We studied the effect of ectopic AtCBF over-expression on physiological alterations that occur during cold exposure in frost-sensitive Solanum tuberosum and frost-tolerant Solanum commersonii. Relative to wild-type plants, ectopic AtCBF1 over-expression induced expression of COR genes without a cold stimulus in both species, and imparted a significant freezing tolerance gain in both species: 2°C in S. tuberosum and up to 4°C in S. commersonii. Transgenic S. commersonii displayed improved cold acclimation potential, whereas transgenic S. tuberosum was still incapable of cold acclimation. During cold treatment, leaves of wild-type S. commersonii showed significant thickening resulting from palisade cell lengthening and intercellular space enlargement, whereas those of S. tuberosum did not. Ectopic AtCBF1 activity induced these same leaf alterations in the absence of cold in both species. In transgenic S. commersonii, AtCBF1 activity also mimicked cold treatment by increasing proline and total sugar contents in the absence of cold. Relative to wild type, transgenic S. commersonii leaves were darker green, had higher chlorophyll and lower anthocyanin levels, greater stomatal numbers, and displayed greater photosynthetic capacity, suggesting higher productivity potential. These results suggest an endogenous CBF pathway is involved in many of the structural, biochemical and physiological alterations associated with cold acclimation in these Solanum species.

Nitrogen fixation and colonization by associative cyanobacteria in the aerial roots (pneumatophores) of black mangrove trees was evaluated in situ at Balandra lagoon, Baja California Sur, Mexico, for 18 consecutive months. Year-round vertical zonation of cyanobacterial colonization was determined along the pneumatophores. The bottom part close to the sediment was colonized mainly by nonheterocystous, filamentous cyanobacteria resembling Lyngbya sp. and Oscillatoria sp. The central zone was colonized mainly by filaments resembling Microcoleus sp. and the upper part was colonized by coccoidal cyanobacteria within defined colonies resembling Aphanothece sp. mixed with undefined filamentous cyanobacteria. Two of the cyanobacteria (Microcoleus sp. and Anabaena sp.) isolated from the pneumatophore were diazotrophs. Massive biofilm production along the pneumatophores was evident throughout the observation period. The surrounding sediment was seasonally dominated by heterocystous Anabaena sp. Glass and dead-wood surfaces incubated for 18 months in the pneumatophore vicinity showed no zonation in the colonization pattern, although they were heavily colonized. In situ N2fixation showed seasonal and diurnal fluctuations. N2fixation was low during winter, increased in early summer, and reached its peak in midsummer. N2fixation in the summer showed diurnal peaks: one in the morning until midday and the second in the late afternoon. N2fixation was at its lowest levels near midnight. Light and water temperature are probably primary environmental factors governing N2fixation on the pneumatophores.Key words: Avicennia germinans, black mangrove, diazotrophic cyanobacteria, nitrogen fixation, pneumatophore.

An isolate of the filamentous cyanobacterium Microcoleus sp. was obtained from black mangrove aerial root (pneumatophore) and inoculated onto young mangrove seedlings to evaluate N2-fixation and root-colonization capacities of the bacterium under in vitro conditions in closed-system experiments. N2 fixation (acetylene reduction) gradually increased with time and reached its peak 5 days after inoculation. Later, it decreased sharply. The level of N2 fixation in the presence of the plant was significantly higher than the amount of nitrogen fixed by a similar quantity of cyanobacteria on a N-free growth medium. The main feature of this root colonization was the gradual production of a biofilm in which the cyanobacterial filaments were embedded. Visible biofilm production increased with time until it completely covered the entire root system of the plant. The in-and-out movement of cyanobacterial filaments from the biofilm probably allows colonization of uncolonized portions of the root several days after the initial inoculation. This is, to the best of our knowledge, the first report of the artificial inoculation of cyanobacteria on marine mangroves.Key words: Avicennia germinans, beneficial bacteria, biofilm, black mangrove, diazotrophic cyanobacteria, Microcoleus sp., nitrogen fixation, plant growth-promoting bacteria, root colonization.

Morphological differences between old-growth trees and saplings of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) may extend to differences in needle anatomy. We used microscopy with image analysis to compare and quantify anatomical parameters in cross sections of previous-year needles of old-growth Douglas-fir trees and saplings at the Wind River Canopy Crane site in Washington and at three sites in the Cascade Mountains of Oregon. We also compared needle anatomy across a chronosequence of 10-, 20-, 40- and 450-year-old Douglas-fir trees from the Wind River site. Anatomy differed significantly between needles of old-growth trees and saplings at all sites, suggesting a developmental change in needle anatomy with increasing tree age. Compared with needles of old-growth trees, needles of saplings were longer and had proportionately smaller vascular cylinders, larger resin canals and few hypodermal cells. Astrosclereids, which sequester lignin in their secondary cell walls and occupy space otherwise filled by photosynthetic cells, were scarce in needles of saplings but abundant in needles of old-growth trees. Needles of old-growth trees had an average of 11% less photosynthetic mesophyll area than needles of saplings. The percentage of non-photosynthetic area in needles increased significantly with increasing tree age from the chronosequence of 10-, 20-, 40- and 450-year-old trees at the Wind River site. This reduction in photosynthetic area may contribute to decreased growth rates in old trees.

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