Subhash C. Minocha

Molecular biology of woody plants

Section A. 1. Gene transfer techniques and their relevance to woody plants S.C. Minocha, J.C. Wallace. 2. Selection of marker-free transgenics plants using the oncogenes (ipt, rol A, B, C) of Agrobacterium as selectable markers H. Ebinuma, et al. 3. Agrobacterium rhizogenes for rooting recalcitrant woody species H.M. Haggman, T.S. Aronen. 4. Genetic engineering of conifers for plantation forestry Pinus radiata transformation C. Walter, L.J. Grace. 5. Transformation of Picea species D.H. Clapham, et al. 6. Transgenic in Larix M.A. Lelu, G. Pilate. 7. Genetic transformation of Populus toward improving plant performance and drought tolerance T. Tzfira, et al. 8. Progress on genetic engineering in four tropical Acacia spp. M. Quoirin, et al. 9. Genetic engineering of rose (Rosa species) M.R. Davey, et al. 10. Transformation of Actinidia species (kiwifruit) E. Rugini, et al. 11. Genetic transformation in Citrus G.A. Moore, et al. 12. Olive (Olea europaea var. sativa) transformation E. Rugini. 13. Transformation of Malus F.A. Hammerschlag. 14. Genetic transformation of Hevea brasiliensis (rubber trees) and its applications towards crop improvement and production of recombinant proteins of commercial value P. Arokiaraj. 15. Production of Transgenic oil palm (Elaeis guinensis JACQ.). using biolistic techniques G. Kadir, A. Parveez. Section B. 16. Molecular characterization of the mycorrhizas of woody plants S. Hambleton, R.S. Currah. 17. Molecular epidemiology tree pathogens R.C. Hamelin. 18. Development of insect resistance in fruit and nut tree crops M. Escob, A.M. Dandekar. 19. Structural and biochemical aspects of cold hardiness in woody plants M. Wisniewski, R. Arora. 20. Herbicide tolerant forest trees D.J. Llewellyn. 21. Cloning of defense related genes against pathogens in forest trees G. Lakshmi Sita, et al. Section C. 22. Research Ethics for Molecular Silviculture P.B. Thompson, S.H. Strauss.

Polyamines and abiotic stress in plants: a complex relationship.

The physiological relationship between abiotic stress in plants and polyamines was reported more than 40 years ago. Ever since there has been a debate as to whether increased polyamines protect plants against abiotic stress (e.g., due to their ability to deal with oxidative radicals) or cause damage to them (perhaps due to hydrogen peroxide produced by their catabolism). The observation that cellular polyamines are typically elevated in plants under both short-term as well as long-term abiotic stress conditions is consistent with the possibility of their dual effects, i.e., being protectors from as well as perpetrators of stress damage to the cells. The observed increase in tolerance of plants to abiotic stress when their cellular contents are elevated by either exogenous treatment with polyamines or through genetic engineering with genes encoding polyamine biosynthetic enzymes is indicative of a protective role for them. However, through their catabolic production of hydrogen peroxide and acrolein, both strong oxidizers, they can potentially be the cause of cellular harm during stress. In fact, somewhat enigmatic but strong positive relationship between abiotic stress and foliar polyamines has been proposed as a potential biochemical marker of persistent environmental stress in forest trees in which phenotypic symptoms of stress are not yet visible. Such markers may help forewarn forest managers to undertake amelioration strategies before the appearance of visual symptoms of stress and damage at which stage it is often too late for implementing strategies for stress remediation and reversal of damage. This review provides a comprehensive and critical evaluation of the published literature on interactions between abiotic stress and polyamines in plants, and examines the experimental strategies used to understand the functional significance of this relationship with the aim of improving plant productivity, especially under conditions of abiotic stress.

Polyamines and cellular metabolism in plants: transgenic approaches reveal different responses to diamine putrescine versus higher polyamines spermidine and spermine

Distribution of biogenic amines—the diamine putrescine (Put), triamine spermidine (Spd), and tetraamine spermine (Spm)—differs between species with Put and Spd being particularly abundant and Spm the least abundant in plant cells. These amines are important for cell viability and their intracellular levels are tightly regulated, which have made it difficult to characterize individual effects of Put, Spd and Spm on plant growth and developmental processes. The recent transgenic intervention and mutational genetics have made it possible to stably alter levels of naturally occurring polyamines and study their biological effects. We bring together an analysis of certain metabolic changes, particularly in amino acids, to infer the responsive regulation brought about by increased diamine or polyamine levels in actively growing poplar cell cultures (transformed with mouse ornithine decarboxylase gene to accumulate high Put levels) and ripening tomato pericarp (transformed with yeast S-adenosylmethionine decarboxylase gene to accumulate high Spd and Spm levels at the cost of Put). Our analysis indicates that increased Put has little effect on increasing the levels of Spd and Spm, while Spd and Spm levels are inter-dependent. Further, Put levels were positively associated with Ala (α and β), Ile and GABA and negatively correlated with Gln and Glu in both actively growing poplar cell cultures and non-dividing tomato pericarp tissue. Most amino acids showed positive correlations with Spd and Spm levels in actively growing cells. Collectively these results suggest that Put is a negative regulator while Spd–Spm are positive regulators of cellular amino acid metabolism.

Increased Putrescine Biosynthesis through Transfer of Mouse Ornithine Decarboxylase cDNA in Carrot Promotes Somatic Embryogenesis.

Carrot (Daucus carota L.) cells were transformed with Agrobacterium tumefaciens strains containing 3[prime]-truncated mouse ornithine decarboxylase (ODC) cDNA under the control of a cauliflower mosaic virus 35S promoter. A neomycin phosphotransferase gene linked with a nopaline synthase promoter was used to select transformed cell lines on kanamycin. Although the nontransformed cells contained no ODC, high amounts of mouse-specific ODC activity were observed in the transformed cells. Transgenic cells showed a significant increase in the cellular content of putrescine compared to control cells. Spermidine, however, remained unaffected. Not only did the transformed cells exhibit improved somatic embryogenesis in the auxin-free medium, they also regenerated some embryos in the presence of inhibitory concentrations of 2,4-dichlorophenoxyacetic acid. These cells acquired tolerance to [alpha]-difluoromethylarginine (a potent inhibitor of arginine decarboxylase) at concentrations that inhibit growth as well as embryogenesis in nontransformed carrot cells, showing that the mouse ODC can replace the carrot arginine decarboxylase for putrescine biosynthesis in the transgenic cells.

High-performance liquid chromatographic method for the determination of dansyl-polyammines☆

Abstract This paper describes a fast reliable, and a sensitive technique for the separation and quantification of dansylated polyamines by high-performance liquid chromatography. Using a small 33 × 4.6 mm I.D., 3 μm particle size, C18 reversed-phase cartridge column and a linear gradient of acetonitrile—heptanesulfonate (10 mM, Ph 3.4), at a flow-rate of 2.5 ml/min, the retention time for different polyamines was: N8-acetyl-spermidine, 1.79 min; N1-acetylspermidine, 1.82 min;putrescine, 2.26 min; cadaverine, 2.43 min; heptanediamine, 2.83 min; spermidine, 3.42 min; and spermine, 4.41 min. With an additional column regeneration time of 3—4 min, the complete cycle per sample took less than 8 min at room temperture. Using a fluorescence detector, the lower limit of detection was less than 1 pmol per 6 μl injection volume. The fluorescence response was linear up to 200 pmol per 6 μl for each polyamine. The method is suitable for separation of polyamines from animal, plant and fungal sources.

Modulation of cellular polyamines in tobacco by transfer and expression of mouse ornithine decarboxylase cDNA

In an attempt to modulate the metabolism of polyamines in plants, Agrobacterium tumefaciens strains were produced which contained either a full-length or a 3′-truncated mouse ornithine decarboxylase (ODC) cDNA under the control of the cauliflower mosaic virus 35S promoter. Plants of Nicotiana tabacum cv. Xanthi were used for transformation with these two strains of Agrobacterium. Transformations were confirmed by Southern hybridization and amplification by polymerase chain reaction. Two plants containing the full-length cDNA (ODC-12 and ODC-30) and two containing the truncated cDNA (12701-2 and 12701-31) were selected for further experiments. Northern blot analysis indicated that transcription was occurring and western blot analysis detected a polypeptide of ca. 50 kDa that was unique to the plants transformed with truncated ODC cDNA. In order to distinguish between the native and the mouse ODC in the transformed tissues, enzyme activity was assayed at pH optima for the two enzymes, i.e. pH 8.2 and 6.8, respectively.Substantially higher levels of ODC activity were seen at pH 6.8 (optimum for mouse ODC) in the transformants as compared to the controls. This ODC activity was inhibited by α-difluoromethylornithine and anti-mouse ODC antisera in a manner consistent with that reported for the mouse ODC. Analysis of cellular polyamines showed significantly elevated levels (4–12-fold) of putrescine in callus derived from transformed plant tissues as compared to the controls. The modulation of polyamine biosynthesis in plants by these techniques should allow us to further analyze the role of these ubiquitous compounds in plant growth and development.

A Rapid and Reliable Procedure for Extraction of Cellular Polyamines and Inorganic Ions from Plant Tissues

A fast and reliable method for the extraction of cellular polyamines and major inorganic ions (Ca, Mg, Mn, K, and P) from several plant tissues is described. The method involves repeated freezing and thawing of samples instead of homogenization. The efficiency of extraction of both the polyamines and inorganic ions by these two methods was compared for 10 different tissues. In each case, the freeze-thaw procedure resulted in a precise and quantitatively equal, or greater, yield than homogenization. Freeze-thawing not only eliminates the need for various tissue homogenizers (such as polytrons, tissumizers, and mortars and pestles), but it is so simple that a large number of samples can be processed simultaneously. We routinely processed 50–80 samples for quantitation of polyamines and inorganic ions. Freeze-thawing was equally useful for the extraction of polyamines from liver, spleen, and kidney tissues of mice.

Genetic Manipulation of the Metabolism of Polyamines in Poplar Cells. The Regulation of Putrescine Catabolism

We investigated the catabolism of putrescine (Put) in a non-transgenic (NT) and a transgenic cell line of poplar (Populus nigra × maximowiczii) expressing a mouse (Mus musculus) ornithine (Orn) decarboxylase (odc) cDNA. The transgenic cells produce 3- to 4-fold higher amounts of Put than the NT cells. The rate of loss of Put from the cells and the initial half-life of cellular Put were determined by feeding the cells with [U-14C]Orn and [1,4-14C]Put as precursors and following the loss of [14C]Put in the cells at various times after transfer to label-free medium. The amount of Put converted into spermidine as well as the loss of Put per gram fresh weight were significantly higher in the transgenic cells than the NT cells. The initial half-life of exogenously supplied [14C]Put was not significantly different in the two cell lines. The activity of diamine oxidase, the major enzyme involved in Put catabolism, was comparable in the two cell lines even though the Put content of the transgenic cells was severalfold higher than the NT cells. It is concluded that in poplar cells: (a) exogenously supplied Orn enters the cells and is rapidly converted into Put, (b) the rate of Put catabolism is proportional to the rate of its biosynthesis, and (c) the increased Put degradation occurs without significant changes in the activity of diamine oxidase.

Polyamines and somatic embryogenesis in carrot. I. The effects of difluoromethylornithine and difluoromethylarginine

Abstract 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used and a very effective inhibitor of somatic embryogenesis in carrot. This growth regulator not only suppresses differentiation of cultured cells but it can also cause a reversion of the developing embryos to undifferentiated callus. Data presented here show that the addition of 1–10 mM DFMO (difluoromethylornithine) to the medium allowed the normal development of somatic embryos to continue even in the presence of inhibitory concentrations of 2,4-D. DFMO caused a significant increase in ADC activity, an increased accumulation of polyamines in the cells, and inhibited the accumulation of ethylene in cell cultures both in the presence or the absence of 2,4-D. Difluoromethylarginine (DFMA) at 0.1–1.0 mM concentration completely inhibited embryogenesis even in the absence of 2,4-D. DFMA also inhibited ADC activity and caused a reduction in the cellular polyamine levels. ODC activity was detected only when fully mature somatic embryos appeared in the cultures. It is suggested that auxin-induced ethylene biosynthesis plays an important role in the development of somatic embryos in carrot. The promotion of polyamine biosynthesis (by DFMO in the present case) may cause a reduction in the cellular pools of S -adenosylmethionine, which in turn may cause a reduction in ethylene biosynthesis, thus allowing embryogenesis to occur in the presence of an auxin.

Expression of a human S-adenosylmethionine decarboxylase cDNA in transgenic tobacco and its effects on polyamine biosynthesis

S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key regulatory enzyme in the polyamine biosynthetic pathway. Numerous studies have shown that the enzyme activity and polyamine levels are generally correlated with cellular growth in plants, animals and bacteria. In order to gain more insight into the role of polyamines in plants, human SAMDC cDNA under control of the 35S promoter of cauliflower mosaic virus, along with a neomycin phosphotransferase gene, was transferred to tobacco (Nicotiana tabacum cv. Xanthi) viaAgrobacterium tumefaciens. Transgenic plants showed the presence of human SAMDC mRNA and a 2-4-fold increase in SAMDC activity. In the transformed tissues, putrescine levels were significantly reduced, while spermidine content was 2–3 times higher than the control tissues. Cellular spermine content was either increased or remained unchanged. Excised leaf segments from transformed plants frequently produced shoots even on callus inducing medium.