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The effects of xylooligosaccharides isolated from the cell walls of Betula platyphylla var. japonica on cells and protoplasts of Pinus radiata were examined. The addition of a semi-purified mixture of xylooligosaccharides at a concentration of 5µg.ml−1 promoted elongation of cultured cells, whereas the neutral fraction of this mixture had no effect; a similar effect was seen in the presence of conditioned medium. The unfractionated mixture of xylooligosaccharides was also found to enhance the viability of protoplasts prepared from cell cultures of Pinus radiata in a concentration dependent manner, highly similar to the effect provided by addition of medium conditioned by pine cells. Such effects are considered to be due to the addition of components that play a structural role in the cell wall of pines. It is inferred that the acidic components of the xylooligosaccharide mixture derived from t Betula are responsible for this effect in the distant pine species. It is speculated that acidic xylooligosaccharides operate either by replacing, or mimicking, the natural cell wall components required for growth and development of pine cultured cells.

Sorting nexin 1 (SNX1), a peripheral membrane protein, has previously been shown to regulate the cell-surface expression of the human epidermal growth factor receptor [Kurten, Cadena and Gill (1996) Science 272, 1008-1010]. Searches of human expressed sequence tag databases with SNX1 revealed eleven related human cDNA sequences, termed SNX2 to SNX12, eight of them novel. Analysis of SNX1-related sequences in the Saccharomyces cerevisiae genome clearly shows a greatly expanded SNX family in humans in comparison with yeast. On the basis of the predicted protein sequences, all members of this family of hydrophilic molecules contain a conserved 70-110-residue Phox homology (PX) domain, referred to as the SNX-PX domain. Within the SNX family, subgroups were identified on the basis of the sequence similarities of the SNX-PX domain and the overall domain structure of each protein. The members of one subgroup, which includes human SNX1, SNX2, SNX4, SNX5 and SNX6 and the yeast Vps5p and YJL036W, all contain coiled-coil regions within their large C-terminal domains and are found distributed in both membrane and cytosolic fractions, typical of hydrophilic peripheral membrane proteins. Localization of the human SNX1 subgroup members in HeLa cells transfected with the full-length cDNA species revealed a similar intracellular distribution that in all cases overlapped substantially with the early endosome marker, early endosome autoantigen 1. The intracellular localization of deletion mutants and fusions with green fluorescent protein showed that the C-terminal regions of SNX1 and SNX5 are responsible for their endosomal localization. On the basis of these results, the functions of these SNX molecules are likely to be unique to endosomes, mediated in part by interactions with SNX-specific C-terminal sequences and membrane-associated determinants.

Potassium is an essential macronutrient in higher plants. It plays an important physiological role in stoma movements, osmoregulation, enzyme activation and cell expansion. The demand for potassium can be substantial, especially when the plant concerned is a Eucalyptus tree in excess of 50 m tall. We have isolated two cDNAs, EcHKT1 and EcHKT2, from Eucalyptus camaldulensis (river red gum) which are expressed in leaves, stems and roots. These encode potassium transporter polypeptides with homology to the wheat K+-Na+ symporter, HKT1. EcHKT1 and EcHKT2 both complemented the K+-limited growth of an Escherichia coli K+-uptake-deficient triple mutant. EcHKT1 and EcHKT2 also mediated Na+ and K+ uptake when expressed in Xenopus oocytes. A comparison of the EcHKT1 and EcHKT2 sequences and their transport properties indicated that these cDNAs represent two K+ transporters with distinct functional characteristics. The functional and structural conservation between these two E. camaldulensis genes and the wheat HKT1 suggests that they play an important, albeit elusive, physiological role.

The development of forest biotechnology is now at a stage where application is a reality. We have placed considerable emphasis on the use of molecular markers for efficient production of genetic maps and detection of correlations with quantitative trait loci. These studies are complicated by the use of families that are small, unbalanced, or contaminated by rogue trees. Nevertheless, a growing number of correlations are being detected between markers and major genes controlling valuable poly genetic traits including wood density, volume growth, branching patterns, and insect resistance. The direct benefits of marker assisted selection are significant and will continue to increase through guided breeding to fix genes, and through further selection. Such genetic gains will inevitably exceed the potential of rejuvenation and cryogenic storage strategies, so that pursuit of these goals is now of dubious benefit. The efficient use of markers depends not only on the value of the correlated traits, but also on the cost of screening which increases exponentially with the number of loci, and the ability to efficiently propagate elite genotypes as large clones. We are using robotic automation of both marker analyses and micropropagation processes to reduce costs and meet massively increasing demands in both these areas.

Protoplasts were prepared from exponentially growing cell suspension cultures derived from Pinus taeda seedlings. The cell suspension was derived from callus that had been initiated on solid Murashige and Skoog medium supplemented with 2mg/1 2,4-D, and maintained in liquid LM medium (D.C. Verma et al, Proceedings of the 5th International Congress of Plant Tissue and Cell Culture, Tokyo, Japan, 1982, p. 59–60). Evaluation of a range of enzyme formulations showed that almost all cells could be converted to viable protoplasts in 9 hours with the combination of 0.5% each of Onozuka R-10, Macerozyme R-10, Hemicellulase, Driselase, and bovine serum albumin in osmoticum (0.4 M mannitol, 0.2 M sorbitol, 1.5 mM CaCl2, 0.7 mM KCl). Protoplast purification required only simple washing which was achieved without damaging centrifugation. When cultured in LM medium supplemented with 1.5mM CaCl2, 10 mM arginine, 10mM glutamine and 5 mM asparagine, cell wall formation was observed after 48 hours. The cultured protoplasts produced numerous colonies in 3 weeks which continued to grow in liquid medium.

The specific growth rates of suspension cultured Loblolly pine (Pinus taeda) cells were used as a physiological response to copper deficiency, thereby circumventing difficulties incurred through cell wall copper adsorption and trace background contamination which complicate alternative experi mental approaches. Variation in specific growth rate with copper concentration is assumed to be hyperbolic, a behaviour consistent with theoretically derived models. Growth.response data are thereby interpreted to estimate background copper contamination, plus the copper dissociation constant of the uptake system, and the inhibition constant for competition by a combination of transition metal ions. Analysis of the time course of growth when cells are transferred from copper sufficiency to deficiency conditions indicates an insignificant endogenous pool of copper. Similar consideration of growth under toxicity conditions indicates that no diffusional barrier to copper influx exists. These results are interpreted to indicate that copper uptake is not mediated by a membrane carrier, but freely diffuses across the plasmalemma to bind directly to a high affinity intracellular acceptor.

SUMMARY An important factor in plant availability of copper is its anomalous binding to soil humic substances, for which aggregation phenomena have been implicated. Two alternative models of copper-induced humate aggregation are theoretically explored to characterize binding behaviour. In the first model the copper modifies the humic acid so that it self-associates indefinitely. Copper binding is found to be dramatically enhanced, except at the very low concentrations corresponding to copper-deficiency. In the second model bivalent copper forms bridges between different humate molecules to yield an array of large cross-linked complexes. In the latter cross-linking model, binding of copper is markedly enhanced at low copper concentrations, a behaviour still found when only relatively few sites sterically permit cross-linking. The cross-linking model accounts, at least in part, for the low availability and immobility of copper in some problem soils and is consistent with several items of experimental evidence.

A steady-state analysis is given of an important double-tuned coupled circuit to determine the input admittance when the primary Q is high and the secondary Q is low. Particular emphasis is placed on the phase variation of the admittance with frequency. Dimensionless curves of magnitude and phase illustrate the effect of varying the coefficient of coupling and the secondary Q. Mathematical expressions are obtained for the points where the phase passes through zero, and are used in a discussion of bandwidth. The effect of detuning is also considered.