Dan Pelah

Water-stress response in aspen (Populus tremula): Differential accumulation of dehydrin, sucrose synthase, GAPDH homologues, and soluble sugars

Summary In a previous study we identified a novel 66-kD boiling-stable protein (BspA) that accumulates in cultured shoots of aspen ( Populus tremula L.) in response to gradual water stress and ABA application, as well as to osmotic and cold stresses (Altman et al., 1996; Pelah et al., 1995). BspA was found to be the major heat-stable water stress-responsive protein in aspen. Here, the expression of other water-stress responsive proteins — DSP 16 (dehydrin), cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and sucrose synthase — was studied in aspen roots and shoots. These proteins were identified immunologically using polyclonal antibodies isolated from the resurrection plant Craterostigma plantagineum . A 43-kD homologue and a 31-kD homologue of dehydrin were found to accumulate in roots and shoots of aspen after water stress in a tissue specific manner. Shoots and roots of intact plantlets subjected to ABA application accumulated a 33-kD dehydrin homologue. A sucrose synthase homologue (90 kD) accumulated in water-stressed shoots and was constitutively present at high levels in control and water-stressed roots harvested from intact plandets. GAPDH, a key enzyme in the glycolysis and gluconeogenesis pathways, exhibited some accumulation in shoots in response to water stress. Sugar analysis revealed that water stress results in increased sucrose and decreased glucose levels in the aspen leaves. The data presented here suggest that the water-stress-response mechanisms present in herbaceous plants are also present in woody plants. Woody plants subjected to water stress accumulate dehydrin-like proteins and specific enzymes such as sucrose synthase and GAPDH, which may play a dominant role in sugar metabolism under these conditions.

Tomato yellow leaf curl virus DNA in callus cultures derived from infected tomato leaves

Callus cultures were induced from leaves of a tomato plant infected with tomato yellow leaf curl virus (TYLCV) and analyzed for viral DNA presence during successive subcultures. No TYLCV DNA was detected in calli sampled after eight months of culture. Considerable differences in the presence of TYLCV DNA were found within sectors of a callus culture and between different callus cultures, throughout the entire eight months period. Infected calli which were cultured at sub-optimal temperature (15°C) retained the viral DNA longer than at 25 °C. The results suggested that TYLCV disappearance during callus culture was due to a disruption of some of the cell-to-cell connections, resulting in islands of infected cells in the midst of uninfected tissue and/or to the competition between the rate of cell division and that of viral DNA replication.

TOWARDS WATER STRESS-TOLERANT POPLAR AND PINE TREES: MOLECULAR BIOLOGY, TRANSFORMATION AND REGENERATION

Novel procedures for the improvement of current breeding, selection and forestation techniques can only be achieved by combining conventional breeding and selection procedures, rapid and efficient propagation, new planting and land use approaches, and novel biotechnological methodologies. An integrated biotechnological approach should be useful both for determining the physiological and molecular basis of tree tolerance, and for rapid selection and tree improvement. In the following, we present data from our studies on micropropagation, molecular analysis of water-stress tolerance, and transformation of Populus spp. and Pinus halepensis. Axillary bud break was induced in Populus stem and leaf cultures, and adventitious buds and roots were formed, resulting in acclimatized plants. Procedures for adventitious shoot formation in root cultures of aspen (Populus tremula) are currently under study. Expiants of mature zygotic embryos, seedlings, and cytokinin-treated mature trees ofP. halepensis are being studied for in vitro clonal propagation. Efficient procedures for the massive induction of first- and second-cycle adventitious shoots from embryos were established. Buds were also induced on pine seedling expiants and on detached needles from long-term embryo culture. Fascicular buds were induced in 3-year-old pine trees, and they were cultured in vitro for further regeneration. Somatic embryogenesis is also being studied. Drought-specific proteins/genes were studied in several Populus species and in P. halepensis. A novel 66 kDa boiling-stable protein was highly expressed in P. tremula shoots, as early as 1 h after gradual water loss and ABA application, as found by Pelah et al. [19]. Using Populus clones which differed in their drought tolerance, we found a good correlation between the expression of dehydrin-like proteins and sucrose synthase and the degree of drought tolerance and ion leakage [21]. cDNAs of the 66 kDa and related proteins are being employed for additional molecular characterization. Here we present additional evidence for the effect of cold and osmotic stresses on protein expression. The 66 kDa boiling-stable protein was highly expressed in aspen callus cultures subjected to osmotic stress in a medium containing 6 or 12% mannitol, simultaneously with a decrease in cellular water potential. The same protein accumulated in response to cold stress, upon culture of aspen callus and shoots at 4°C. A highly efficient transformation and regeneration procedure of aspen, which does not require a pre-selection stage on antibiotics, was established. P. halepensis embryos, seedlings and mature buds were also efficiently transformed, as monitored by root formation and/or GUS expression. These are fully described by Tzfira et al. [29, 31].

In Vitro Organogenesis, Transformation and Expression of Drought-Related Proteins in Forest Tree Cultures

A procedure for aseptic in vitro culture of Populus spp. and Pinus halepensis buds from adult trees was developed, and bud cultures have been used to elucidate the hormonal control of bud growth and development. Populus cultures served as well for establishing procedures for in vitro propagation. Axillary bud break was induced, and adventitious buds and roots were formed, resulting in acclimatized plants. Adventitious shoot formation in root cultures of Populus is being studied. Expiants of zygotic embryos, seedlings and rejuvenated mature trees of Pinus halepensis are examined for in vitro clonal propagation. Efficient procedures for massive induction of adventitious shoots from mature pine embryos were established, and good rooting was achieved. Efforts are being made to induce somatic embryogenesis. A novel 66 kD boiling-stable protein was highly expressed in Populus tremula shoot cultures and in callus, in response to gradual desiccation, cold and osmotic stress, and ABA application. Populus clones differing in their drought tolerance are characterized by different expression patterns of this protein. This protein shows a high degree of homology with similar proteins form other species. Similar boiling-stable proteins were differentially expressed in germinating pine seeds. Populus shoots and Pinus embryos were transformed using A rhizogenes. as monitored by root formation and GUS expression, and complete transgenic aspen plants were established.