Water Relations, Gas Exchange, Chlorophyll Fluorescence and Electrolyte Leakage of Ectomycorrhizal Pinus halepensis Seedlings in Response to Multi-Heavy Metal Stresses (Pb, Zn, Cd)

The success of mine site restoration programs in arid and semi-arid areas poses a significant challenge and requires the use of high-quality seedlings capable of tolerating heavy metal stresses. The effect of ectomycorrhizal fungi on different physiological traits was investigated in Pinus halepensis seedlings grown in soil contaminated with heavy metals (Pb-Zn-Cd). Ectomycorrhizal (M) and non-ectomycorrhizal (NM) seedlings were subjected to heavy metals stress (C: contaminated, NC: control or non-contaminated) soils conditions for 12 months. Gas exchange, chlorophyll fluorescence, water relations parameters derived from pressure–volume curves and electrolyte leakage were evaluated at 4, 8 and 12 months. Ectomycorrhizal symbiosis promoted stronger resistance to heavy metals and improved gas exchange parameters and water-use efficiency compared to the non-ectomycorrhizal seedlings. The decrease in leaf osmotic potentials (Ψπ100: osmotic potential at saturation and Ψπ0: osmotic potential with loss of turgor) was higher for M-C seedling than NM-C ones, indicating that the ectomycorrhizal symbiosis promotes cellular osmotic adjustment and protects leaf membrane cell against leakage induced by Pb, Zn and Cd. Our results suggest that the use of ectomycorrhizal symbiosis is among the promising practices to improve the morphophysiological quality of seedlings produced in forest nurseries, their performance and their tolerance to multi-heavy metal stresses.

Bark Transpiration Rates Can Reach Needle Transpiration Rates Under Dry Conditions in a Semi-arid Forest

Drought can cause tree mortality through hydraulic failure and carbon starvation. To prevent excess water loss, plants typically close their stomata before massive embolism formation occurs. However, unregulated water loss through leaf cuticles and bark continues after stomatal closure. Here, we studied the diurnal and seasonal dynamics of bark transpiration and how it is affected by tree water availability. We measured continuously for six months water loss and CO2 efflux from branch segments and needle-bearing shoots in Pinus halepensis growing in a control and an irrigation plot in a semi-arid forest in Israel. Our aim was to find out how much passive bark transpiration is affected by tree water status in comparison with shoot transpiration and bark CO2 emission that involve active plant processes, and what is the role of bark transpiration in total tree water use during dry summer conditions. Maximum daily water loss rate per bark area was 0.03–0.14 mmol m−2 s−1, which was typically ~76% of the shoot transpiration rate (on leaf area basis) but could even surpass the shoot transpiration rate during the highest evaporative demand in the control plot. Irrigation did not affect bark transpiration rate. Bark transpiration was estimated to account for 64–78% of total water loss in drought-stressed trees, but only for 6–11% of the irrigated trees, due to differences in stomatal control between the treatments. Water uptake through bark was observed during most nights, but it was not high enough to replenish the lost water during the day. Unlike bark transpiration, branch CO2 efflux decreased during drought due to decreased metabolic activity. Our results demonstrate that although bark transpiration represents a small fraction of the total water loss through transpiration from foliage in non-stressed trees, it may have a large impact during drought.

Drought Drives Growth and Mortality Rates in Three Pine Species under Mediterranean Conditions

Drought constrains tree growth in regions with seasonal water deficit where growth decline can lead to tree death. This has been observed in regions such as the western Mediterranean Basin, which is a climate-warming hotspot. However, we lack information on intra- and inter-specific comparisons of growth rates and responses to water shortage in these hotspots, considering tree species with different drought tolerance. We sampled several sites located in north-eastern Spain showing dieback and high mortality rates of three pine species (Pinus sylvestris, Pinus pinaster, Pinus halepensis). We dated death years and reconstructed the basal area increment of coexisting living and recently dead trees using tree ring data. Then, we calculated bootstrapped Pearson correlations between a drought index and growth. Finally, we used linear mixed-effects models to determine differences in growth trends and the response to drought of living and dead trees. Mortality in P. sylvestris and P. pinaster peaked in response to the 2012 and 2017 droughts, respectively, and in sites located near the species’ xeric distribution limits. In P. halepensis, tree deaths occurred most years. Dead trees showed lower growth rates than living trees in five out of six sites. There was a strong growth drop after the 1980s when climate shifted towards warmer and drier conditions. Tree growth responded positively to wet climate conditions, particularly in the case of living trees. Accordingly, growth divergence between living and dead trees during dry periods reflected cumulative drought impacts on trees. If aridification continues, tree drought mortality would increase, particularly in xeric distribution limits of tree species.

Cell-wall fluorescence highlights the phases of xylogenesis

The monitoring of xylogenesis makes it possible to follow tree growth responses to stress factors in real-time, by observing the course of wood cell division and differentiation. Proper microscopy techniques are of key importance to exactly identify the xylem cells during the different phases of differentiation. We aimed to apply epifluorescence microscopy to follow the lignification process during the different phases of xylogenesis in Mediterranean softwood and hardwood. Microcores from trees of Pinus halepensis Mill. and Arbutus unedo L. were collected at a site in southern Italy, during the period June-December. Fluorescence imaging of sections stained with a water solution of safranin and Astra blue clearly highlighted the contrast between lignified and un-lignified tissue. The proposed methodology is useful to quickly and unambiguously detect the different stages of cell differentiation, as well as the progress in the lignification process. Moreover, it proved to be easily applied to demanding wood materials, such as Mediterranean woods and can be helpful to better track stress responses and the development of anomalies during wood formation, such as intra-annual density fluctuations.

The Chemical Environment at Maturation Stage in Pinus spp. Somatic Embryogenesis: Implications in the Polyamine Profile of Somatic Embryos and Morphological Characteristics of the Developed Plantlets

Changes in the chemical environment at the maturation stage in Pinus spp. somatic embryogenesis will be a determinant factor in the conversion of somatic embryos to plantlets. Furthermore, the study of biochemical and morphological aspects of the somatic embryos could enable the improvement of somatic embryogenesis in Pinus spp. In the present work, the influence of different amino acid combinations, carbohydrate sources, and concentrations at the maturation stage of Pinus radiata D. Don and Pinus halepensis Mill. was analyzed. In P. radiata, the maturation medium supplemented with 175 mM of sucrose and an increase in the amino acid mixture (1,100 mgL–1 of L-glutamine, 1,050 mgL–1 of L-asparagine, 350 mgL–1 of L-arginine, and 35 mgL–1 of L-proline) promoted bigger embryos, with a larger stem diameter and an increase in the number of roots in the germinated somatic embryos, improving the acclimatization success of this species. In P. halepensis, the maturation medium supplemented with 175 mM of maltose improved the germination of somatic embryos. The increase in the amount of amino acids in the maturation medium increased the levels of putrescine in the germinated somatic embryos of P. halepensis. We detected significant differences in the amounts of polyamines between somatic plantlets of P. radiata and P. halepensis; putrescine was less abundant in both species. For the first time, in P. radiata and P. halepensis somatic embryogenesis, we detected the presence of cadaverine, and its concentration changed according to the species.