AB, Canada T6G 2P5
c Dep. of Biological Sciences, Univ. of Calgary, Calgary, AB, Canada T2N 1N6

* Corresponding author (jafranklin{at}utk.edu)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Canola (Brassica napus L.) is planted in early spring and must survive both low soil temperatures and periods of wet weather. Shoot effects result from both low root temperature and low aeration, but little is known about the interaction between these two environmental factors, particularly with respect to changes in gene products. In this study, canola plants (‘46A65’) were treated in solution culture. Shoot temperatures were maintained at day/night temperatures of 24/18°C, while roots were maintained at an ambient temperature of 24/18°C, or cooled to 10°C. Roots were either aerated, or not aerated to create hypoxic treatments. Plants with roots in cold and hypoxic solution accumulated starch in the root, and had greater reductions in fresh weight and leaf area than those in either cold or hypoxic treatments alone. Twenty-one changes in protein expression were also found in the cold hypoxic treatment, 17 of which were not found in either cold or hypoxic treatments alone. Gene products up-regulated in leaves included cytochrome oxidase Subunit I (COX1) in response to hypoxia, elongation factor eEF1 {gamma} chain in plants with cooled roots, and chaperonin 10 when roots were cooled without aeration. Results demonstrate the interaction between multiple stresses on a molecular level, and suggest that flooding under cool soil temperatures will be more detrimental to canola than that which occurs at warmer temperatures.

Abbreviations: ABA, abscisic acid • COX1, cytochrome oxidase Subunit I • DTT, dithiothreitol • IEF, isoelectric focusing • IPG, immobilized pH gradient • MS/MS, tandem mass spectrum • SDS, sodium dodecyl sulfate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SPRING CANOLA is planted as early as possible in the spring to minimize periods of heat and drought during the sensitive stages of flowering and seed-filling, and plantings after 15 May generally produce decreased yields. When planted early, low soil temperatures delay germination and inhibit water and nutrient uptake. But seedlings are cold-tolerant, and can survive temperatures as low as –5°C. The period of highest yearly rainfall occurs during May and June in much of the Canadian canola-growing region (Environment Canada, 2003), and may lead to transient flooding of young plants. It is, therefore, necessary to understand the interaction between soil temperature and flooding, and their effects on the early growth of canola.

Often, the plant shoot may be growing under one set of environmental conditions, while the root is growing under a very different set of conditions. In the case of low soil aeration, or low soil temperature, the shoot growing under optimum conditions may yet show a notable response such as visible injury or reduced growth. Previous studies of grasses and legumes have found greater injury resulting from flooding at high soil temperatures than at low temperatures (Huang, 2000). Shoot responses resulting from root hypoxia are potentially the result of changes in water relations (Jackson et al., 1996), carbohydrate relations with the root, or chemical or hormonal message (Armstrong et al., 1994; Reid and Wample, 1985). Changes in the shoot as a result of root temperature are often reported in terms of growth and yield, but temperature is also known to alter water and nutrient uptake (Bowen, 1991), and there is evidence of chemical signaling under low root temperature conditions (Atkin et al., 1973). Protein changes in tissues subjected to stress have been well characterized for temperature (Guy, 1999) and somewhat studied for hypoxia (Baxter-Burrell et al., 2003), but relatively little is known of changes in shoot proteins in response to these root stresses, or of their interaction on gene products.

This study was undertaken to determine the relationship of root temperature and hypoxia in canola. Growth parameters, pigment concentration, transpiration rates, and nonstructural carbohydrate concentrations were measured in plants at a constant root temperature of 10°C or ambient temperature of 24/18°C day/night, under aerated or hypoxic conditions. To characterize the similarities and possible interactions between temperature and flooding treatments, proteomic changes in the shoots were determined and selected proteins were identified by mass spectroscopy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material and Treatments
Beginning 10 Sept. 2002, seeds of canola (46A65, Pioneer Hi-Bred Ltd, Chatham, ON) were germinated at 24/18°C (day/night) in an environmental chamber in a mixture of coarse sand and fine vermiculite (2:1 volume) which retained moisture but easily washed free of the roots. Fourteen days later, seedlings were transferred to 10-cm-diam. pots, one plant per pot, filled with the above mixture. Plants were grown in a controlled environment chamber, with a temperature of 24/18°C (day/night), relative humidity of 32 ± 5%, and an 18-h photoperiod with a photosynthetically active radiation of 275 ± 10 µmol m–2 s–1 provided by cool white fluorescent lights.

Twenty-eight days after sowing, the roots were gently washed free of the substrate, and placed in full strength Hoagland's solution (Hoagland and Arnon, 1948) aerated by an Optima aquarium pump (model 807, Rolf C. Hagen, Inc., Montreal, QC, Canada). Plants remained in aerated room temperature nutrient solution for 3 d before the imposition of treatment to allow roots to recover. One plant was placed in each of 24 individual 300-mL containers of nutrient solution that were then placed into one of two water baths. Using a split-plot design, half of the containers in each bath were aerated as described above. The water temperature in one bath was then lowered to 10°C during a period of 48 h (Days 1 and 2). Water temperature was regulated by means of a copper coil at the bottom of the container through which water was pumped from a refrigerated circulating bath (Model D1, Thermo Haake, Karlsruhe, Germany). In control containers, water temperature remained the same as air temperature (24/18 ± 1°C). The nutrient solution was changed after 1 wk, and solution levels were maintained as needed by the addition of deionized water. Throughout the experiment all plants were grown in the same environmental chamber, and the experiment was repeated with sowing dates of 24 Dec. 2002 and 14 Jan. 2003.

Physiological Measurements
Transpiration was measured between 1200 and 1400 on the uppermost fully expanded leaf, using a steady state porometer (LI-1600, Li-Cor Inc., Lincoln, NE). Twelve days after the beginning of the treatment period plants were harvested, measured, and weighed. Leaf area was measured using a {Delta}T area meter. One half of the root and shoot tissue was immediately frozen in liquid nitrogen, and stored at –70°C until protein and pigment analysis. The remaining tissue was oven-dried at 50°C for 72 h, weighed to determine water content, and then analyzed for nonstructural carbohydrates. Soluble sugar content of ethanol extracts was determined using the anthrone method (Ashwell, 1957). The extracted tissue was then analyzed for starch content by enzyme conversion of starches to sugars (Hendrix, 1993), and analysis of sugars using the anthrone method. Chlorophyll a and b concentrations were measured by spectrophotometric analysis of 80% acetone extracts, and calculated using the equations of MacKinney (Sestak et al., 1971). Total carotenoid concentration was determined in acetone extracts as described by Davies (1976). A general linear model with repeated measures was used to analyze transpiration data, and all other data were analyzed using a linear mixed model (SPSS, 2002).

Protein Extraction
The shoot tissue from the control and treated canola plants were ground in liquid nitrogen to a fine powder. One tenth of a gram of the ground tissue was resuspended with 1 mL ice-cold 10% (w/v) trichloroacetic acid (TCA), 0.07% (w/v) dithiothreitol (DTT)–acetone solution, after which the samples were incubated at –20°C for 1 h. The samples were then centrifuged in a Sorvall RC-5B Refrigerated Superspeed Centrifuge with a Sorvall SA-600 rotor (Mandel Scientific Co., Ltd., Guelph, ON, Canada) at 24460 x g for 10 min. The supernatants were discarded and the pellets were washed with 1 mL ice-cold 0.07% (w/v) DTT–acetone solution and incubated at –20°C for 1 h to remove excess TCA. The samples were centrifuged as before and the wash step was repeated. Following the final wash step, the pellets, which contained the precipitated proteins, were vacuum-dried for 30 min and then were resolubilized with 0.5 mL rehydration/sample buffer which contained 8 M urea, 2% 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate, 40 mM DTT, 0.2% Bio-Lyte 3/10 (ampholytes), and 2 mM tributylphosphine (TBP). The samples were mixed thoroughly and then incubated at 4°C overnight, after which they were centrifuged as before. The supernatants were collected and the protein concentrations of the protein extracts were determined using a modified Bradford assay with bovine serum albumin as the protein standard. The protein extraction was repeated using a different set of canola plants to ensure that any observed proteomic changes were reproducible.

Two-Dimensional Electrophoresis Visualization of Protein Spots
The protein extract solutions were used to rehydrate passively 7 cm, pH 3 to 10 nonlinear, immobilized pH gradient (IPG) strips overnight in a final volume of 125 µL containing 75 µg protein per strip. The rehydrated strips were transferred to the 7-cm focusing tray and submerged in mineral oil to prevent dehydration of the strips during the isoelectric focusing (IEF) step. The provided interlocking lid was placed on top of the IPG strips and once the focusing tray was securely positioned within the PROTEAN IEF Cell (Bio-Rad Laboratories, Hercules, CA), the isoelectric focusing of the proteins was commenced using a preprogrammed four-step method. The first steps consisted of a conditioning step, where 250 V was applied to the strips for 15 min to remove undesirable salt ions and charged contaminants, and a voltage ramping step, where the applied voltage was increased in a linear fashion during 2 h to reach the required 4000 V. The focusing of the proteins was completed at 4000 V for 20000 V-h, after which the voltage was rapidly decreased to and maintained at 500 V. Upon completion of the IEF protocol, the strips were placed gel-side up in rehydration/equilibration trays and stored at –20°C overnight. The IPG strips were allowed to thaw at room temperature before being equilibrated in preparation for the second dimension separation. Initially, the strips were submerged twice for 10 min each in the first equilibration buffer, which contained 6 M urea, 2% sodium dodecyl sulfate (SDS), 0.375 M Tris-HCl, pH 8.8, 20% glycerol, and 130 mM DTT. The strips were then saturated twice for 10 min each in a second equilibration buffer that differed from the first only by the fact that 135 mM iodoacetamide was present instead of 130 mM DTT. The equilibrated strips were embedded on top of 1 mm 13% SDS–polyacrylamide gel electrophoresis gels and then immobilized in place with molten agarose. The subsequent electrophoresis step was completed with a Mini-PROTEAN 3 system (Bio-Rad Laboratories, Hercules, CA) set for 80 min at 150 V. Following electrophoresis, the gels were submerged in a 0.05% (w/v) Coomassie Blue R250 (Sigma-Aldrich, Oakville, ON, Canada) staining solution containing 50% (v/v) methanol and 10% (v/v) glacial acetic acid, for 45 min with gentle agitation. The gels were then destained overnight in 5% (v/v) methanol, 10% (v/v) glacial acetic acid to remove excess Coomassie blue dye (Sigma-Aldrich, Oakville, ON, Canada), and then silver stained using the Silver Stain Plus kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions.

Image Analysis and Mass Spectrometry
The double-stained gels were scanned using the GS-800 Calibrated Densitometer (Bio-Rad Laboratories, Hercules, CA) to create images that could be compared using the PDQuest 2D analysis software (Bio-Rad Laboratories, Hercules, CA). This software facilitated the automated detection and matching of protein spots between selected gels as well as the determination of differences in the densities of corresponding spots. However, manual correction of the results produced by the automated functions was sometimes necessitated by the fact that the software misidentified or mismatched protein spots. Following the comparisons of the gel images, protein spots that showed significant and reproducible changes after exposure to the various stresses studied were noted and selected protein spots were excised from the gels with sterile surgical blades and then analyzed by electrospray ionization-quadrupole-time of flight mass spectrometry at the Institute for Biomolecular Design, University of Alberta.

The protein spots were prepared using a MassPREP Station (Micromass, Manchester, UK), which produced peptide samples that were destained, reduced, alkylated, and trypsin-digested. Liquid chromatography/Mass Spectrometry/Mass Spectrometry analysis was performed using a capillary HPLC system (CapLC system, Waters Corporation, Milford, MA) which used a PepMap C18 column (LC Packings, Sunnyvale, CA) for loading and desalting in conjunction with a PicoFrit capillary reversed-phase column (New Objectives, Inc., Cambridge, MA) for the separation of the peptides, coupled to a Quadrupole Time of Flight 2 mass spectrometer (Micromass, Manchester, UK). The tandem mass spectrum (MS/MS) data were used to search protein databases using the Mascot search engine (Matrix Science, Inc., Boston, MA), which uses the following sequence databases: dbEST, MSDB, NCBInr, OWL, and Swiss-Prot, to determine the expected identities of the corresponding protein spots.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
By the end of the treatment period, the pH in all treatments was lower than in the control (Table 1). Oxygen content of the nonaerated treatment solutions was reduced, but not anoxic. Neither tissue water contents, nor leaf pigment concentrations, were significantly altered by 10 d of treatment. Shoot fresh weight and leaf area were significantly reduced by cold temperatures, but the effect of aeration within temperature was not significant at {alpha} = 0.05 (Fig. 1). Root fresh weight was significantly reduced by lack of aeration at both temperatures, although the temperature effect was not significant. The effects of hypoxia under cold temperatures were generally greater than those of temperature alone. By day three, 24 h after the final treatment temperature had been reached, transpiration rates of plants in the cold treatments had decreased significantly, and remained so for the remainder of the experiment (Fig. 2). The effect of aeration on transpiration rate was insignificant. Soluble sugars increased in leaves and roots of all treated plants, with sugar content in those receiving the cold hypoxic treatment being four times that of controls (Fig. 3a). The effect of temperature on sugars was significant for both roots and leaves, while the effect of aeration was significant only in roots. Starch content was not significantly affected by treatment (Fig. 3b).


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  		Table 1. Mean pH and oxygen content of treatment solutions after 10 d of treatment, tissue water content, and concentrations of chlorophylls a and b, and total carotenoids in leaves of canola plants with roots under cold or hypoxic conditions. Standard errors are shown in parentheses. Means of water content and pigment concentrations do not differ significantly between treatments. n = 12.

 


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  		Fig. 1. (a) Shoot and root fresh weights and (b) leaf area of canola plants treated for 12 d with roots aerated at 24/18°C (day/night; control) or 10/10°C (cold), or not aerated at 24/18°C (hypoxic) or 10/10°C (cold + hypoxic). Standard errors are indicated. n = 16.

 


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  		Fig. 2. Midday transpiration rates of canola plants treated for 12 d with roots aerated at 10/10°C (day/night), or nonaerated at 24/18 or 10/10°C. Upper and lower limits of control (24/18°C) 95% confidence interval are shown. n = 12.

 


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  		Fig. 3. Shoot and root concentrations of (a) total soluble sugars and (b) starch of canola plants treated for 12 d with roots aerated at 24/18°C (day/night; control) or 10/10°C (cold), or not aerated at 24/18°C (hypoxic) or 10/10°C (cold + hypoxic). Standard errors are indicated. n = 14.

 
Silver-stained images of two-dimensional gels used to separate proteins extracted from the shoots of control canola plants and those whose roots were subjected to hypoxia, cold temperature, and a combination of hypoxia and cold temperatures are shown in Fig. 4. Overall, the two-dimensional profile of the proteins extracted from these shoot tissues were quite similar. Exceptions are those spots indicated by arrows and numbers which were altered reproducibly in the treatments when compared with the control gel and are summarized in Table 2.



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  		Fig. 4. Sodium dodecyl sulfate (13%)–polyacrylamide gel electrophoresis gel images showing the protein profiles of (A) the control, and (B, C, D) the treated (hypoxic, cold, and hypoxic + cold, respectively) canola samples. The numbers indicate the protein spots that were reproducibly affected by exposure to the stresses.

 

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  		Table 2. Summary of proteomic changes resulting from exposure to stress.

 
Hypoxia alone downregulated one spot (Spot 1, indicated in Fig. 4A) and up-regulated three others (Spots 2–4, Fig. 4B), whereas cold treatment by itself up-regulated four spots (Spots 5–8, Fig. 4C). When hypoxia was combined with the cold treatment, Spots 1 to 3 exhibited a similar trend as observed with hypoxia alone (Spots 15, 17, and 19, Fig. 4A and 4D) and Spots 7 and 8 (Fig. 4C) exhibited a trend similar to cold treatment by itself (Spots 28 and 29, Fig. 4D). In addition to the five spots described above that were common to all the three treatments, the combination of hypoxia and cold treatment also affected the levels of 16 other spots with six of them being downregulated and the remaining up-regulated (Table 2). The identities of selected spots that showed reproducible changes in their levels were established by ESI-Q-TOF-MS/MS analysis and are shown in Table 3 along with the sequence information that was generated from the MS/MS data.


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  		Table 3. Summary of the identities of selected proteins.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Hypoxia
Effects on plants that are commonly reported in response to flooding include reduced root and shoot growth, reduced leaf area, chlorosis, reduced water uptake and leaf water content, and an increased sugar accumulation in roots (Huang, 2000). Our results show reductions in shoot and root fresh weight, leaf area, and transpiration rate in response to hypoxia. Reduced leaf area as a result of root hypoxia may result from the accumulation of ethylene (Lee and Reid, 1997), or reduced cell-wall extensibility in the leaves (Smit et al., 1989). Increased leaf abscisic acid (ABA) concentrations under root hypoxia may also be responsible for reductions in leaf area (Else et al., 1996) and transpiration rate (Armstrong et al., 1994). Ethanol has been shown to be translocated from flooded roots to shoots of sunflower (Helianthus annuus L. cv. Delgren 131) (Fulton and Erickson, 1964; Jayasekera et al., 1990) and, along with ethylene and ABA, may act as a long-distance signaling mechanism.

The survival of roots under hypoxia is reliant on the supply of carbohydrates, and reductions in both photosynthesis and translocation in flooded plants may limit carbohydrate availability to roots (Huang, 2000). Such limitations are evident in flood-intolerant white oak (Quercus alba L.) in which an increase in leaf starch concentration and a decrease in root starch concentration was found after 32 d of flooding (Gravatt and Kirby, 1998). Our results show no evidence of reduced carbohydrate availability in canola roots after 12 d of flooding. Of the four changes in proteins occurring in leaves of hypoxic plants, one was identified as COX1 (Spot 4). While no effect of hypoxia on the expression of COX1 has been previously reported in plants, COX1 mRNA has been found to increase in the cerebral cortex of rats exposed to hypoxic conditions (Tan et al., 2002). In response to hypoxia, cytochrome oxidase activity has been found to decrease in roots of flood-intolerant, but not flood-tolerant, species (Pearson and Havill, 1988).

Root Temperature
Many low-temperature-responsive genes and proteins have been described in plants and have been reviewed by Guy (1999). However, unlike other studies of cold-induced changes in proteins, we have analyzed leaf tissue that was not directly exposed to cold, but was exposed to the altered water and carbohydrate relations induced by root cooling, and to any chemical signals that may have been transported from the root. Root growth generally decreases with decreasing soil temperature in the 5 to 30°C range, and a reduced shoot growth and photosynthetic rate may be a result of reduced water and nutrient uptake at low soil temperatures (Bowen, 1991). The lack of a temperature effect on root growth in this study may be related to the relative cold tolerance of canola. Similar to our findings, a root temperature of 15°C reduced the relative growth rate of tomato (Lycopersicon esculentum Mill.) shoots compared to plants with roots at 25°C, when shoots were maintained at 25°C, although this study found little impact on tissue carbohydrate concentrations (Durenkamp et al., 2000). Leaves that were visibly smaller and fewer in the 10°C treatments accounted for the reduction in leaf area. A greatly reduced translocation of cytokinin and gibberellin has been found at low root temperatures (Atkin et al., 1973) and may play a role in growth inhibition. There is also growing evidence that ABA is involved in cold-temperature signaling (Tahtiharju et al., 2001), and so may also be a means of long-distance root-to-shoot signaling in plants with cooled root systems.

Acclimation to cold temperatures involves an increase in total solutes. The increase in soluble sugars in both roots and shoots in response to cold is likely related to osmotic adjustment. Acidification of the nutrient solution was observed under both cold and hypoxic conditions and suggests an increased activity of proton pumps that would lead to increased uptake of inorganic nutrients. An increased synthesis of compatible solutes such as proline is well documented. The expression of many regulatory proteins in plants is altered by cold temperatures (Guy, 1999), and there are many reports of increased levels of elongation factors associated with low temperatures in microorganisms. The expression of elongation factor 1Bbeta has been found to be induced in barley (Hordeum vulgare L.) exposed to cold temperatures (Baldi et al., 2001). We found an up-regulation of elongation factor eEF1 {gamma} chain (Spot 8) in leaves of plants with cooled roots, suggesting that it is regulated through hydraulic or chemical signaling rather than by the signal-transduction pathway proposed by Dhindsa et al. (1998) for some cold acclimation responses.

Hypoxia under Cold Temperatures
We found an additive response of flooding and low temperature on growth, carbohydrate concentrations, and protein expression. The four-fold increase in soluble sugars in shoots and roots may be the result of a reduced rate of translocation from shoot to root, as has been reported for bean (Phaseolus vulgaris L.) (Schumacher and Smucker, 1985). Accumulation of sugar in the roots of treated plants could result from a root respiration rate depressed by low temperatures, an adaptive response to cold or hypoxia, or simply the result of a reduced growth rate. However, a depression of root respiration cannot be inferred from our data. Flooding has been shown to increase soluble sugar content in roots and starch in leaves of wax apple [Syzygium samarangense (Blume) Merr. & L.M. Perry] plants flooded for 14 d (Hsu et al., 1999). No temperature and flooding interaction was noted by Vu and Yelenosky (1992), who found an increased concentration of starch in leaves and decreased starch in roots of flooded citrus trees (Citrus jambhiri Lush. and C. amara Link) with little difference in response between plants grown at 10 and 30°C.

In wheat (Triticum aestivum L.) (Trought and Drew, 1982), red fescue (Festuca rubra L.) (Beard, 1970), and alfalfa (Medicago sativa L.) (Meek et al., 1986), an interaction between flooding and soil temperature has been demonstrated with flooding injury increasing as temperatures increase. Because root respiration is often reduced under lower temperatures, less injury to flooded plants may occur under cold temperatures simply due to a lower metabolic rate and delay in root mortality. However, in a growth chamber study similar to our own, where shoot and root temperatures were individually controlled, there was a greater reduction in shoot weight in flooded alfalfa at a root temperature of 13 than at 25°C, and a high survival rate after 14 d (Heinrichs, 1972). This study also demonstrated that the response is species specific, as flooded sainfoin (Onobrychis viciifolia Scop.) showed little growth response to temperature, but a high degree of root decay and mortality that decreased with decreasing temperature. In contrast to our results, these flooded legumes had a visible yellowing of leaves after 14 d. Reductions in chlorophyll content have also been reported for flooded tobacco (Nicotiana tabacum L.) (Hurng and Kao, 1993) and tomato (Railton and Reid, 1973). It is possible that canola is able to avoid losses of pigmentation through an increased production of components of the photosynthetic system. We identified two such components, the Photosystem II oxygen-evolving complex protein 2 precursor (Spot 17) that, interestingly, was also up-regulated under hypoxic conditions alone (Spot 2), and Photosystem I reaction center Subunit II chloroplast precursor (Spot 27), to be up-regulated in response to hypoxia at low temperatures.

Members of the chaperonin family have been shown to increase in response to heat shock and oxidative stresses and aid in protein folding (Nishimura, 1998). Low temperatures have been found to alter expression of several molecular chaperones in plants (Guy, 1999), including an increase in hsp60 mRNA in all tissues of canola exposed to temperatures of 5°C (Krishna et al., 1995). Chaperonin 10 (Spot 10), found to be up-regulated in leaves of plants subjected to cool roots and hypoxia, is a calmodulin-binding protein (Yang and Poovaiah, 2000) and so may be involved in cold-signaling, as importance of calcium in cold sensing and signaling is well known (Lee et al., 2002; Monroy and Dhinsda, 1995).

In the case of canola, spring flooding at cold soil temperatures could result in a greater growth reduction than would flooding at warmer temperatures. Aside from growth inhibition, however, canola was quite tolerant of both flooding and low root temperatures. The large number of proteins up-regulated by treatments, and the identity of some of these, suggest that plants acclimated to these treatments. Plants did not exhibit any chlorosis or depletion of carbohydrate reserves that would have a lasting impact on growth after release from the stress. However, plant recovery was not tested, and oxidative stress resulting from the alleviation of flooding could result in damage. It is interesting to find a large number of proteins whose expression in the leaves was altered by stress imposed not to the leaves directly, but to the root system.


   ACKNOWLEDGMENTS
 
Financial assistance from the Natural Sciences and Engineering Research Council of Canada and the University of Alberta are gratefully acknowledged.

Received for publication June 10, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 


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Crop Science 2005 45: vii. [Full Text]  




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