Progress 10/01/01 to 09/30/04
Outputs
Chilling (0~10C) injury reduces maize yield and quality. For example, the chilling stress, occurred in August 2004, resulted in an estimated crop loss more than 100 million dollors in Minnesota alone. After encountering numerous obstacles, we finally succeeded in obtaining several lines of transgenic maize with codA gene, which encodes choline oxidase. This enzyme is responsible for the synthesis of glycinebetaine (GB) in bacteria. We found GB, by root feeding, prevents fatty acid oxidation under chilling stress, resulting in increased chilling tolerance in maize, but most lines of maize lacks the ability to synthsize GB under stress. Our transgenic maize plants, therefore, may posses the potential to be used to produce inbred seeds for the improvement of crop production in cold region like MN. MN Corn Growers Association currently sponsors for a 3-year research to characterize the usefulness of the transgenic maize stock with codA gene.
Impacts
Seedlings/plants, which raised from seeds obtained from transgenic plants, would be more tolerance with better growth in a chilling environment, and thus would result in an improved crop production.
Publications
- Li, P. H and E. T. Palva (eds.) 2002 PLANT COLD HARDINESS - Gene Regulation and Genetic Engineering. Kluwer Academic/Plenum Publishers, New York, Boston, Dordrecht, London, Moscow. PP. 294
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Progress 01/01/03 to 12/31/03
Outputs
We have obtained ca. 20 transgenic corn plants with codA gene grown in the greenhouse. codA gene, which was cloned from bacteria, encodes choline oxidase. Choline oxidase catalizes the synthesis of glycinebetaine (GB). In a chilling environment, GB content increases in plants and thus enables the plants to tolerate more chilling stress. In most corn lines , unfortunately, this synthesis process was lost during million years evolution in the tropical/subtropical region. We have planned to characterize the transgenic plants in terms of DNA, mRNA, choline oxidase, GB content and chilling tolerance. Hopefully, desirable plants will be obtained for seed production after the characterization .
Impacts
Seedlings/plants, which raised from seeds obtained from transgenic plants, would be more tolerance with better growth in a chilling environment, and thus would result in an improved crop production.
Publications
- Li, P. H. and E. T. Palva, eds. 2002. PLANT COLD HARDINESS - Gene Regulation and Genetic Engineering. Kluwer Academic/Plenum Publishers, New York, N.Y., pages 294.
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Progress 01/01/02 to 12/31/02
Outputs
Due to low transformation efficiency with immature embryos of Black Mexican Sweet corn as target tissue for transformation, we added 2 additional inbred lines (A188 & B37) as alternatives for the transformation. Attempts were then made to transform codA gene with Agrobacterium-mediated method into embryos of these 2 inbreds according to a procedure recommended by the Plant Transformation Facility at Iowa State U. Inclusion of cysteine in the co-cultivation medium clearly reduced hypersensitive response that often causes cell death of the infected calli. Thus far, we have not yet be able to obtain calli from 360 transformation events with A188 linbred that survive antibiotic selection. We now are using GUS reporter gene trying to pinpoint the possible difficulty in our procedure that caused this low transformation efficiency.
Impacts
High content of glycinebetaine (GB) improves corn chilling tolerance, but many inbreds like A188, B37, etc., are non-GB accumulators. codA gene controls GB synthesis. Transgenic corn with codA would thus be able to transfer a non-GB accumulator into a GB accumulator. The inbred line with the ability to accumulate GB could then be used as a breeding stock to produce seeds for cultivation.
Publications
- LI, P.H. and E. T. Palva, ed. 2002. Plant Cold Hardiness: Gene regulation and genetic engineering. Kluwer Academic/Plenum Publishers, New York. P. 294.
- Chen, W.P. and Li, P.H. 2002. Membrane stabilization by ABA under cold aids proline in alleviating chilling injury in maize cultured cells. Plant Cell Environ. 25:955-962.
- Chen, W.P. and Li, P.H. 2002. Attenuation of reactive oxygen production during chilling in ABA-treated maize cultured cells. In: Li and Palva, eds, Plant Cold Hardiness: gene regulation and genetic engineering, Kluwer Academic/Plenum Publishes, New York, pp.223-233.
- Chen, W.P. and Li, P.H. 2002. Chilling-induced programmed cell death in maize cultured cells. Int. Hort. Congress, S04-P-37, pp.143.
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Progress 01/01/01 to 12/31/01
Outputs
This report covers the summary of CM6 research for improving maize production in cold and/or drought environments. CM6 is an agent designed for alleviating maize cold (and/or drought) adverse effect, developed in Minnesota and consisting of [6] chemical ingredients. It was developed theoretically for maintaining the integrity of cell membranes under stress based on what we understood of the mechanisms of plant cold hardiness. Seeds can be soaked or coated with the agent prior planting. It improves seedling early vigor and always aids the plant to develop a much larger root system. As a consequence, the effect of CM6 can, therefore, last the whole season. One field (3 acres) experiment, held at the Sand Plain Research Farm, Becker, MN, showed 10% increase in yield with CM6 when seasonal rainfall at this location was 4.07"/June,3.19"/July,1.82"/August and 0.37/September when compared with nontreated plants.
Impacts
CM6 is now US Patent pending (filed 4/28/00)and PCT filed in April, 2001. Monsanto and Syngenta expressed an interest in it. Pioneer Hi-Bred International is currently preparing to sign a material acquisition agreement with U of M for further testing.
Publications
- Chen, W. P. and P. H. Li. 2001. Chilling-induced Ca2+ overload enhances production of active oxygen species in maize (Zea mays L.) cultured cells: the effect of abscisic acid treatment. Plant, Cell and Environment 24:791-800
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Progress 01/01/00 to 12/31/00
Outputs
How abscisic acid (ABA) is able to improve the maize's chilling tolerance: (1). When maize cultured cells were treated with ABA for 1 d at 26C, the treatment improved the cell's chilling tolerance and was able to prevent chilling-induced Ca influx and overload in the treated cells. Ultrastructure of the treated cells was also altered similar as those when cold-insensitive plants were cold acclimated. (2). Chilling enhanced active oxygen species (AOS) production,which resulted in membrane lipid peroxidation, one of the major chilling injury mechanisms. Such enhancement of AOS production was significantly reduced in the ABA-treated cultured cells. (3). The ABA-treated cells were found having a higher activity of the cyanide-resistant alternative pathway, which is believed to play an important role in lowering AOS production at both chilling exposure and recovery from chilling. ABA-enhaced higher respiratory activity may provide a better protection strategy against
chilling-induced injurious oxidative stress, thus resulting in improved tolerance.
Impacts
Chilling injury reduces maize crop production. Information of the progress report may provide clues on how to improve maize chilling tolerance by molecular biology strategy.
Publications
- Jian,L.C.,Li,J.H.,Li,P.H. and Chen,T.H.H. 2000. Intercellular communication channels and intracellular calcium levels involved in the dormancy development of poplar (Populus deltoides) plants. p. 291-312. In: Viemont,J. D. and Crabbe,J.eds. DORMANCY IN PLANTS. CABI Publishing,Oxon,UK and New York,USA.
- Chen, W.P. 2000. Regulation of calcium influx, free radical production and alternative pathway activity is associated with abscisic acid-improved chilling tolerance in maize (Zea mays L.) cultured cells. Ph.D. thesis, University of Minnesota, St. Paul, MN.
- Chen,W.P.,Li,P.H., and Chen,T.H.H. 2000. Glycinebetaine increases chilling tolerance and reduces chilling-induced lipid peroxidation in Zea mays L. Plant, Cell and Environment 23:609-618.
- Jian,L.C.,Li,J.H. and Li,P.H. 2000. Seasonal alteration in amount of Ca in apical bud cells of mulberry (Morus bombciz Koidz): an electron microscopy-cytochemical study. Tree Physiology 20:623-628.
- Jian,L.C.,Li,J.H.,Li,P.H. and Chen, T.H.H. 2000. An electron microscopic-cytochemical localization of plasma membrane Ca-ATPase activity in poplar apical bud cells during the induction of dormancy by short-day photoperiods. Cell Research 10:103-114.
- Jian,L.C.,Li, J.H.,Li,P.H. and Chen, T.H.H. 2000. Structural association of endoplasmic reticulum with other membrane systems in Populus deltoides apical bud cells and its alterations during the short day-induced dormancy. Acta Botanica Sinica 42:803-810.
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Progress 01/01/99 to 12/31/99
Outputs
Chilling tolerance was increased in cultured cells and seedlings of maize, cv Black Mexican Sweet, grown in media containing glycine betaine (GB). Treatment with 2.5 mM GB resulted in maximum chilling tolerance improvement (40%) as compared to the controls. The degree of increased chilling tolerance was positively correlated with the level of GB accumulated in the cells. However, the accumulation of GB in the cells was saturated at a concentration of 240 umol (gDW)-1 without the GB toxicity. Lipid peroxidation, a type of chilling injury which is caused by chilling-induced active oxygen, was significantly reduced in the GB-treated cells as compared to the control. Evidence suggests that the increased chilling tolerance by GB seems due, in part, to the reduction of lipid peroxidation of the membrane. Most chilling sensitive lines of corn do not have the gene(s) for choline oxidase, which is responsible for the synthesis of glycine betaine (GB). High GB content aids
plants to alleviate chilling injury. A clone for choline oxidase is available, so that screening for improved chilling tolerance of maize from transgenic plants is highly possible.
Impacts
Maize is a chilling sensitive crop. Its chilling injury, which occurs often in the field, reduces crop production. The GB information suggests a strategy which can be used for plant hardiness improvement, which in turn would result in better crop production.
Publications
- Li, P.H., et al. 1999. Cytochemical localization of calcium and Ca2+-ATPase activity in plant cells under chilling stress: a comparative study between the chilling-sensitive maize and the chilling-insensitive winter wheat. Plant, Cell Physiology. 40:1061-1071.
- Li, et al. 2000. Seasonal alteration of Ca2+ concentration in apical bud cells of mulberry: an EM-cytochemical study. Tree Physiology (In press).
- Li, et al. 2000. Ca2+-homostasis differs between plant species with different cold-tolerance at 4C chilling. Acta Botanica Sinica (In press.)
- Li, et al. 2000. Glycine betaine increases chilling tolerance and reduces chilling-induced lipid peroxidation in Zea mays L. Plant, Cell Environment (In press).
- Li, et al. 2000. Intercellular communication channels and intracellular calcium levels involved in the dormancy development of poplar plants. In: The proceedings of the 2nd Internatinal symposium of Woody Plant Dormancy, July 19-23, 1999, Angers, Farnce, (In press).
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Progress 01/01/98 to 12/31/98
Outputs
Relationship between ABA-induced chilling tolerance and alternative respiratory pathway was investigated in maize suspension-cultured cells. Mitochondria isolated from 26 C ABA-treated cells displayed a two-fold, from 50 to 90 nmole oxygen/mg protein/minute, increase in the capacity with succinate as substrate as compared to the mitochondria isolated from non-ABA-treated control cells Cycloheximide inhibited the increased alternative pathway capacity induced by ABA treatment. Antibodies against alternative oxidase (AOX) protein revealed that cells, treated at 26 C with ABA possessed 2 times higher content of AOX protein than those of control. Results suggest that ABA-induced chilling tolerance in maize is associated with the increased activity of the alternative respiratory pathway which plays a role as an antioxidant defense system under chilling stress. CM6 agent increased soybean production by 5-8% under mild drought stress in the field as compared to the control
when crop seeds were coated with the agent prior to planting.
Impacts
(N/A)
Publications
- Jian, L.C. and P.H. Li. 1997. Is Ca 2+ homeostasis essential in the cold acclimation of winter wheat seedlings? P. 69-71. In: Z. Bedo, ed. Cereal Adaptation to Low Temperature Stress in Controlled Environments. Agricultural Research Institute, Hungarian Academy of Sciences, Martonvasar, Hungary.
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Progress 01/01/97 to 12/31/97
Outputs
This report covers: (1) effects of different Nitrogen sources on the chilling tolerance of maize cells, and (2) improvement of soybean production with CM6 agent. (1) N starved and NO3-grown cells for 1 day at 26C had survival rates of more than 70% after 7-day chilling at 4C, similar to NH4/NO3-grown cells after ABA treatment. In contrast, survival of NH4-grown and NH4/NO3-grown cells was less than 10%. Notably, NH4-grown cells could not increase chilling tolerance even with ABA treatment. Results suggest that NH4 ion in maize cells may be a critical factor that affects cell survival under cold stress, and the NO3 assimilation pathway where it involves NH4 ion may play an important role in ABA-induced maize chilling tolerance. (2) Soybean seeds were coated with CM6 before planting. There was no difference in emergence rates from cold soil between the control (non-coated seeds) and CM6-coated seeds. However, CM6 does enhance plant growth, specifically, the development
of rather large rooting systems. 4 parameters (per unit area, per plant, 1000-seed weight, and 100-seed volume) have been used to measure yield. All parameters indicated an improvement of yield among varieties of Parker, Hardin, and McCall. Based on a 3-meter row, Parker, Hardin, and McCall had 20, 9, and 10%, respectively, increases in yield from CM6 plants as compared to the control. In general, all varieties had larger seed size from CM6 plants as compared to the control.
Impacts
(N/A)
Publications
- LI, P.H. and T.H.H. CHEN, eds. 1997. Plant Cold Hardiness: molecular biology, biochemistry and physiology. Plenum, New York-London. pp.
- JIAN, L.C. AND P.H. LI. 1997. Is Ca2+ homeostasis essential in the cold acclimation of winter wheat seedlings. p. 69-71. In: Z. Bedo, ed. Cereal adaptation to low temperature stress in controlled environments. Agr. Res. Inst. of the Hungarian Academy of Sciences,
- JIAN, L.C., P.H. LI AND T.H.H. CHEN. 1997. Alteration in ultrastructure and subcellular localization of Ca 2+ in poplar apical bud cells during the induction of dormancy. J. Expt. Bot.
- LI, P.H., etc. 1997. Abscisic acid-induced chilling tolerance in maize. p. 215-236. In: P.H. LI and T.H.H. CHEN, eds, Plant Cold Hardiness: molecular biology, biochemistry, physiology. Plenum, New
- VALVERDE, R., T.H.H. CHEN AND P.H. LI. 1997. Frost hardiness and cold acclimation in Solanum species. p. 57-66. In: P.H. LI and T.H.H. CHEN, eds. Plant Cold Hardiness: Molecular biology, biochemistry, physiology. Plenum, New York-London.
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Progress 01/01/96 to 12/30/96
Outputs
Vegetative dormancy is an important strategy for the survival and growth of temperate perennial plants. In poplar, bud dormancy and freezing tolerance can be concomitantly induced by short days (SD) at room temperature. Poplar plants stop growing by the 20th day of SD, but changes in neither dormancy nor freezing tolerance can be detected. After 77 days of SD, plants become deep-dormant and freezing tolerance increases from -6 to -18C. During the development of dormancy, the thickness of cell walls and the number of starch granules increase. The most striking change is the constriction and blockage of the plasmodesmata. When dormancy is initiated after 20 days of SD, Ca2+ deposits decrease in intercellular spaces, whereas some deposits can be found in the cytosol. From the 28th to 49th day of SD, large numbers of deposits are in the cytosol. When deep dormancy is reached, deposits decrease in cytosol, whereas deposits are again observed in cell walls and intercellular
spaces. The elevation in cytosolic Ca2+ may trigger the synthesis and deposition of such callose in cell walls, leading to thickened cell wall & blockage constriction of plasmodesmata. The blockage constriction of plasmodesmata may stop symplastic transport and signal transduction between adjacent cells, which in turn may lead to events associated with growth cessation & dormancy development.
Impacts
(N/A)
Publications
- ZHU, B. L., T.H.H. Chen and P.H. LI. 1996. Analysis of late-blight disease resistance and freezing tolerance in transgenic potato plants expressing sense and anti-sense genes for an omotin-like protein. Planta 198:70-77.
- SCHWAB, P.M., D. K. BARNES, C.C. SHEAFFER and P.H. LI. 1996. Factors affecting a laboratory evaluation of alfalfa cold tolerance. Crop Sci, 36:318-324. JIAN L.
- C., P.H. LI and T.H.H. CHEN. 1996. Alterations in ultrastructure and subcellular localization of Ca2+ in poplar apical bud cells during the inductionof dormancy. J. Expt. Botany, In press.
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Progress 01/01/95 to 12/30/95
Outputs
Evidence suggests that the increase in low temperature tolerance is closely related to the cytosolic Ca distribution. ABA at 28C can increase maize chilling tolerance. Calcium distributions in ABA-treated maize cultured cells at 28C and 4C were investigated using antimonate precipation techniques. At 28C, non-treated controls had their calcium deposits located mainly in the vacuoles and rarely in the cytosol and nucleus. In contrast, ABA-treated cells redistribute their vacuolar calcium to the outside of tonoplasts by 1 hour. Meanwhile, there was a separation of the plasma membrane from cell wall with Ca deposited vesicles formed in between. At 4C, the controls responded to chilling with redistribution of the vacuolar calcium to the tonoplast and with high levels of calcium in the cytosol and nucleus. The cells retained this similar pattern of calcium distribution as chilling was prolonged until death. There were few calcium deposits localized in the cytosol when
ABA-treated cells were chilled. Observations suggest that ABA stimulates a transient increase in cytosolic calcium at warm temperature and subsequently leads cells to remove excess calcium from the cytosol to the outside of cells. Maintenance of a low concentration of calcium in the cytosol of ABA-treated cells at chilling temperature is therefore possible, and is proposed as an important strategy for maize cells to survive at the low temperature.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
Treatment with ABA at 28 C for 1-d increases the chilling tolerance of chilling sensitive suspension-cultured cells. No chilling tolerance is induced when cells are chilled at the inception of treatment. Calcium distribution under these conditions has been examined. Vacuoles are the Ca reservoir in cultured cells. After 1-h treatment at 28 C, Ca deposits were seldom seen in vacuoles, whereas plasmolysis occurred. During this 1-h period, many vesicles with Ca desposits on them were observed outside of the plasma membrane. After 1-d treatment, cells became deplasmolyzed with abundant starch grains. After 1-d chilling (4 C), Ca in non-ABA-treated cells was significantly re-distributed from vacuoles to the cytosol and nucleus. Cells chilled at the inception of ABA treatment essentially had a similar Ca distribution pattern as the controls. For ABA-treated cells, few Ca deposits were seen in cytosol and nucleus after 1-d chilling. After 5-d chilling, cells were
plasmolysized again with abundant starch grains. Evidence suggests that ABA treatment at room temperature causes cells to go through some kind of preparation (or reorganization) which in turn enables the cells to be acclimated upon chilling exposure, and the high content of Ca in cytosol caused by chilling has an adverse impact on the cell's ability to cold acclimate.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
We isolated pA13, pA35 and pA81 cDNAs of ABA-responsive genes from Solanum commersonnii cell cultures. These three cDNAs encode osmotin(OSM) like proteins. These cDNAs have extensive identity in the coding regions and may encode three isoforms of potato pathogenesis-related (PR) type 5 proteins. RNA gel blot using gene-specific probes showed that the expression of OSM-like genes is subjected to complex environmental controls. Constitutive expression of these genes was found in leaves, stems, roots, and flowers, with high levels in roots and flowers. ABA, low temperature, and NaCl increased the accumulation of all three mRNAs in S. commersonii cell cultures and plants grown in vitro. Infection with fungus Phytophthora infestans induced strong and localized expression of all three OSM-like genes. OSM-like proteins are encoded by at least six members of a multigene family in Solanum commersonii. We isolated a genomic clone ((lambda)pGEM2a-7) that contains two OSM-like
genes (pOSML13, pOSML81) arranged in the same transcriptional orientation. Restriction map and sequence analysis indicated that these two intron-less genes correspond to the characterized pA13 and pA81 cDNAs encoding osmotin-like proteins. Chimeric gene expression in transgenic potato plants showed that both 5' flanking DNA sequences were sufficient to impart reported gene inducibility by environmental cues and fungal infection.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
ABA-induced gene expression during the induction of cold hardiness of S. commersonii cultured cells was investigated. The cold hardiness of ABA-treated cells increased from -6(degree)C (control) to -11(degree)C in 12h, declined after 48 h of ABA treatment, and returned to the control level by 72 h. By differential screening, a cDNA (pA13) corresponding to an ABA-responsive gene was isolated. This cDNA corresponds to a transcript of ca. 1.0-Kb, has one open reading frame, and encodes a polypeptide of 246 amino acids with a predicted M.W. of 26,655 and pI 6.7. The deduced amino acid sequence of pA13 cDNA shares 89% and 91% identity with tobacco osmotin and tomato NP24 protein, respectively. Northern blot analysis indicated that a low level of transcript corresponding to pA 13 cDNA was detected in the controls. An increase in a steady state level of mRNA corresponding to pA13 cDNA was detected after 3 h of ABA treatment, was maintained at this high level for up to 12 h,
and then declined to the initial control level by 24 h. Tobacco osmotin and tomato NP24 increase in abundance during the adaptation to low water potentials. It is well known that freezing, drought, and salt stresses exert a similar stress on cells, i.e. dehydration. ABA seems to be involved in the acquisition of tolerance to these stresses through the regulation of some common sets of genes. This observation supports Levitt's suggestion that a common mechanism may be associated with the development of tolerance to various stresses.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
To study the role of ABA in the regulation of gene expression associated with freezing tolerance induction, we isolateon associated with freezing tolerance induction, we isolated 6 cDNA clones (ACR8, ACR13, ACR32, ACR35, ACR72, and ACR81) of ABA responsive genes from S.commersonii cell suspension and studied their expression in response ABA and cold tratments. RNA gel analysis indicated that genes representedthat genes represented by ACR8, ACR13, ACR35, and ACR81 were transiently expressed in response to ABA (75 mu), coincidng with ABA induced freezing tolerance. The 6 ABA responsive genes were also cold regulated, and their expression wasdependent which was associated with the development of cold induced freezing tolerance. Results suggest that ABA plays crucial role in hardiness induction during cold acclimation presumably by the regulation of gene expression. Southern analysis indicated these ABA and cold modulated genes belong to multigene families. Primary
sequencing data of ACR13 indicated that it shared a high holology with tobacco osmotin, which accumulated in a NaC1-adapted tobacco cell line.
Impacts
(N/A)
Publications
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Progress 01/01/90 to 12/30/90
Outputs
Freezing tolerance in plantlets of Solanum commersonii stem-culture increased from -3.5(degree)C to -8.5(degree)C during either 14 days of 5(degree)C day/night acclimation or 7 days of ABA-treatment (15 mg/1) at 20(degree)/15(degree)C day/night. Changes in polypeptide synthesis and in RNA populations were investigated during the induction of cold hardiness. About 20 either cold- or ABA-induced polypeptides were identified. The synthesis of one group of polypeptides is prominent and stable throughout the treatment period. The other group is transient. The most prominent/stable polypeptides have molecular weights of 21, 22, 31 and 83 kDa. Both cold acclimation and ABA treatment alter translatable mRNA populations during the development of cold hardiness. Two mRNAs, encoding translation products at 26 and 27 kDa were identified. These proteins may play important roles in the programming for the development of cold hardiness in potato. 2. Chilling tolerance in Zea mays
cell suspension cultures increased significantly with ABA treatment (10-(superscript 4 )M). However, when treatment was held at the inception of chilling, no induction of chilling tolerance was observed. At least 3 new polypeptides with molecular weights of 24, 27 and 65 kDa were synthesized during treatment. These proteins were lacking when the treatment was held at the inception of chilling. Whether these proteins are relevant to the development of tolerance remains unknown. 3.
Impacts
(N/A)
Publications
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Progress 01/01/89 to 12/30/89
Outputs
This report covers three aspects of crop hardiness. Freezing - Low temp (4 degrees C) induces cold hardiness in cultured cell suspension of Solanum commersonii. Hardiness peaked in 2 days of acclimation instead of 14 days which are required for stem-cultured plantlets and pot-grown plants. Floridone, a ABA synthesis inhibitor, blocks the development of cold hardiness at 4 degrees C. This observation provides additional evidence that the synthesis of ABA during cold acclimation required in the development of cold hardiness. Chilling - ABA induces chilling tolerance in maize cell suspension. The induction occurs only at warm temp regime, and the induced tolerance is transient in nature. The ABA-induced tolerance can be prevented by cycloheximide, a protein synthesis inhibitor, and actinomycin D, a RNA synthesis inhibitor. Results indicate that ABA alters gene expression which leads to increased tolerance and these alterations only occurs at warm temp regime. High temp -
A study with Phaseolus bean species, to determine whether differential root sensitivity to heat is an important factor determining differences in plant heat tolerance, suggests that decrease in photosynthetic rate (a parameter used to measure plant heat tolerance) was not due to heat injury to the mesophyll, and differences in plant heat tolerance were not solely determined by root sensitivity to high temp.
Impacts
(N/A)
Publications
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Progress 01/01/88 to 12/30/88
Outputs
This report covers freezing, chilling & high temperature hardiness research. Freezing-Low con. mefluidide (M) increased freezing tolerance in actively growing plants of potato & winter wheat. The content of free ABA was peaked 12-h after M treatment, whereas conjugated ABA decreased. The time lag between peaked ABA & peaked hardiness was about 2-3 days which was similar as that in naturl cold acclimation. These observations support a previous conclusion that increasing free ABA content is vital for hardiness induction. M treated wheat seedlings showed higher contents of 18:2 & 18:3 fatty acids in the lipid portion of the plasma membrane than in the control. These alterations suggest that the composition of plasma membranes altered & thus resulted in increased hardiness. Chilling - ABA induced chilling tolerance (CT) in corn cell suspension & the induction occurred only at warm temperature. When cycloheximide, a protein synthesis inhibitor, was added to the system
with ABA at the same time, it inhibited the ABA-induced Ct but failed to do so if applied 6-h after ABA application. Evidence suggest that active protein synthesis is involved in the ABA-induced CT. High-Temp - Leaf heat tolerance (HT) & leaf heat acclimation potential (HAP) were examined in 74 bean genotypes. Positively significant correlation was observed between leaf HAP & post-stress performances in plant dry weight, pod set, pod weight & seeds/plant among genotypes.
Impacts
(N/A)
Publications
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Progress 01/01/87 to 12/30/87
Outputs
This report covers three areas: 1) freezing stress-mefluidide induces cold hardiness of seedlings of winter and spring wheat 2-3 days after a foliar spray at 2ppm. A 3-4C hardiness was increased, and the increased tolerance was sustained for about 4-5 days. Concentrations of 5 ppm or higher increased tolerance but they inhibited post-application growth. Mefluidide also increased cold hardiness of crab apple flowers 1-2 days after application of 10 or 15 ppm. The increased hardiness was about 2-3C, and it lasted 4-5 days. 10 or 15 ppm had no apparently adverse growth effects on leaves, twigs and fruits. The model of action of mefluidide on increased cold hardiness is being studied. 2) Chilling stress-mefluidide may alter plasma membrance properties of rice seedlings and thus lead to increased chilling tolerance. Significant protection was apparent at least for 72 hours after a single treatment at 5-10 ppm. Higher concentrations students growth, even though it
showed protection. Mefluidide also induces protection in corn cell suspensions. Alterations of gene expression are being studied in mefluidide-induced chilling protected corn cells. 3) Heat stress-Based on heat acclimation potential (HAP), 67 bean genotypes were classified into high, intermediate and low categories. HAP is defined as changes in leaf heat tolerance after month-old plants have been exposed to 37C for 24 hours.
Impacts
(N/A)
Publications
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Progress 01/01/86 to 12/30/86
Outputs
Protein profiles were examined by 2-D gel electrophoresis in Solanum commersonii(Sc) stem-cultured plantlets after 1, 3, 5, 7, and 14 days of 5C cold acclimation or of ABA treatment at 20C. Proteins wre labeled in vivo and in vitro with S-methionine. Newly synthesized proteins were initiated in Sc after 1 day of ABA treatment or 3 days of cold acclimation. Similarity of protein profiles was found between cold-acclimated and ABA-treated plants. These were not observed in non-cold-acclimated or non-ABA-treated plants. These proteins disappeared after 1 day of deacclimation. Proteins with molecular sizes of 26-27 Kd in vitro synthesis and 20-22 Kd in vivo synthesis that are good candidates for a role in induction of cold hardiness in the potato are identified. Properties of these proteins are being studied. Mefluidide increases not only chilling resistance in chilling sensitive plants such as corn, but also freezing resistance in chilling insensitive plants such as
potato. It also reduces chilling injury of rice seedlings. Evidence obtained from a fluridone study supports the hypothesis that mefluidide-induced low temperature tolerance is due to its ability to increase endogenous ABA content when plants grow in a non-temperature stress environment with adequate water supply.
Impacts
(N/A)
Publications
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Progress 01/01/85 to 12/30/85
Outputs
Rates of acclimation and deacclimation were determined for three Chinese cultivars and one American cultivar of spinach. The effect of air & soil temp. on freezing tolerance and water relations during acclimation was also investigated. All 4 cultivars acclimated and de-acclimated very rapidly. A 2 to 4C increase and a 3 to 5C decrease in freezing tolerance were found after 1 day of acclimation and deacclimation, respectively. After 3 days, the rates of acclimation and deacclimation declines dramatically. Root hydraulic conductivity decreased at low root temp., inducing a water stress in plants with a high shoot temp. but not plants with a low shoot temp. Freezing tolerance, however, was increased only by low shoot temp. treatment. Evidence indicates that water stress is not solely responsible for hardiness increase in spinach. Mefluidide was capable of triggering ABA increase when corn plants were grown in a non-chilling (26C) and non-water-stress regime. It
is suggested that the ABA increase may activate a protecting system that enables corn to minimize the injury during chilling exposure. ABA increase prior to chilling seems more important for the protection than the high level of ABA that occurred during chilling. Mefluidide should be applied at least 12 hr. prior to chilling exposure to be sure of protection. Low concentrations of mefluidide treatments also promoted corn radicle elongation at chilling temperatures.
Impacts
(N/A)
Publications
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Progress 01/01/83 to 12/30/83
Outputs
A computerized open-top field exposure chamber system was used to examine the effects of ozone, sulfur dioxide and their mixture on soybean yield. The pollutant exposure regimes simulated the ambient patterns in Minnesota. Numerical analyses showed that log, exponent, sine, arcsine transformations and other related approaches could not satisfactorily explain the relationships between the pollutant exposures and the measured plant parameters. In cooperation with Alberta Government and Scientists, a polynomial - fourier model was successfully developed to explain the pollutant - plant response relationships. For the first time in the literature, this model accounts for the number of pollutant episodes (any concentration above the detection limits); the size (mathematical area under the exposure) of each individual episode and the peak pollutant concentration in each episode. Using this model, soybean pod number, pod weight, seed number, and seed weight were
successfully accounted (ry = 0.90) for the various pollutant treatments.
Impacts
(N/A)
Publications
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Progress 01/01/82 to 12/30/82
Outputs
Potato species have a tendency to be supercooled to -3.5 degrees C or lower in nature. Lab evidence indicates that supercooling may not be a desirable characteristic for survival in frost-hardy potatoes like Solanum acaule. For example, leaves of acaule can tolerate intercellular freezing up to -5/-6 degrees C if tissue ice initiates at -1 degree C. When ice occurs in the supercooled tissue at any temperature below -2.0 degrees C, leaves are killed at the moment of ice initiation. It could be at any temperature below -2.0 degrees C. Through a laborious search, we have found that fluorophlogopite, a synthetic mica, is effective to initiate ice formation -l degree C after spray (1% aqueous suspension). We believe that the lab findings may bear a potential for application in reducing the late fall or early spring frost damage to crops by maximizing the plant hardiness potential through extracellular freezing mechanisms. At the 1981 V-130 annual meeting, fruit
people from several states decided to test our findings on deciduous fruit flower buds in the field to see if spring frost killing can be prevented/reduced.
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Progress 01/01/81 to 12/30/81
Outputs
Evidence from recent study indicates that an elevation of ABA during cold acclimation may induce the protein synthesis which is responsible for the increase of frost hardiness in the potato. Specifically, it has been observed that: Increase in frost hardiness was initiated after endogenous ABA levels peaked and protein synthesis had been initiated, ABA can substitute for exposure of plants to low temp in inducing frost hardiness and induced levels of frost hardiness are positively correlated with the amount of ABA added, ABA and low temp did not induce any increase of frost hardiness when plants were exposed to a cytoplasmic protein synthesis inhibitor at the beginning of acclimation, and the inhibitor did not reverse or inhibit cold acclimation if it was supplied after the endogenous ABA in plants had peaked and protein synthesis had been initiated. It is conceivable that when plants of cold acclimatable potato species are exposed to low temperature (2 degrees C),
the low temperature triggers an elevation of endogenous ABA needed to induce the synthesis of specific proteins which are responsible for the increase of frost hardiness.
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Progress 01/01/80 to 12/30/80
Outputs
Triphenyl tetrazolium chloride reduction test in addition to the conductivity test can be used to evaluate heat tolerance in crop plants. The degree of heat tolerance could be expressed by either the heat killing temperature or the heat killing time, the latter being the more sensitive indicator. When breeding for high temperature conditions, we believe one should select those genotypes with high adaptability to heat stress rather than those genotypes with high pre-acclimation level of heat hardiness. This heat adaptability characteristic cannot be distinguished among genotypes unless plants are subjected to temperature about 30 degrees C for about 12-14 hours - a process of heat acclimation. Whether crops are for seeds, fruits or tubers, the leaf organs could serve as the testing tissue for screening heat tolerant genotypes with plants as young as about one month old.
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Progress 01/01/79 to 12/30/79
Outputs
Frost hardiness levels in potatoes bear no relation to cold acclimation. TwelveC and 2 C are the critical and optimum acclimating temperatures, respectively. Net soluble-protein is correlated with net frost hardiness after acclimation. Increases in both heat tolerance and frost resistance are seen in acclimatable species following subjection to low temperatures. We hypothesize such increases are due to the low temperature treatment increasing the capacity of proteins to withstand denaturation at high temperatures and aggregation at freezing. Membrane protein particle aggregation during acclimation treatment indicates an adaptive fluidity mechanism which is likely responsible for hardening. Initial freeze-injury mechanism appears different in hardy and non-hardy potatoes. Structure and function of RuBPCase from Solanum tuberosum (St; non-hardy) was compared with that from S. commersonii (Sc; hardy). Before cold acclimation (CA) Sc RuBPCase had fewer surface SH
groups than St RuBPCase; both had same no. of total SH groups. Sc less susceptible than St RuBPCase to freeze-thaw-induced aggregation caused by intermolecular S-S formation. Low-temp activity higher for Sc than for St RuBPCase. Long exposure of rye to 4 degrees/2 degrees D/N temp increased both mesophyll cell size and leaf thickness 1.5-fold, changed both stomatal frequency and distribution, increased leaf dry wt and sap conc. Hardened rye leaves have 2-shouldered water freezing curve, non-hardened have a single peak.
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Progress 01/01/78 to 12/30/78
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Hardy or tender potatoes with or without cold hardening ability were identified. Abilities of frost resistance and for hardening are independent. Lower temp achieved greater hardiness. Stem-cultured plants and leaf callus can be cold hardened. ABA can increase hardiness in both hardy and tender potatoes. TTC reduction and electrical conductivity can measure true heat tolerance in addition to evaluate freeze tolerance. Tender potatoes resist more heat stress than hardy ones. Cold hardening increases heat resistance in potatoes. Phase transition in phospholipid vesicles from non-hardy potato is at minus 3 degrees C, the killing temp. Activation energy for H(2)O permeability in dogwood stem tissue Delta E(a)(,)(H)(2)(0)) stable during SD, warm temp cold acclimation (CA) but rose in 1st 3 wk of H(2)O stress, implying increase in fatty acid unsaturation not essential to dogwood CA. Delta E (a)(,)(H)(2)(0) was 5.5 kcal/mol for tissue killed by membrane rupture, 8.1
when slowly frozen to minus 9 degrees C (a lethal freeze), and 8.3 for healthy tissue, indicating membrane rupture not cause of freeze dehydration death. Most H(2)O stress induced frost hardiness is lost 7 days after rewatering in LD. Frost increased hardiness of mod. stressed dogwood but decreased it in severely stressed plants. During acclimation, Alaska clone decreased H(2)O content and closed stomates 3-5 wk before Oregon. Alaska clone equal to 10 degrees C hardier than Ore but H(2)O cont was similar.
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Progress 01/01/77 to 12/30/77
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Leaves of hardy potato varieties had 2 layers palisade cells; non-hardy ones had1; 1 hardy hybrid also had 2 layers; this may be a selection marker for breeding hardy clones. Cell membrane active transport pumps are the sites of primary freezing injury. Sequence of events to death in freeze injured cells are: protoplast swelling-then to chloroplast and mitochondira swelling-then to tonoplast disruption-then to protoplasm disorganization-then to death. Stem cultured potatoes can be cold acclimated. Low temp-water stress-short day trigger independent hardening mechanisms in dogwood initially; total hardiness expression is an additive effect. No H(2)O moved in arborvitae in frozen soil but moved after thawing. Activation energy for H(2)O permeability in dogwood stem tissue rose significantly after 3 wk severe H(2)O stress. A scanning calorimeter was built for examining molecular changes during hardening. When dogwood was rewatered after water and freeze stresses,
LD plants lost 9 degrees hardiness while SD plants lost only 4 degrees C hardiness. Stomatal resistance (r(s)) was similar in all treatments during recovery from water stress. Water stress accelerates hardening but only maintains hardiness under SD. Cycling in dogwood root resistance could not be eliminated by growing plants at low temp and continuous light. Leaf ABA content followed r(s) trends with LD plants having a larger quantity of ABA than SD.
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Progress 01/01/76 to 12/30/76
Outputs
67 Solanum species were evaluated for frost hardiness. Killing temp ranges from-2.5 to -6.5c. S. acaule (hardy potato) had leaf cell wall 200mu thick. S. tuberosum (tender sp.) had 100mu. After cold acclimation (acclim), acaule chloroplast had increased osmiophilic globuli and few starch grains. Unchanged status of tuberosum chloroplasts after cold treatment may be associated with inability to cold acclimate. Freeze-etch procedure developed for potato membrane study. Potato hybrids could be acclimated. Cool growing temp caused higher GDH activity which was due to less enz. inhibitor present. Tuber had insig GDH activity. Highest activity was in root. Low temp for 2 nights, resulted in more frost hardiness in H(2)- Oxtressed LD plant. Isolation & GC-MS det'n of yHHO in plants perfected to follow H(2)Oy uptake from frozen soil. 6 days after start of acclim in SD, dogwood transpiration (Ts) rate became higher & stomatal resistance became lower than LD control.
5-8 wks after start of SD, stomata closed tightly & Ts rate dropped below LD level. No difference found in leaf water potential (Y) until late in acclim when SD Y increased above LD values. Stem relative water content decreased during acclim. Field results on clones from different climates were similar to lab results. Root resistance (RR) to water uptake increased during acclim but in fall, RR increased 40 days sooner than in summer. Different coc of 0(2) have no effect on degree of freezing damage or plasmolysis in onion cells.
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Progress 01/01/75 to 12/30/75
Outputs
Increase of frost hardiness in H(2)O stressed dogwoods is due to increase of tolerance and avoidance of freeze-induced dehydration; SD induced hardiness is also due to both mechanisms; H(2)O stress may override SD in induction of 1st stage acclimation; low temp reduces acclimation in LD grown dogwood. Wood and flower buds of many hort and forest trees are killed at -40 when deep supercooled H(2)O freezes. Low temp exotehrms, homogeneous nucleation and cold hardiness are closely related. NMR, DTA and microscopic studies show supercooling required specific morphology. S. tuberosum doesn't have the physiological bases for developing frost hardiness; wild S. species can cold acclimate. Potato dogwood, turf, wheat survive by tolerating ice; frost hardiness not related to bound H(2)O, and in potato, tissue H(2)O or cell solutes. CCC increases hardiness in wild S. species. Callus formation can be used for evaluating viability after freezing. Onion cells are killed at
ca. -20C; damage in onion and potato cells are not reduced by freezing in low/high 0(2) partial pressure; developing bean seeds have high H(2)O and osmotic potentials which rapidly decline 35-40 days after anthesis. An Environmental Stress Symposium was organized which included 24 papers during 4-day on low/hightemp, H(2)O, radiation and other stresses.
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Progress 01/01/70 to 12/30/73
Outputs
Studies completed: Dogwood's first phase of cold acclimation was phytochrome controlled - Far north races acclimated most rapidly in short days - One frost induced second acclimation phase - Acclimation changed H(2)O relations; Rhododendron & Prunus floral primordia were killed by vital fraction of H(2)O freezing - Hardiness mechanism was supercooling; Blackheart injury resulted fromfreezing vital supercooled H(2)O - Freezing & death were inevitable before -40C was reached; Hardy apple varieties had most liquid water below OC; growth regulators had limited success delaying bud break; Rapid screening method of potato tuber protein was not found - Carbohydrates interfered in Udy dye-bindingmethod - Neotec Grain Quality Analyzer was not reliable for protein; Simazine & cytokinins did not effect potato protein - did increase yield - Low Temperature stimulated nitrogen.
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Progress 01/01/70 to 12/30/72
Outputs
Woody: The intital stage of cold acclimation in dogwood is phytochrome mediated- Typical red forms and is formed from for red reversiblityt evident - End of day red lite exposure enhances acclimation; water stress (2 wks) induces severaldegrees of hardening at any daylength - Reducing agents induce hardening in plants preconditioned under short days; acclimation accompanied by increased unsaturation of fatty acly moieties of membrane lipids; nuclear magnetic resonance indicated decreased hydration and increased H(2)O binding early in hardening; problem of asmotic stress resolved in permeability studies - tritiated water technique applicable for studying H(2)O permeability. Climate modification studies in fruit and nut crops indicate possible commercial applications - and apple rootstock/scion hardiness interactions characterized. Herbaceous: Low growing-temperature raises tuber protein in some genotypes of potato - Simazine treatment has similar effect; RNA synthesis
is faster under short days - Polysome levels are high at low temperatures; Phosphatidyl choline appears to be the exclusive potato ribosome phospholipid.
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Progress 06/01/68 to 09/30/70
Outputs
Electrical measurements can predict hardiness before frost exposure - non-lethalfrosts induce a sharp increase in frost resistance and a change in electrical properties - conductance increases following freezing death in non-hardened but not in hardy stems. Data suggest that frost induced hardening involves a major physiological change, and that freezing death is due to plasmalemma destruction in non-hardened but not in hardened stems. Exotherm profiles of blueberry stemssupport this concept; the lethal temperature and cause of death in various distinct tissues are different. Boxwood hardening is accompanied by synthesis of proteins and nucleic acids - there is a large increase in rRNA and a decreasein RNase activity - rapid destruction of all RNA species following freezing death - and rapid metabolism during temp induced dehardening.
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Progress 01/01/69 to 12/30/69
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Hydration influences hardiness in woody stems. Artificial dehydration (4-14%) increased resistance 12C in autumn and 6 in spring. Apple bark hardiness is related to air temp of previous day. Bark dehardens 15 per day and rehardens 5 per day. Short day induced hardiness factors are translocated; move via phloem; and are not genotype specific. Low temp induction is not translocated. RNAse increases rapidly after freezing. An aseptic culture technique was developed for acclimating excised stems - exogenous sugars and abscisic acid don't enhance acclimation - nucleic acid and ox.phosphorylation inhibitors enhance growth on short days. A 5S rRNA species found in potato leaves for firsttime in higher plants. Day-length and temp interact reversibly to regulate tRNA and frost resistance in potato-tRNA changes may reflect endogenous regulatory system.
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Progress 01/01/68 to 12/30/68
Outputs
Apple leaves produced a translocatable hardiness promoter under short days; an inhibitor under long days. Defoliated plants under long days harden more, undershort days less, than plants with foliage. Frost induction of hardening is not translocatable. The maturity of apple rootstock may have more effect on the hardiness of the scions than the relative hardiness of the rootstock itself. Apple twigs showed rapid hardening in autumn, with a 15 C increase from Oct. 20to 27. This rapid hardiness was immediately preceeded by a 40 percent increase in tRNA and rRNA. During cold hardening a 3rd freezing point appears in stems of Forsythia, Acer, Rhododendron, and Malus subjected to controlled freezing stress.
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Progress 01/01/67 to 12/30/67
Outputs
Progress has been made in two areas of the project: The development of analytical techniques for studying woody plant tissues, and the development of aworking hypothesis to use as a basis for elucidating the physiology of cold acclimation and for ultimately devising means of attenuating winter injury to woody plants. Methods have been developed for woody tissues to effectively extract total RNA and DNA, to separate proteins by acrylamide gel electrophoresis, to separate specific nucleic acids on MAK columns, and to culture callus and meristems. The following sequence of events are hypothesizedto be involved in cold acclimation: (A phytochrome system detects short days in autumn) Yields (P(R) phytochrome induces formation of hormones involved in growth cessation and stage I of hardening) Yields (Autumn hormones activate DNA operons) Yields (New RNAs regulate synthesis of proteins) Yields (Enzymatic and/or structural proteins bring about the physical and biochemical
changes involved in Stage II of hardening). All phases of this hypothesis are under investigation. Results show that tRNA is regulated by photoperiod and temperature, that water content influences frost resistance, that long days or lack of frost inhibit harding, and that decenyl succinic acid does not increase water permeability of plant cells.
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