Manipulating freezing tolerance in transgenic plants

Winterhardiness is a composite of tolerances to freezing, desiccation, ice-encasement, flooding and diseases. From one point of view, winterhardiness may not be easily manipulated by genetic engineering technology because many different genes are involved in the tolerance of these diverse stresses. However, these various stresses have similarities. They promote formation of activated forms of oxygen, promote membrane lipid and protein degradation, cause similar biophysical changes in membrane structure, and culminate with increased leakage of cytoplasmic solutes and loss of cellular membrane functions. These similarities led to the hypothesis that winter injury might be reduced in crop plants if their tolerance of oxidative stress was increased. Towards that objective we created transgenic alfalfa (Medicago sativa L.) plants that overexpress either Mn-SOD or Fe-SOD cDNA (provided by Dirk Inzé, Universiteit Gent). Petiole explants were transformed using Agrobacterium tumefaciens and plants were regenerated by somatic embryogenesis. The primary transgenic plants were screened using PCR (polymerase chain reaction), Southern hybridization and native PAGE for SOD activity. Greenhouse and laboratory studies showed a minimal difference in stress tolerance between the primary transgenic and non-transgenic plants. In the first field trial, four primary transgenic plants expressing two forms of the Mn-SOD cDNA had greater survival after two winters than the non-transgenic RA3. Similar results were obtained in a second field trial, comparing 18 independent transformants with Mn-SOD targeted to the mitochondria, 11 independent transformants with Mn-SOD targeted to the chloroplast and 39 independent transformants with Fe-SOD targeted to the chloroplast, expressed in three different non-transgenic plants. The transgenic plants averaged over 25% higher survival than the non-transgenic controls after one winter. There was no effect of subcellular targeting or SOD type on field survival, but there was variation among independent transformants containing the same SOD construct. Activated oxygen therefore appears to be one of the possible causes of winter injury, and it should be possible to reduce winter injury in transgenic plants by constitutive overexpression of SOD.     

Iron-superoxide dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance

To determine whether overexpression of Fe-superoxide (SOD) dismutase would increase superoxide-scavenging capacity and thereby improve the winter survival of transgenic alfalfa (Medicago sativa L.) plants, two genotypes were transformed with the vector pEXSOD10, which contains a cDNA for Arabidopsis Fe-SOD with a chloroplast transit peptide and cauliflower mosaic virus 35S promoter. A novel Fe-SOD was detected by native PAGE in both greenhouse- and field-grown transgenic plants, but activity varied among independent transgenic plants. The increased Fe-SOD activity was associated with increased winter survival over 2 years in field trials, but not with oxidative stress tolerance as measured by resistance of leaves to methyl viologen, a superoxide generator. Total shoot dry matter production over 2 harvest years was not associated with Fe-SOD activity. There was no detectable difference in the pattern of primary freezing injury, as shown by vital staining, nor was there additional accumulation of carbohydrates in field-acclimated roots of the transgenic alfalfa plants. We did not detect any difference in growth of one transgenic plant with high Fe-SOD activity compared with a non-transgenic control. Therefore, the improvement in winter survival did not appear to be a consequence of improved oxidative stress tolerance associated with photosynthesis, nor was it a consequence of a change in primary freezing injury. We suggest that Fe-SOD overexpression reduced secondary injury symptoms and thereby enhanced recovery from stresses experienced during winter.     

Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.).

Activated oxygen or oxygen free radicals have been implicated in a number of physiological disorders in plants including freezing injury. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide into O2 and H2O2 and thereby reduces the titer of activated oxygen molecules in the cell. To further examine the relationship between oxidative and freezing stresses, the expression of SOD was modified in transgenic alfalfa (Medicago sativa L.). The Mn-SOD cDNA from Nicotiana plumbaginifolia under the control of the cauliflower mosaic virus 35S promoter was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation. Two plasmid vectors, pMitSOD and pChlSOD, contained a chimeric Mn-SOD construct with a transit peptide for targeting to the mitochondria or one for targeting to the chloroplast, respectively. The putatively transgenic plants were selected for resistance to kanamycin and screened for neomycin phosphotransferase activity and the presence of an additional Mn-SOD isozyme. Detailed analysis of a set of four selected transformants indicated that some had enhanced SOD activity, increased tolerance to the diphenyl ether herbicide, acifluorfen, and increased regrowth after freezing stress. The F1 progeny of one line, RA3-ChlSOD-30, were analyzed by SOD isozyme activity, by polymerase chain reaction for the Mn-SOD gene, and by polymerase chain reaction for the neo gene. RA3-ChlSOD-30 had three sites of insertion of pChlSOD, but only one gave a functional Mn-SOD isozyme; the other two were apparently partial insertions. The progeny with a functional Mn-SOD transgene had more rapid regrowth following freezing stress than those progeny lacking the functional Mn-SOD transgene, suggesting that Mn-SOD serves a protective role by minimizing oxygen free radical production after freezing stress.     

Water-Deficit Tolerance and Field Performance of Transgenic Alfalfa Overexpressing Superoxide Dismutase

Transgenic alfalfa (Medicago sativa) expressing Mn-superoxide dismutase cDNA tended to have reduced injury from water-deficit stress as determined by chlorophyll fluorescence, electrolyte leakage, and regrowth from crowns. A 3-year field trial indicated that yield and survival of transgenic plants were significantly improved, supporting the hypothesis that tolerance of oxidative stress is important in adaptation to field environments.     

Accumulation of overproduced ferritin in the chloroplast provides protection against photoinhibition induced by low temperature in tobacco plants

Wild-type tobacco plants (Nicotiana tabacum L. cv. Petit Havanna line SR1) and plants transformed with full-length alfalfa ferritin cDNA with the chloroplast transit peptide under the control of a Rubisco small subunit gene promoter (C3 and C8) were cold-treated at 0 degrees C with continuous light (250mumolm(-2)s(-1)). These transgenic plants had higher chlorophyll content and higher F(v)/F(m) chlorophyll-a fluorescence induction parameters than wild-type plants after 2 or 3d of cold treatment in C3 and C8 transgenic plants, respectively. Thermoluminescence studies on the high-temperature bands suggest that these plants suffered less oxidative damage in comparison to the wild-type genotype. The present experiments provide evidence that transgenic tobacco lines overexpressing alfalfa ferritin, which is accumulated in the chloroplasts, may show higher tolerance to various stress factors, generating ROS including low temperature-induced photoinhibition.     

Effects of water stress on antioxidant enzymes of leaves and nodules of transgenic alfalfa overexpressing superoxide dismutases

The antioxidant composition and relative water stress tolerance of nodulated alfalfa plants ( Medicago sativa L. ×  Sinorhizobium meliloti 102F78) of the elite genotype N4 and three derived transgenic lines have been studied in detail. These transgenic lines overproduced, respectively, Mn-containing superoxide dismutase (SOD) in the mitochondria of leaves and nodules, MnSOD in the chloroplasts, and FeSOD in the chloroplasts. In general for all lines, water stress caused moderate decreases in MnSOD and FeSOD activities in both leaves and nodules, but had distinct tissue-dependent effects on the activities of the peroxide-scavenging enzymes. During water stress, with a few exceptions, ascorbate peroxidase and catalase activities increased moderately in leaves but decreased in nodules. At mild water stress, transgenic lines showed, on average, 20% higher photosynthetic activity than the parental line, which suggests a superior tolerance of transgenic plants under these conditions. However, the untransformed and the transgenic plants performed similarly during moderate and severe water stress and recovery with respect to important markers of metabolic activity and of oxidative stress in leaves and nodules. We conclude that the base genotype used for transformation and the background SOD isozymic composition may have a profound effect on the relative tolerance of the transgenic lines to abiotic stress.     

A novel cold-regulated gene from Phlox subulata, PsCor413im1, enhances low temperature tolerance in Arabidopsis

Low temperature stress adversely affects plant growth, development, and crop productivity. Analysis of the function of genes in the response of plants to low temperature stress is essential for understanding the mechanism of chilling and freezing tolerance. In this study, PsCor413im1, a novel cold-regulated gene isolated from Phlox subulata, was transferred to Arabidopsis to investigate its function under low temperature stress. Real-time quantitative PCR analysis revealed that PsCor413im1 expression was induced by cold and abscisic acid. Subcellular localization revealed that PsCor413im1-GFP fusion protein was localized to the periphery of the chloroplast, consistent with the localization of chloroplast inner membrane protein AtCor413im1, indicating that PsCor413im1 is a chloroplast membrane protein. Furthermore, the N-terminal of PsCor413im1 was determined to be necessary for its localization. Compared to the wild-type plants, transgenic plants showed higher germination and survival rates under cold and freezing stress. Moreover, the expression of AtCor15 in transgenic plants was higher than that in the wild-type plants under cold stress. Taken together, our results suggest that the overexpression of PsCor413im1 enhances low temperature tolerance in Arabidopsis.     

转柽柳eIF1A基因烟草的耐盐性分析

目的:验证柽柳eIF1A基因的功能,为通过基因工程手段培育耐盐植物提供基础资料。方法:对转eIF1A基因的烟草和对照烟草进行不同浓度NaCl胁迫实验,测定其相对电导率、SOD活性和丙二醛含量,统计生根率、生长量和盐害程度。结果:转基因烟草的相对电导率、丙二醛含量均随盐浓度的增加而增大,但都较非转基因对照烟草低。SOD活性随着盐浓度的升高而升高,相同浓度NaCl胁迫下各转基因烟草的SOD活性均高于对照烟草的SOD活性。NaCl浓度为240mmol/L时,非转基因对照烟草不能生根,盐害指数高达67.7%;而转基因烟草均能生根,大部分转基因株系的生根率大于50%。结论:柽柳eIF1A基因的转化提高了烟草的耐盐性。     

Effects of salicylic acid and cold on freezing tolerance in winter wheat leaves

The effects of salicylic acid (SA) (0.01, 0.1 and 1 mM) and cold on freezing tolerance (freezing injury and ice nucleation activity) were investigated in winter wheat (Triticum aestivum cv. Dogu-88) grown under control (20/18 °C for 15, 30 and 45-day) and cold (15/10 °C for 15-day, 10/5 °C for 30-day and 5/3 °C for 45-day) conditions. Cold acclimatisation caused a decrease of injury to leaf segments removed from the plants and subjected to freezing conditions. Exogenous SA also decreased freezing injury in the leaves grown under cold (15/10 °C) and control (15 and 30-day) conditions. Cold conditions (10/5 and 5/3 °C) caused an increase in ice nucleation activity by apoplastic proteins, which were isolated from the leaves. For the first time, it was shown that exogenous SA caused an increase in ice nucleation activity under cold (15/10 and 10/5 °C) and control conditions. These results show that salicylic acid can increase freezing tolerance in winter wheat leaves by affecting apoplastic proteins.     

Modification of reactive oxygen species scavenging capacity of chloroplasts through plastid transformation

Reactive oxygen species (ROS), including superoxide anions, hydrogen peroxide and hydroxyl radicals are generated through normal biochemical processes, but their production is increased by abiotic stresses. The prospects for enhancing ROS scavenging, and hence stress tolerance, by direct gene expression in a vulnerable cell compartment, the chloroplast, have been explored in tobacco. Several plastid transformants were generated which contained either a Nicotiana mitochondrial superoxide dismutase (MnSOD) or an Escherichia coli glutathione reductase (gor) gene. MnSOD lines had a three-fold increase in MnSOD activity, but interestingly a five to nine-fold increase in total chloroplast SOD activity. Gor transgenic lines had up to 6 times higher GR activity and up to 8 times total glutathione levels compared to wild type tobacco. Photosynthetic capacity of transplastomic plants, as measured by chlorophyll content and variable fluorescence of PSII was equivalent to non-transformed plants. The response of these transplastomic lines to several applied stresses was examined. In a number of cases improved stress tolerance was observed. Examples include enhanced methyl viologen (Paraquat)-induced oxidative stress tolerance in Mn-superoxidase dismutase over-expressing plants, improved heavy metal tolerance in glutathione reductase expressing lines, and improved tolerance to UV-B radiation in both sets of plants.     

Enhanced salt tolerance of transgenic poplar plants expressing a manganese superoxide dismutase from <Emphasis Type="Italic">Tamarix androssowii</Emphasis>

Superoxide dismutases (SODs) play important role in stress tolerance of plants. In this study, an MnSOD gene (TaMnSOD) from Tamarix androssowii, under the control of the CaMV35S promoter, was introduced into poplar (Populus davidiana × P. bolleana). The physiological parameters, including SOD activity, malondialdehyde (MDA) content, relative electrical conductivity (REC) and relative weight gain, of transgenic lines and wild type (WT) plants, were measured and compared. The results showed that SOD activity was enhanced in transgenic plants, and the MDA content and REC were significantly decreased compared to WT plants when exposed to NaCl stress. In addition, the relative weight gains of the transgenic plants were 8- to 23-fold of those observed for WT plants after NaCl stress for 30 days. The data showed that the SOD activities that increased in transgenic lines are 1.3–4-folds of that increased in the WT plant when exposed to NaCl stress. Our analysis showed that increases in SOD activities as low as 0.15-fold can also significantly enhance salt tolerance in transgenic plants, suggesting an important role of increased SOD activity in plant salt tolerance.     

Phylogenetic distribution of superoxide dismutase supports an endosymbiotic origin for chloroplasts and mitochondria

Three isozymes of superoxide dismutase (SOD) have been identified and characterized. The iron and manganese isozymes (Fe-SOD and Mn-SOD, respectively) show extensive primary sequence and structural homology, suggesting a common evolutionary ancestor. In contrast, the copper/zinc isozyme (CuZn-SOD) shows no homology with Fe-SOD or Mn-SOD, suggesting an independent origin for this enzyme. The three isozymes are unequally distributed throughout the biological kingdoms and are located in different subcellular compartments. Obligate anaerobes and aerobic diazotrophs contain Fe-SOD exclusively. Facultative aerobes contain either Fe-SOD or Mn-SOD or both. Fe-SOD is found in the cytosol of cyanobacteria while the thylakoid membranes of these organisms contain a tightly bound Mn-SOD. Similarly, most eukaryotic algae contain Fe-SOD in the chloroplast stroma and Mn-SOD bound to the thylakoids. Most higher plants contain a cytosol-specific and a chloroplast-specific CuZn-SOD, and possibly a thylakoid-bound Mn-SOD as well. Plants also contain Mn-SOD in their mitochondria. Likewise, animals and fungi contain a cytosolic CuZn-SOD and a mitochondrial Mn-SOD. The Mn-SOD found in the mitochondria of eukaryotes shows strong homology to the prokaryotic form of the enzyme. Taken together, the phylogenetic distribution and subcellular localization of the SOD isozymes provide strong support for the hypothesis that the chloroplasts and mitochondria of eukaryotic cells arose from prokaryotic endosymbionts.     

Differential remodeling of the lipidome during cold acclimation in natural accessions of Arabidopsis thaliana

Freezing injury is a major factor limiting the geographical distribution of plant species and the growth and yield of crop plants. Plants from temperate climates are able to increase their freezing tolerance during exposure to low but non‐freezing temperatures in a process termed cold acclimation. Damage to cellular membranes is the major cause of freezing injury in plants, and membrane lipid composition is strongly modified during cold acclimation. Forward and reverse genetic approaches have been used to probe the role of specific lipid‐modifying enzymes in the freezing tolerance of plants. In the present paper we describe an alternative ecological genomics approach that relies on the natural genetic variation within a species. Arabidopsis thaliana has a wide geographical range throughout the Northern Hemisphere with significant natural variation in freezing tolerance that was used for a comparative analysis of the lipidomes of 15 Arabidopsis accessions using ultra‐performance liquid chromatography coupled to Fourier‐transform mass spectrometry, allowing the detection of 180 lipid species. After 14 days of cold acclimation at 4°C the plants from most accessions had accumulated massive amounts of storage lipids, with most of the changes in long‐chain unsaturated triacylglycerides, while the total amount of membrane lipids was only slightly changed. Nevertheless, major changes in the relative amounts of different membrane lipids were also evident. The relative abundance of several lipid species was highly correlated with the freezing tolerance of the accessions, allowing the identification of possible marker lipids for plant freezing tolerance.