Progress 07/01/02 to 06/30/08
Outputs
OUTPUTS: The study focuses on understanding the nature and mechanism of freezing tolerance in strawberry and developing methods to improve freezing tolerance in this crop. The components of cold acclimation have been characterized especially in relation to their contribution to freezing tolerance in strawberry. The molecular response to these components of cold acclimation has been characterized, including the activation of orthologs cold responsive genes. Several approaches involving modification of membranes were examined with the purpose of improving freezing tolerance in Arabidopsis and strawberry. The results have been shared with the scientific community and researchers through several forums including professional meetings and workshops, and through publications in journals and books. PARTICIPANTS: C. Rajashekar, PI; Myung Min Oh, graduate student and Manasa Panda, graduate student. TARGET AUDIENCES: Researchers, extension personnel, and growers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The two components of cold acclimation, namely water stress and low temperature, were identified during cold acclimation in strawberry. Plants acclimated fully at 3/1 C (day/night) with in 1 week to survive -21C. Water stress was an integral part of cold acclimation; water stress was induced readily when strawberry plants were exposed to low temperature. The leaf water potential decreased to approximately -1.6 MPa after two weeks of cold acclimation treatment. Water stress and low temperature can independently induce freezing tolerance in plants with water stress being a dominant factor. However, both these components act additively and are needed for maximum freezing tolerance. Cold acclimation in strawberry plants was associated with the activation of cold responsive genes, orthologs of COR, including FaCOR15a, FaCOR47, and FaCOR78. The two components of cold acclimation, water stress and low temperature, also activated the orthologs of COR, suggesting that pathways for induction of freezing tolerance by these factors are perhaps independent. To improve freezing tolerance of strawberry, a number of approaches involving medication of membrane properties were explored. Phospholipase D (PLD) is a major enzyme involved in breakdown of membrane phospholipids and is known to contribute to freezing injury in plants. Arabidopsis plants with antisense suppression of PLD gene were developed to study the role of PLD in their freezing injury. Suppression of PLD resulted in significant increase in freezing tolerance in both non-acclimated and acclimated plants. Exposing PLD-deficient plants to cold acclimating conditions resulted in a stronger expression of cold responsive genes including COR47 and COR78. Also, in response to cold acclimation, PLD-deficient plants accumulated a number of osmolytes such as raffinose, sucrose, glucose and proline more than did the wild-type plants. These results explain the superior freezing tolerance in PLD-deficient plants. Furthermore, PLD suppression appears to affect the growth and development of Arabidopsis plants, resulting in shorter plants but with larger leaf area and slightly early bolting. However in strawberry, we inhibited the activity of PLD by application of lysophosphatidylethanolamine (LPE). Similar to PLD suppression in Arabidopsis, the application of LPE resulted in significant increase in freezing tolerance of strawberry plants. Modifying membrane fluidity by treating strawberry plants with various alcohols resulted in improving their freezing tolerance. The results show that application of LPE and ethanol has potential practical application in improving freezing tolerance of strawberry crop under field conditions.
Publications
- Rajashekar, C.B., Zhou, Han-E, Wang, Y., Li, W., and Wang, X. 2006, Suppression of phospholipase Da1 in Arabidopsis: Response of cold-responsive genes and osmolyte accumulation, J. Plant Physiol. 163:916-926.
- Rajashekar, C.B. 2006, Molecular responses and mechanisms of plant adaptation to cold and freezing stress, In Plant Environmental Interactions, 3rd edition- B. Huang (Ed.), CRC-Taylor and Francis, Boca Raton, FL, p. 47-67.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The study focuses on understanding the nature and mechanism of freezing tolerance in strawberry and developing methods to improve freezing tolerance in this crop. The specific objectives of this study were: 1. Determine the role of various components involved in cold acclimation in strawberry, 2. Understand molecular response, especially cold responsive genes in contributing to freezing tolerance, 3. Compare the molecular responses in relation to low temperature and dehydration, both of which can increase freezing tolerance in strawberry, and 4. Examine the involvement of membranes in freezing tolerance and to develop strategies to improve freezing tolerance in strawberry by two approaches, a) by characterizing the role of phospholipase D, which breaks down membrane phospholipids, on the freezing tolerance of strawberry by use of inhibitor and anti-sense gene suppression, and b) by modifying the bilayer fluidity using various alcohols.
PARTICIPANTS: Myung Min Oh and Manasa Panda, Departmental Graduate Students
TARGET AUDIENCES: Researchers, extension personnel and growers.
Impacts
Water stress was induced when strawberry plants were exposed to low temperature (3/1 C; day/night) as part of cold acclimation treatment. The leaf water potential decreased to approximately -1.6 MPa after two weeks of cold acclimation treatment. Plants fully acclimate in one week, reaching a freezing tolerance of -21 C. The results indicate that both low temperature and the water stress contribute to the induction freezing tolerance in an additive fashion, with water stress being dominant. These two components can independently induce freezing tolerance, water stress can increase freezing tolerance by more than two-fold. However, for maximum freezing tolerance, both components are needed. In response to cold acclimation, many cold responsive genes (orthologs of COR), FaCOR15a, FaCOR47, and FaCOR78. These genes were induced within one hour of exposure to cold acclimation treatment. In response to low temperature (without water stress) the expression of both FaCOR47 and
FaCOR78 was strong compared to that of FaCOR15a. However, there was strong activation of all these genes by water stress. Anti-sense suppression of phospholipase D (PLD) in Arabidopsis resulted in activation cold responsive genes and in significant increase in freezing tolerance of plants. We inhibited the activity of PLD in strawberry by application of lysophosphatidylethanolamine (LPE), which resulted in significant increase in freezing tolerance of plants. Modifying membrane fluidity by treating strawberry plants with various alcohols resulted in improving their freezing tolerance. The results of this study show that application of LPE and ethanol has potential practical application in improving freezing tolerance of strawberry crop under field conditions.
Publications
- C. B. Rajashekar, Han-E Zhou, Y. Wang, W. Li and X. Wang, 2006, Suppression of phospholipase Da1 in Arabidopsis: Response of cold-responsive genes and osmolyte accumulation, J. Plant Physiol. 163:916-926.
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Progress 01/01/06 to 12/31/06
Outputs
Cold acclimation of strawberry plants (cv. Surecrop) by exposing them to low temperature at 3/10C (day/night) up to 2 weeks increased their cold tolerance to about -20C, a 3-fold increase over that in the control plants. Exposure of strawberry plants to cold induced gradual water stress resulting in an increase in their cold tolerance. Water stress and low temperatures are two integral parts of cold acclimation in that each can independently induce cold tolerance in strawberry plants, but complement each other during cold acclimation. Exposure of plants to mild water stress (-1.2 MPa) at 22C could partially substitute cold acclimation treatment. Water stressed strawberry plants without cold acclimation survived -14C. Thus, water stress was more effective in inducing cold tolerance than low temperatures, but low temperatures are needed to induce cold tolerance to the maximum level. Similar results have been observed in Arabidopsis plants. In response to low temperature
the leaves reached a water potential of -2 MPa after exposing the plants to 2C for 10 days. If water stress was prevented during cold acclimation, the plants did not acquire cold tolerance, indicating the importance of water stress during typical cold acclimation. In strawberry, both low temperatures and water stress activate orthologs of cold-responsive genes, namely FaCOR15a, FaCOR47 and FaCOR78. The membrane integrity is an important factor that favors plant survival against low temperatures and is typically compromised by membrane-destabilizing phospholipases, of which phospholipase D (PLD) is known to be involved in freezing injury of plants. In this study we treated plants with various levels of a PLD inhibitor, lysophosphotidylethanolamine (LPE), ranging in concentration from 50 to 200 mg/L. LPE at 100 mg/L was most effective in improving the cold tolerance of plants. Other treatments that improved the stability of membranes (alcohols) also increased the cold tolerance. These
treatments also activated the cold-responsive orthologs such as FaCOR15a, FaCOR47 and FaCOR78. The results suggest that factors such as typical cold acclimation and other treatments that stabilize membranes and induce cold tolerance appear to activate cold-responsive genes in strawberry.
Impacts
Studies are aimed at understanding the nature of cold acclimation and molecular responses in strawberry. The results show that a number of membrane-modifying methods are useful in increasing freezing tolerance in strawberry, which may help protect the crop against frost injury in early spring.
Publications
- C. B. Rajashekar, Han-E Zhou, Y. Wang, W. Li and X. Wang, 2006, Suppression of phospholipase Da1 in Arabidopsis: Response of cold-responsive genes and osmolyte accumulation, J. Plant Physiol. 163: 916-926
- X. Zhao, E. E. Carey, W. Wang and C. B. Rajashekar, 2006, Does organic production enhance phytochemical content of fruit and vegetables? Current knowledge and prospects for research, HortTech. 16: 449-456
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Progress 01/01/05 to 12/31/05
Outputs
Cold acclimation in strawberry is triggered by two distinct components, namely low temperatures and water stress. Typically, low temperatures induced water stress, with leaf water potential of -1.6 MPa after 2 weeks of cold acclimation. These two components independently can induce freezing tolerance in strawberry and water stress alone can play a dominant role in inducing freezing tolerance in strawberry, and was found to increase the freezing tolerance by more than two-fold over the control plants. The results show that both water stress and low temperatures complement each other and both are needed for full cold acclimation. Typically, water stress contributed to about 56% of the increase in freezing tolerance during cold acclimation while the remainder was due to the low temperatures. Although these tow components of cold acclimation act independently, they appear to produce similar response with regard to the activation of otrhologs of cold responsive
transcription factors, CBF1, CBF2 and CBF3, and COR genes. As membrane stability plays an important role in tolerance to freezing, we examined the effect of phospholipase D inhibitor, lysophosphatidylethanolamine (LPE) on the freezing tolerance of strawberry plants. Significant increase in freezing tolerance in strawberry plants (1.4C) was found by the foliar application of LPE (50mg/L). Similarly, various alcohols which are known to maintain membrane fluidity were tested for their effects on freezing tolerance. Some primary alcohols were effective in increasing freezing tolerance of strawberry plants. These membrane-modifying agents also activate a number of cold responsive genes in strawberry.
Impacts
Studies are aimed at understanding the nature of cold acclimation and molecular responses in strawberry. The results show that a number of membrane-modifying methods are useful in increasing freezing tolerance in strawberry, which may help protect the crop against frost injury in early spring.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Studies are underway to investigate the role of water stress in cold acclimation of strawberry and to develop methods to improve their freezing tolerance. Strawberry (Fragaria x ananassa, cv. Surecrop) plants were cold acclimated at 3/1C (day/night) with a 9 hour photoperiod for 3 weeks and were also acclimated under above conditions with 100 percent relative humidity and by keeping the soil at field capacity to prevent water stress. During cold acclimation, leaf water potential decreased to -1.24 MPa within 4 hours of cold treatment and decreased continuously over the cold acclimation period. However, there was no loss of water during cold acclimation of plants under 100 percent relative humidity. In response to cold acclimation, plants increased their freezing tolerance from -6.7 C to -20 C, however when water stress was prevented during cold acclimation, the increase in freezing tolerance was only to -11.6 C. These results suggest that water stress plays a key role
in inducing freezing tolerance in strawberry, and low temperature alone accounts for a small increase in freezing tolerance during cold acclimation. To determine if water stress alone can induce freezing tolerance, plants were water stressed at 22C for up to 10 days (leaf water potential of -2.5 MPa) and freezing tolerance was evaluated. Water stress without low temperature could increase the freezing tolerance to -14.5 C. Taken together, the results suggest that both low temperature and water stress are important during cold acclimation while the latter playing a greater role in inducing freezing tolerance. Expressions of orthologs of CBFs and cold responsive genes were studied in plants during cold acclimation with and without water stress. Although all the COR gene orthologs are expressed during cold acclimation under both conditions, they seem to be activated much later in response to cold acclimation without the water stress. The expression of cold responsive genes in response to
water stress was similar to that in response to cold acclimation. To improve freezing tolerance in strawberry, we used foliar sprays of lysophosphatidylethanolamine (LPE), which an inhibitor of phospholipase D that typically damages membranes during plant stress, and ethanol, a membrane fluidizer. Both treatments resulted in a significant increase freezing tolerance of strawberry plants. To explain the increase in freezing tolerance, expressions of orthologs of CBFs and cold responsive genes in response to LPE and ethanol treatments are being characterized.
Impacts
The study characterizes cold acclimation in strawberry plants and shows that water stress plays an important role during this process. Water stress appears to be an integral part of plants exposed to cold and is essential to induce freezing tolerance in strawberry. Water stress during cold acclimation plays a major part in inducing freezing tolerance. Strawberry plants can be made freezing tolerant by inducing water stress. The results show a great deal of similarities in molecular responses between water stress and cold. Use of approaches to modify membrane characteristics, such as use of LPE and ethanol, can increase freezing tolerance in strawberry, and is likely to have potential filed applications.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
The primary objective of this study was to examine the effects of blocking phospholipase D (PLD) and altering membrane stability on the freezing tolerance in strawberry. PLD is a catabolic enzyme that breaks down membrane phospholipids, which can affect the stability and integrity of membranes. Furthermore, the membrane stability is affected by one of the immediate products of PLD enzyme action, namely phosphatidic acid which typically favors non-bilayer formation. Therefore, in order to block the PLD activity in plants, they were treated with lysophophotidylethanolamine (LPE), a PLD inhibitor, at various concentrations. LPE at 100 mg/L was very effective in inducing freezing tolerance in strawberry plants. The freezing tolerance increased from -5C to -10C in non-acclimated plants. However, LPE did not have a significant effect at higher concentrations or in cold acclimated plants. We also used ethanol and butanol which are known affect the fluidity of membrane
lipids. Exogenous butanol gave inconsistent results while ethanol showed positive results. Both 1 and 2% ethanol applications significantly increased freezing tolerance similar to LPE application. Ethanol application can keep membrane in a fluid state even at low temperatures, which can reduce injury during freezing. During cold acclimation, a number of osmolytes such as proline, glycine betaine and sucrose accumulated in the leaves. As glycine betaine is metabolically stable, we used exogenously application in strawberry plants. A significant increase in freezing tolerance was observed with foliar application of 10 mM glycine betaine in unhardened plants. These studies are presently being conducted under field conditions.
Impacts
The primary objective is to develop strategies to improve freezing tolerance in strawberry. Exogenous applications of osmolytes, and substances which can improve the stability of membranes are being used to improve freezing tolerance in marginally cold hardy plants like strawberry. These approaches are particularly useful in early spring when most temperate fruit species are susceptible to frost injury.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Primarily two approaches are being used to improve the freezing tolerance of strawberry (Fragaria X ananassa), a) by using appropriate compatible osmolytes and b) by suppressing the membrane metabolizing enzyme, phospholipase D (PLD). Accumulation of a number of osmolytes including sucrose, raffinose, glucose, proline, and glycine betaine in the leaves during cold acclimation ( 2 C for 5 weeks) was monitored. With the exception of raffinose, the levels of osmolytes increased significantly in response to cold acclimation. As glycine betaine is metabolically stable, we chose to use this osmolyte for exogenous application. Plants were treated with 10 and 20 mM glycine betaine through root application. A significant increase in freezing tolerance was observed both in unhardened and cold-hardened plants with 10 mM glycine betaine application. The freezing tolerance increase due to glycine betaine application was more dramatic in unhardened plants. Glycine betaine
application at 20 mM was not as effective as that at10 mM. Following the application of glycine betaine we monitored plants for oxidative burst, as this signals the first step of cold acclimation in the plants. A sharp increase in hydrogen peroxide and superoxide anion levels was noted within 4 hours of plant exposure to low temperatures. A similar response in hydrogen peroxide was observed when glycine betaine was applied to unhardened plants within 2 hours of application. This suggests that the process of freezing induction by glycine betaine is similar to that which occurs during the natural cold acclimation.
Impacts
The primary objective has been to develop methods and plant types to increase the freezing tolerance in strawberry. Exogenous application of osmolytes is useful for providing cold protection in late winter and early spring. However, as a long range goal, metabolic engineering is more beneficial/cost-effective in developing freezing tolerant plants.
Publications
- R. Welti, W. Li, Y. Sang, H. Biesiada, H. Zhou, C. B. Rajashekar, T.D. Williams, X. Wang, 2002, Profiling membrane lipids in plant stress responses, J. Biol. Chem. 31994-32002.
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