Paul H. Li

Analysis of late-blight disease resistance and freezing tolerance in transgenic potato plants expressing sense and antisense genes for an osmotin-like protein

The expression patterns of plant defense genes encoding osmotin and osmotin-like proteins imply a dual function in osmotic stress and plant pathogen defense. We have produced transgenic potato (Solanum commersonii Dun.) plants constitutively expressing sense or antisense RNAs from chimeric gene constructs consisting of the cauliflower mosaic virus 35S promoter and a cDNA (pA13) for an osmotin-like protein. Transgenic potato plants expressing high levels of the pA13 osmotin-like protein showed an increased tolerance to the late-blight fungus Phytophthora infestans at various phases of infection, with a greater resistance at an early phase of fungal infection. There was a decrease in the accumulation of osmotin-like mRNAs and proteins when antisense transformants were challenged by fungal infection, although the antisense transformants did not exhibit any alterations in disease susceptibility. Expression of pA13 sense and antisense RNAs had no effect on the development of freezing tolerance in transgenic plants when assayed under a variety of conditions including treatments with abscisic acid or low temperature. These results provide evidence of antifungal activity for a potato osmotin-like protein against the fungus P. infestans, but do not indicate that pA13 osmotin-like protein is a major determinant of freezing tolerance.

Anatomical Changes in Leaves of Puma Rye in Response to Growth at Cold-Hardening Temperatures

Rye plants were cold hardened by growth at 4/2 C day/night (D/N); unhardened control plants remained at 25/20 C D/N. Leaves from hardened plants contained 33% less water on a dry weight basis than those from unhardened plants. The osmolar concentration of expressed sap from hardened leaves was 1.5 times greater than that of unhardened leaves. The number of cell layers in fresh leaves remained constant, but leaves from hardened plants were about 1.5 times thicker than those from unhardened plants Increased leaf thickness was caused by increased mesophyll cell size. The structure of the vascular bundle and both stomatal frequency and distribution patterns of the upper and lower epidermal surfaces were altered.

Activation of Two Osmotin-Like Protein Genes by Abiotic Stimuli and Fungal Pathogen in Transgenic Potato Plants

Osmotin-like proteins are encoded by at least six members of a multigene family in Solanum commersonii. A genomic clone ([lambda]pGEM2a-7) that contains two osmotin-like protein genes (OSML13 and OSML81) arranged in the same transcriptional orientation has been isolated. Restriction mapping and sequence analysis indicated that the two intronless genes correspond to the previously characterized pA13 and pA81 cDNAs. To study the transcriptional activation of OSML13 and OSML81 promoters, the 5[prime] flanking DNA sequence (-1078 to +35 of OSML13 and -1054 to +41 of OSML81) was fused to the [beta]-glucuronidase (GUS) coding region, and the chimeric gene fusions were introduced into wild potato (S. commersonii) plants via Agrobacterium-mediated transformation. Analysis of the chimeric gene expression in transgenic potato plants showed that both 5[prime] flanking DNA sequences are sufficient to impart GUS inducibility by abscisic acid, NaCl, salicylic acid, wounding, and fungal infection. Low temperature activated both chimeric genes only slightly. Infection with Phytophthora infestans resulted in strong GUS expression from both chimeric genes primarily in the sites of pathogen invasion, suggesting a limited diffusion of fungal infection-mediated signals. The expression patterns of both osmotin-like protein genes implicate their dual functions in osmotic stress and plant pathogen defense.

EXPRESSION OF THREE OSMOTIN-LIKE PROTEIN GENES IN RESPONSE TO OSMOTIC STRESS AND FUNGAL INFECTION IN POTATO

We have characterized three cDNAs encoding osmotin-like proteins from potato (Solanum commersonii) cell cultures. These cDNAs (pA13, pA35, and pA81) have extensive nucleotide identity in the coding regions but low homology in the 3′ non-coding sequences, and may encode three isoforms of potato pathogenesis-related (PR) type 5 proteins. Using gene-specific probes, RNA gel blot analyses showed constitutive accumulation of osmotin-like protein mRNAs in cell cultures, leaves, stems, roots and flowers, with high abundance in the roots and mature flowers. Treatments with abscisic acid (ABA), low temperature, and NaCl increased the accumulation of all three mRNAs in S. commersonii cell cultures and plants grown in vitro. Salicylic acid (SA), and wounding resulted in a moderate increase in the levels of pA13 and pA81 but not pA35 mRNAs. Infection with the fungus Phytophthora infestans activated strong and non-systemic expression of all three osmotin-like protein genes. The accumulation of osmotin-like proteins, however, was detected only in P. infestans-infected tissues but not in plants treated with ABA, SA, NaCl, low temperature, or wounding.

Expression of an ABA-responsive osmotin-like gene during the induction of freezing tolerance in Solanum commersonii.

We have isolated a cDNA (pA13) of an ABA-responsive gene from suspension cultures of Solanum commersonii. The deduced amino acid sequence of pA13 cDNA revealed 89 and 91% identity with tobacco osmotin and tomato NP24 protein, respectively. The accumulation of the transcript corresponding to pA13 cDNA was regulated by ABA, cold temperature, and low water potential treatments. Cold-induced accumulation of the pA13 transcript was partially suppressed by fluridone, an ABA synthesis inhibitor, and the suppression was restored by exogenous ABA application. The transcript corresponding to pA13 also accumulated in an organ-specific manner in response to ABA or cold treatment.

Plant cold hardiness and freezing stress. Mechanisms and crop implications.

Plant cold hardiness and freezing stress :mechanisms and crop implications , Plant cold hardiness and freezing stress :mechanisms and crop implications , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Relationship between Proline and Abscisic Acid in the Induction of Chilling Tolerance in Maize Suspension-Cultured Cells.

Both proline and abscisic acid (ABA) induce chilling tolerance in chilling-sensitive plants. However, the relationship between proline and ABA in the induction of chilling tolerance is unclear. We compared the time course of the increase in chilling tolerance induced by proline and ABA, and the time course of the uptake of both into the cultured cells of maize (Zea mays L. cv Black Mexican Sweet) at 28[deg]C. The plateau of proline-induced chilling tolerance preceded by 12 h the plateau of ABA-induced chilling tolerance. The uptake of exogenous ABA into the cells reached a plateau in 1 h, whereas the uptake of exogenous proline gradually increased throughout the 24-h culture period. Although the proline content in ABA-treated cells was 2-fold higher than in untreated cells at the end of the 24-h ABA treatment at 28[deg]C, the correlation between the endogenous free proline content and the chilling tolerance in the ABA-treated cells was insignificant. Isobutyric acid treatment, which resulted in a larger accumulation of proline in the cells than ABA treatment, did not increase chilling tolerance. The induction of chilling tolerance by proline and ABA appeared to be additive. Cycloheximide inhibited ABA-induced chilling tolerance, but it did not inhibit proline-induced chilling tolerance. Newly synthesized proteins accumulate in ABA-treated cells at 28[deg]C while the chilling tolerance is developing (Z. Xin and P.H. Li [1993] Plant Physiol 101: 277–284), but none of these proteins were observed in the proline-treated cells. Results suggest that proline and ABA induce chilling tolerance in maize cultured cells by different mechanisms.

Frost killing temperatures of 60 tuber-bearingSolanum species

Environmental factors affect frost hardiness development in many plant species. Plants which are given long days and warm temperatures grow and fail to harden. Short days and warm temperatures stop growth and enhance hardiness. Low temperatures and sufficient light also increase hardiness, regardless of photoperiod. Solanum tuberosum possesses very little or no frost tolerance while a number of wild species (S. acaule, S. chomatophilum, S. commersonii, S. multidissectum, etc.) are considered to be frost tolerant. The major difference between a tender and hardy species is the ability of the hardy species to tolerate more frozen water at frost killing temperatures. Low temperature appears to be the primary environmental factor in the development of frost hardiness in potato plants, and S. tuberosum apparently does not have the physiological bases for developing frost hardiness. It is generally agreed among plant stress physiologists that plants which tolerate cold are probably also tolerant of drought, heat and, perhaps, salt stress. Information concerning the frost killing temperatures of the following 60 tuber-bearing Solanum species may, therefore, be interesting to those who are working in the area of environmental stress. Tests were conducted in a controlled freezing apparatus and data were collected from leaf tissues of plants grown in a greenhouse under a regime of long days and warm temperatures.

Classification of plant cell cryoprotectants

Current theories of freezing injury to plant cells and interpretations of cryopreservation do not take the role of the cell wall into consideration. Freezing injury relative to the role of the cell wall is discussed. A classification of plant cell cryoprotectants into the following three categories is proposed based on their penetrating ability as determined by molecular weight: namely, cryoprotectants which are (1) penetrable through the cell wall and into the protoplast, (2) penetrable through the cell wall only, and (3) unable to penetrate through the cell wall. Each category of cryoprotectants has a different role in cryopreservation, and a proper combination of the three should, therefore, provide the optimum cellular cryoprotection environment of plant cells.

Relationship between heat and frost resistance of tuber-bearing Solanum species : effect of cold acclimation on heat resistance.

A possible relationship between heat and freezing-stress resistance was investigated in four potato species. These species differed in degree of freezing-stress resistance and ability to acclimate to cold. The 2, 3, 5-triphenyl tetrazolium chloride reduction and the conductivity test were used to evaluate the relative heat and freezing-stress resistance of excised leaflets. Data from both tests failed to show a systematic relationship between heat and freezing-stress resistance in the four potato species. The cultivated species, Solanum tuberosum, had the lowest level of freezing-stress resistance and one of the highest levels of heat-stress resistance. One noncultivated accession had a high degree of frost and heat resistance, whereas the other three accessions had a high degree of frost but poor heat resistance. Fifteen days at 5/2 C day/night did not increase heat- and freezing-stress resistance of S. tuberosum but increased both heat- and freezing-stress resistance in S. commersonii and other species capable of cold acclimation. These results show that (1) resistance to heat and freezing stress is not always mutually exclusive; (2) if a plant is capable of increasing freezing-stress resistance during cold acclimation, it also seems to have the ability to increase heat-stress resistance; and (3) the enzymes that participate in oxidation-reduction reactions and active transport of ions are sensitive to heat injury.