Mohammed Saleem

Winter Flounder Antifreeze Protein Improves the Cold Hardiness of Plant Tissues

Summary Exposure of plant tissues to the winter flounder antifreeze protein (AFP) has revealed three novel properties by which plant cold hardiness may be improved. Firstly, vacuum infiltration of the protein into leaves of potato, canola ( Brassica napus ) and Arabidopsis thaliana resulted in a significant depression of the spontaneous freezing temperature relative to water infiltrated controls. In the case of canola, the freezing temperature was decreased by an average of 1.8 °C. These results demonstrated the ability of the AFP to function as an anti-nucleator in plant tissues. Secondly, exposure of suspension cultured cells of bromegrass to the antifreeze protein resulted in a reduction in the amount of freezable water frozen at any given temperature. This showed that the protein could act as a cryoprotectant. Thirdly, the antifreeze protein decreased the rate of ice crystal formation. These results demonstrate the feasibility of improving the cold hardiness of plants by introduction of the antifreeze protein gene.

Role of oxidative stress in cereal protoplast recalcitrance

Cereal leaf protoplasts are often extremely unstable in culture and usually lyse within 24 hours. Using the thiobarbituric acid test and the ferrous thiocyanate test we have shown that corn (Zea mays L. cv. Market Beauty) and wheat (Triticum aestivum L. cv. Benito) leaf protoplasts accumulate peroxides and peroxide degradation products during culture. This increase correlated with an increase in lipoxygenase activity. On the other hand, enzymes involved in detoxification of peroxides such as catalase and peroxidase decreased during culture. The occurrence of lipid peroxidation in leaf protoplasts is likely to be a consequence of a temporary imbalance in the enzymes involved in oxygen metabolism. It has previously been shown that the lipoxygenase inhibitor n-propyl gallate stabilizes the protoplasts in culture and so peroxidation is likely to be the cause of leaf protoplast instability. Protoplasts obtained from suspension cultures are stable in culture and do not undergo lipid peroxidation. This stability is due to a decrease in lipoxygenase activity and increases in catalase and peroxidase activity after protoplast isolation.

Design and cloning of a synthetic gene for the flounder antifreeze protein and its expression in plant cells.

A synthetic gene coding for the winter flounder antifreeze protein (AFP) has been constructed. A new strategy for the synthesis has been employed such that one strand of the duplex was chemically synthesized and the other was produced enzymatically by chain extension. The chemically synthesized blocks were constructed so that the second strand was self-priming. The resulting DNA fragment was incorporated into the vector, pGCS1, which contained a translational fusion of the sequence encoding AFP and the N terminus of cat (encoding chloramphenicol acetyltransferase, CAT), under the control of the cauliflower mosaic virus 35S promoter. This plasmid was introduced into protoplasts of corn (var. Black Mexican Sweet) by electroporation. Production of the fusion peptide was monitored by CAT assay and Western blotting with antisera to AFP and CAT.

Improved fusion of mesophyll and cotyledon protoplasts with PEG and high pH-Ca2+ solutions

Summary Commercial-grade polyethylene glycol has been shown to contain substantial amounts of impurities, mostly acidic in character. These impurities were removed by deionization of PEG with a mixed-bed ion exchange resin. The deionized product was shown to be less toxic to mesophyll and young cotyledon protoplasts. The viability and the efficiency of heterokaryocyte formation was better with the deionized PEG. For the fusion of alfalfa mesophyll protoplasts with protoplasts of young cotyledons of soybeans, it was essential to reduce the ratio of PEG solution (33%) to protoplast culture medium from 3:1 to 1:1, and to increase the pH from 5.5 to 6 and calcium concentration from 500 mg · l-1 to over 1000 mg · l-1 in all solutions and culture media to ensure viability of the heterokaryocyte and parental protoplasts. Frequency of PEG and high pH-Ca2+ solution induced fusion was greatly reduced by increasing the osmolality of the eluting solution and enhanced by eluting the PEG with a hypotonic salt solution. The observations suggested that hypotonic shock is essential in PEG induced fusion.

Cereal protoplast recalcitrance

SummaryCereal leaf protoplasts are extremely difficult to culture (recalcitrant) in vitro. There have been few reports of division and the protoplasts typically exhibit excessive enlargement and vacuolization with reduced cell wall deposition. Inasmuch as leaf base explants are capable of callus formation in vitro, protoplasts derived from this tissue must have lost the ability to divide as a consequence of changes induced by the wall-digestion process. We review evidence suggesting that the inhibition of mitosis in these protoplasts is a consequence of a cascade of events initiated at the plasma membrane. The enzyme treatment necessary for wall removal triggers membrane depolarization and other changes that can lead to the initiation of lipid peroxidation and oxidative stress. Mitotically inactive cereal leaf protoplasts are unable to mount a protective response to these degradative processes. Consequently, the resulting membrane perturbations and permeabilization give rise to secondary effects on the cytoskeleton and the cell wall. These effects include reduced or absent microtubules as well as reduced and uneven wall deposition. Such abnormalities are observed in cereal leaf protoplasts and are sufficient to account for recalcitrance because the occurrence of mitosis is strongly dependent on a normal cell wall and cytoskeleton.

Stabilizing corn leaf protoplasts with n-propyl gallate

Summary Corn mesophyll protoplasts are unstable and rapidly deteriorate in culture. Under normalcircumstances, all protoplasts died within 3 days of isolation. Supplementing the protoplast isolation and culture media with the lipoxygenase inhibitor n-propyl gallate (0.25 mM) significantly reduced protoplast lysis. Many of the surviving protoplasts regenerated a cell wall and remained viable for 2 - 3 weeks.

Preparation and Characterization of Chemically and Osmotically Permeabilized Soybean [Glycine max. (L.) Merr.] Protoplasts

Summary Soybean [ Glycine max . (L.) Merr.] protoplasts have been permeabilized with dimethyl-sulfoxide, detergents and hypotonic shock as visualized by the fluorescent dye calcein. Maximum uptake was observed with hypotonic shock. Treated protoplasts were viable and underwent cell division in culture. The internalized dye was distributed throughout the cell, particularly in the vacuole. On the other hand, treatment of protoplasts with PEG resulted in a vesicular localization of fluorescence, concentrated in the cytoplasm. It was also shown that hypotonic shock results in protein (FITC: BSA) uptake.

A 5.3-kilobase genomic fragment from Arabidopsis thaliana containing kin1 and cor6.6.

Cloning: acclimation have been identified in recent years (Lee and Chen, 1993). The gene k in l from Arabidopsis thaliana is particularly interesting because it codes for a 6.5-kD polypeptide that bears some compositional similanty to the fish Alarich antifreeze proteins (Kurkela and Franck, 1990). This similarity as well as its increased expression during cold acclimation has led to the speculation that the product of kinl might be involved in enhancing plant cold tolerance. A homologous gene was more recently reported by Gilmour et al. (1992) as cor6.6 and by Kurkela and Borg-Franck (1992) as kin2. We are interested in how the expression of stress-related genes is regulated. Therefore, we set out to clone sequences containing the promoters of k in l and cor6.6/kin2. Using nonradioactive approaches, we identified genomic clones, and a 5.3-kb region was sequenced that contained both proteincoding sequences and extensive sequences 5’ and 3’ to the transcribed regions (Table I). In the 5311-bp genomic fragment, almost 4 kb consists of two homologous sequences (direct repeats) (nucleotides 6722628 and 2665-4597) with a short spacer (nucleotides 26292664). From previous reports, k in l (Kurkela and Franck, 1990) can be located in the region 1946 to 2758, and cor6.6 (Gilmour et al., 1992) lies between 4006 and 4739. There are three mismatches between the sequence presented here and that reported previously (Kurkela and Franck, 1990), which has an A (instead of T) at 2065 and an A (instead of C) at 2562 and misses 2 As at 2175 and 2176. The same ecotype (Columbia) was used for both studies. The sequence coinciding with cor6.6 is identical. A sequence comparison of the transcribed regions was made previously by Gilmour et al. (1992) for cor6 6. From the present sequence it is interesting to note that the longest transcribed k in l (Kurkela and Franck, 1990) ends at 2743, which is 79 nucleotides into the 5’ of the second homologous sequence. This region thus has homology to the upper 5’ region of k in l (nucleotides 672-751). The 1.4-kb upstream sequences of both genes are mostly homologous. The regions proximal to the transcriptional start

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of 3H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-α, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca2+ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.

Microtubules in maize leaf protoplasts in relation to donor tissue and in vitro culture

SummaryMaize (Zea mays) leaf protoplasts were isolated from various leaves of two-week (4-leaf) seedlings and from sections of the third leaf blades. Microtubules (MTs) were visualized using immunofluorescence microscopy. Only freshly isolated protoplasts from the third and fourth leaf blades contained MTs, with protoplasts from the fourth leaf containing the most i.e. 13% of fourth-leaf protoplasts contained MTs. In general, protoplasts with fewer and smaller chloroplasts had more MTs. Initially 90–95% of protoplasts from basal portions of leaves had MTs but the percentage decreased slightly during culture particularly after 10 days. The antioxidant n-propyl gallate was beneficial in maintaining MT content. Few protoplasts from older sections intitially contained MTs but in all sections at least some protoplasts regained a significant MT content during culture (e.g., 10% of protoplast from the tip section possessed microtubules after 7 days of culture). Far fewer MTs were observed in individual leaf protoplasts than those isolated from suspension culture.