Salicylic and Jasmonic Acid Synergism during Black Knot Disease Progression in Plums.

Black knot (BK) is a deadly disease of European (Prunus domestica) and Japanese (Prunus salicina) plums caused by the hemibiotrophic fungus Apiosporina morbosa. Generally, phytopathogens hamper the balance of primary defense phytohormones, such as salicylic acid (SA)-jasmonic acid (JA) balance, for disease progression. Thus, we quantified the important phytohormone titers in tissues of susceptible and resistant genotypes belonging to European and Japanese plums at five different time points. Our previous results suggested that auxin-cytokinins interplay driven by A. morbosa appeared to be vital in disease progression by hampering the plant defense system. Here, we further show that such hampering of disease progression is likely mediated by perturbance in SA, JA, and, to some extent, gibberellic acid. The results further indicate that SA and JA in plant defense are not always necessarily antagonistic as most of the studies suggest but can be different, especially in woody perennials. Together, our results suggest that the changes in phytohormone levels, especially in terms of SA and JA content due to BK infection and progression in plums, could be used as phytohormonal markers in the identification of BK-resistant cultivars.

Hormonal Interplay Leading to Black Knot Disease Establishment and Progression in Plums.

Black Knot (BK) is a deadly disease of European (Prunus domestics) and Japanese (Prunus salicina) plums caused by the hemibiotrophic fungus Apiosporina morbosa. After infection, the appearance of warty black knots indicates a phytohormonal imbalance in infected tissues. Based on this hypothesis, we quantified phytohormones such as indole-3-acetic acid, tryptophan, indoleamines (N-acetylserotonin, serotonin, and melatonin), and cytokinins (zeatin, 6-benzyladenine, and 2-isopentenyladenine) in temporally collected tissues of susceptible and resistant genotypes belonging to European and Japanese plums during of BK progression. The results suggested auxin-cytokinins interplay driven by A. morbosa appears to be vital in disease progression by hampering the plant defense system. Taken together, our results indicate the possibility of using the phytohormone profile as a biomarker for BK resistance in plums.

In Vitro Evaluation of Biological and Anticancer Activities Exhibited by Five Varieties of Vitis vinifera L.

Vitis vinifera commonly known as grapevine is one of the most important fruit crops worldwide. Its cultivation started more than 7000 years ago in the Near East, and over the millennia was followed by the development of thousands of cultivars that were further selected and characterized for specific purposes. Its important pharmacological value and its richness in phytoconstituents were the triggers to perform this project. Seven extracts were prepared from five different V. vinifera varieties (V. vinifera 'Black Pearl' (BP), V. vinifera 'Red Glob' (RG), V. vinifera 'Crimson' (CR), V. vinifera 'Beitamouni' (BE) and V. vinifera 'Superior' (SU)) by separating the pulp from the seeds, followed by methanolic extraction. The phytochemical analysis showed that red colored grapes (RE, BP and CR), the seeds from V. vinifera 'Black Pearl' and V. vinifera 'Red Globe' contain higher amounts of primary and secondary metabolites such as polyphenols, anthocyanins and reducing sugars. In addition to their richness in phytoconstituents, these varieties/seeds possess an important antioxidant activity. The results of the cell viability assays showed that the red varieties have a potential anticancer activity against Capan-2 pancreatic cancer and MDA-MB231(TNBC) breast cancer cell lines, with the greatest promise when combined with the seeds.

Maturity Assessment of Different Table Grape Cultivars Grown at Six Different Altitudes in Lebanon.

Table grapes are harvested based on well-known maturity indices that must be monitored after fruit veraison. The aim of this study was to assess these indices across multiple locations and environmental conditions, encompassing different table grape cultivars such as Black Pearl, Crimson Seedless, Superior Seedless, and Red Globe. For this reason, grape sampling was conducted across six distinct locations characterized by varying altitudes above sea level (m asl) and environmental conditions over the ripening season. The main maturity indices, including pH, sugar content, titratable acidity, berry firmness, and other parameters were monitored over the growing season. Moreover, the quantification of total polyphenols, total anthocyanins, and antioxidant activity was determined using spectrophotometric assays at harvesting. The study has examined the effect of the vineyard's location on grape quality and its interaction with the cultivar and environment. Crimson Seedless maintained a relatively high level of acidity with altitude near harvesting. Black Pearl exhibited a notable decline in both sugar content and berry firmness as elevation increased, whereas Red Globe demonstrated contrasting outcomes. The optimal maturity of Superior Seedless was observed at an elevation of 1000 m asl. Black Pearl and Crimson Seedless exhibited better adaptability to intermediate elevations (650 and 950 m asl), while Red Globe and Superior Seedless showed better adaptability to higher elevations (1000-1150 m asl). Among the studied cultivars, Black Pearl exhibited significantly higher levels of total polyphenols and anthocyanins, while close values were noticed between red and green cultivars.

Quality and Phytochemical Composition of Sweet Cherry Cultivars Can Be Influenced by Altitude.

Sweet cherries (Prunus avium L.) are among the most important stone fruits in Lebanon. They are harvested between May and July; however, the introduction of new early varieties in low and medium altitudes (500-1000 m) and late varieties in higher altitudes (1800-2200 m) along with postharvest technologies can extend harvesting season. In this study, physicochemical characteristics along with total phenolic content, total anthocyanin content, and antioxidant activity of the most commercial cherry cultivars were evaluated at different altitudes to determine optimum harvesting time. Findings indicated that some varieties, such as "Teliani" and "Irani", are more significantly impacted by altitude than the other varieties in terms of maturity indices. Duration of fruit development was prolonged with altitude, and in most instances, higher fresh weights and sizes were observed; however, fruit firmness decreased. While total phenolic content (expressed as gallic acid equivalent) did not significantly vary between varieties, the antioxidant activity (FRAP and DPPH assays) showed the lowest value in "Banni" and the total anthocyanin content showed the highest levels in "Irani" and "Feraouni" and the lowest in "Mkahal" and "Banni". Furthermore, total phenolic content and reduction of ferric complex (FRAP) were interestingly influenced by geographical locations, in contrast to total anthocyanin content and radical scavenging activity (DPPH) which were unaffected.

Evaluation of Postharvest Maturity Indices of Commercial Avocado Varieties Grown at Various Elevations Along Lebanon's Coast.

Avocado is known to be a climacteric fruit that must be harvested during the suitable physiological maturity stage to achieve the best edible characteristics and reach the required export standards. It is very hard to visually determine the optimum maturity phases in the different avocado varieties for harvesting, especially because of the limited changes in the external fruit morphology during the maturity phase and because the harvest season is extended throughout several months. Therefore, some laboratory analyses are very crucial to determine the best timing to harvest the fruit. The aim of this study was to evaluate the postharvest maturity indices over 3 harvest stages, mainly dry matter (DM), oil content (OC), fruit firmness, titratable acidity (TA), total soluble solid (TSS/Brix), and fruit weight in commercial avocado varieties in regard to different altitudes and agricultural practices. The varieties in this study were as follows: Hass, Lambhass, Ettinger, Fuerte, Pinkerton, Reed, and Horshim growing at different altitudes that ranged from 50 to 400 m in 7 different regions in Lebanon. Statistical comparison of maturity indices under different locations by variety and harvest stage was performed using one-way ANOVA as well as by principal component analysis (PCA). The results showed a high linear correlation between DM and OC over the different harvest stages. During the late harvest stage, the weight showed a negative correlation between fruit firmness and TSS. The minimum oil content % and dry matter % were recorded for Reed variety (8.2 DM and 9.7 OC%) and the highest oil content % and dry matter % were recorded for Fuerte variety (28.5 DM and 21.6 OC%). The data obtained during this study are used to achieve the best edible characteristics and export standards of commercial avocado varieties growing along the Lebanese coast.

Preharvest Spray Hexanal Formulation Enhances Postharvest Quality in 'Honeycrisp' Apples by Regulating Phospholipase D and Calcium Sensor Proteins Genes.

'Honeycrisp' (Malus domestica Borkh.), a premium applecultivar, is highly susceptible to bitter pit and decline in quality during long-term storage. In order to enhance the quality, an aqueous composition containing hexanal was applied as a preharvest spray. The effects of hexanal were assessed on the treated fruit and compared with HarvistaTM (a sprayable 1-Methylcyclopropene based commercial formulation) applied and control fruit under both cold (2.5 °C; four months) and cold after room temperature storage (20 °C; 14 days) conditions. Color, firmness, and total soluble solids (TSS) did not show a significant change in response to any treatment at harvest, while abscisic acid (ABA) significantly reduced and tryptophan increased in response to hexanal, compared to HarvistaTM and control. The treatment effects on quality traits were observed during storage. Both hexanal and HarvistaTM sprayed apples had higher TSS under both cold and room temperature storage. In addition, both sprays enhanced firmness at room temperature storage. However, the effects of sprays on other quality traits showed a different pattern. Apples sprayed with hexanal had lower phospholipase D enzyme (PLD) activity, lower incidence of bitter pit, and decreased expression of MdPLDα1 compared to HarvistaTM and control. On the other hand, HarvistaTM treated fruit produced lower ethylene. Both sprays decreased the expression of MdPLDα4, MdCaM2, MdCaM4 and MdCML18 genes. Generally, PLD alpha has a direct role in promoting fruit senescence, whereas the calcium senor proteins (CaM/CMLs) may involve in fruit ripening process via calcium and ethylene interactions. Therefore, improved postharvest qualities, including the lower incidence of bitter pit in hexanal treated 'Honeycrisp', may be associated with lower membrane damage due to lower PLD enzyme activity and decreased expression of MdPLDα1 and MdPLDα4 genes throughout the storage period.

Transcriptomics of Improved Fruit Retention by Hexanal in 'Honeycrisp' Reveals Hormonal Crosstalk and Reduced Cell Wall Degradation in the Fruit Abscission Zone.

Apples (Malus domestica Borkh) are prone to preharvest fruit drop, which is more pronounced in 'Honeycrisp'. Hexanal is known to improve fruit retention in several economically important crops. The effects of hexanal on the fruit retention of 'Honeycrisp' apples were assessed using physiological, biochemical, and transcriptomic approaches. Fruit retention and fruit firmness were significantly improved by hexanal, while sugars and fresh weight did not show a significant change in response to hexanal treatment. At commercial maturity, abscisic acid and melatonin levels were significantly lower in the treated fruit abscission zone (FAZ) compared to control. At this stage, a total of 726 differentially expressed genes (DEGs) were identified between treated and control FAZ. Functional classification of the DEGs showed that hexanal downregulated ethylene biosynthesis genes, such as S-adenosylmethionine synthase (SAM2) and 1-aminocyclopropane-1-carboxylic acid oxidases (ACO3, ACO4, and ACO4-like), while it upregulated the receptor genes ETR2 and ERS1. Genes related to ABA biosynthesis (FDPS and CLE25) were also downregulated. On the contrary, key genes involved in gibberellic acid biosynthesis (GA20OX-like and KO) were upregulated. Further, hexanal downregulated the expression of genes related to cell wall degrading enzymes, such as polygalacturonase (PG1), glucanases (endo-ß-1,4-glucanase), and expansins (EXPA1-like, EXPA6, EXPA8, EXPA10-like, EXPA16-like). Our findings reveal that hexanal reduced the sensitivity of FAZ cells to ethylene and ABA. Simultaneously, hexanal maintained the cell wall integrity of FAZ cells by regulating genes involved in cell wall modifications. Thus, delayed fruit abscission by hexanal is most likely achieved by minimizing ABA through an ethylene-dependent mechanism.

Comparative Anatomical Responses of Tolerant and Susceptible European Plum Varieties to Black Knot Disease.

Plums are affected by a cancerous disease called "black knot disease" caused by the fungus Apiosporina morbosa. It affects both Japanese (Prunus salicina) and European (Prunus domestica) plums equally. To understand the spread of the disease, histological analysis was performed in two different European plum cultivars (susceptible and tolerant). Light and scanning electron microscope (SEM) analyses confirmed the presence of the growing hyphae in the internal tissues of the susceptible trees. By using stereoscopic analysis with a fluorescence filter, we were able to detect the hyphae in the visible lesion area. At about 2 inches from above and below the knots, no spore or hypha were visible with the light microscope. However, SEM images showed strong evidence that the fungus is capable of migrating to adjacent vessels in the susceptible plum genotype. In fact, at that distance below and above the knots, conidia were detected inside xylem vessels suggesting a systemic movement of the fungus that has not been shown so far. No symptoms were observed in the resistant genotype. Starch granules, vessel occlusions, and lipid droplets were the main distinguishable characteristics between susceptible and tolerant varieties.

Functional characterization of a gibberellin F-box protein, PslSLY1, during plum fruit development.

Fruit development is orchestrated by a complex network of interactions between hormone signaling pathways. The phytohormone gibberellin (GA) is known to regulate a diverse range of developmental processes; however, the mechanisms of GA action in perennial fruit species are yet to be elucidated. In the current study, a GA signaling gene PslSLY1, encoding a putative F-box protein that belongs to the SLY1 (SLEEPY1)/GID2 (gibberellin-insensitive dwarf2) gene family, was isolated from Japanese plum (Prunus salicina). PslSLY1 transcript abundance declined as fruit development progressed, along with potential negative feedback regulation of PslSLY1 by GA. Subcellular localization and protein-protein interaction assays suggested that PslSLY1 functions as an active GA signaling component that interacts with the ASK1 (Arabidopsis SKP1) subunit of an SCF-ubiquitin ligase complex and with PslDELLA repressors, in a GA-independent manner. By using a domain omission strategy, we illustrated that the F-box and C-terminal domains of PslSLY1 are essential for its interactions with the downstream GA signaling components. PslSLY1 overexpression in wild-type and Arabidopsissly1.10 mutant backgrounds resulted in a dramatic enhancement in overall plant growth, presumably due to triggered GA signaling. This includes germination characteristics, stem elongation, flower structure, and fertility. Overall, our findings shed new light on the GA strategy and signaling network in commercially important perennial crops.

The joint influence of photoperiod and temperature during growth cessation and development of dormancy in white spruce (Picea glauca).

Timely responses to environmental cues enable the synchronization of phenological life-history transitions essential for the health and survival of north-temperate and boreal tree species. While photoperiodic cues will remain persistent under climate change, temperature cues may vary, contributing to possible asynchrony in signals influencing developmental and physiological transitions essential to forest health. Understanding the relative contribution of photoperiod and temperature as determinants of the transition from active growth to dormancy is important for informing adaptive forest management decisions that consider future climates. Using a combination of photoperiod (long = 20 h or short = 8 h day lengths) and temperature (warm = 22 °C/16 °C and cool = 8 °C/4 °C day/night, respectively) treatments, we used microscopy, physiology and modeling to comprehensively examine hallmark traits of the growth-dormancy transition-including bud formation, growth cessation, cold hardiness and gas exchange-within two provenances of white spruce [Picea glauca (Moench) Voss] spanning a broad latitude in Alberta, Canada. Following exposure to experimental treatments, seedlings were transferred to favorable conditions, and the depth of dormancy was assessed by determining the timing and ability of spruce seedlings to resume growth. Short photoperiods promoted bud development and growth cessation, whereas longer photoperiods extended the growing season through the induction of lammas growth. In contrast, cool temperatures under both photoperiodic conditions delayed bud development. Photoperiod strongly predicted the development of cold hardiness, whereas temperature predicted photosynthetic rates associated with active growth. White spruce was capable of attaining endodormancy, but its release was environmentally determined. Dormancy depth varied substantially across experimental treatments suggesting that environmental cues experienced within one season could affect growth in the following season, which is particularly important for a determinate species such as white spruce. The joint influence of these environmental cues points toward the importance of including local constant photoperiod and shifting temperature cues into predictive models that consider how climate change may affect northern forests.

Responses of hybrid aspen over-expressing a PIP2;5 aquaporin to low root temperature.

Aquaporins mediate the movement of water across cell membranes. Plasma membrane intrinsic protein 2;5 from Populus trichocarpa×deltoides (PtdPIP2;5) was previously demonstrated to be a functionally important water conducting aquaporin. To study the relevance of aquaporin-mediated root water transport at low temperatures, we generated transgenic Populus tremula×alba over-expressing PtdPIP2;5 under control of the maize ubiquitin promoter, and compared the physiological responses and water transport properties of the PtdPIP2;5 over-expressing lines (PtdPIP2;5ox) with wild-type plants. We hypothesized that over-expression of PtdPIP2;5 would reduce temperature sensitivity of root water transport and gas exchange. Decreasing root temperatures to 10 and 5°C significantly decreased hydraulic conductivities (Lp) in wild-type plants, but had no significant effect on Lp in PtdPIP2;5ox plants. Recovery of Lp in the transgenic lines returned to 20°C from 5°C was faster than in the wild-type plants. Low root temperature did not induce major changes in transcript levels for other PIPs. When roots were exposed to 5°C in solution culture and shoots were exposed to 20°C, wild-type plants had significantly lower net photosynthetic and transpiration rates compared to PtdPIP2;5ox plants. Taken together, our results demonstrate that over-expression of PtdPIP2;5 in P. tremula×alba was effective in alleviating the effects of low root temperature on Lp and gas exchange.

Differences in defence responses of Pinus contorta and Pinus banksiana to the mountain pine beetle fungal associate Grosmannia clavigera are affected by water deficit.

We tested the hypotheses that responses to the mountain pine beetle fungal associate Grosmannia clavigera will differ between the evolutionarily co-evolved host lodgepole pine (Pinus contorta var. latifolia) and the naïve host jack pine (Pinus banksiana) and that these responses will be influenced by water availability. G. clavigera inoculation resulted in more rapid stem lesion development in lodgepole than in jack pine; water deficit delayed lesion development in both species. Decreased hydraulic conductivity was observed in inoculated lodgepole pine seedlings, likely because of tracheid occlusion by fungal hyphae and/or metabolite accumulation. Drought but not inoculation significantly impacted bark abscisic acid levels. Jasmonic and salicylic acid were implicated in local and systemic responses of both species to G. clavigera, with salicylic acid appearing to play a greater role in jack pine response to G. clavigera than lodgepole pine. Water deficit increased constitutive levels and/or attenuated induced responses to G. clavigera for several monoterpenes in lodgepole but not jack pine. Instead, inoculation of well-watered but not water deficit jack pine resulted in a greater number of xylem resin ducts. These findings reveal mechanisms underlying differences in G. clavigera-induced responses between lodgepole and jack pine hosts, and how water availability modulates these responses.

Overexpression of Laccaria bicolor aquaporin JQ585595 alters root water transport properties in ectomycorrhizal white spruce (Picea glauca) seedlings.

The contribution of hyphae to water transport in ectomycorrhizal (ECM) white spruce (Picea glauca) seedlings was examined by altering expression of a major water-transporting aquaporin in Laccaria bicolor. Picea glauca was inoculated with wild-type (WT), mock transgenic or L. bicolor aquaporin JQ585595-overexpressing (OE) strains and exposed to root temperatures ranging from 5 to 20°C to examine the root water transport properties, physiological responses and plasma membrane intrinsic protein (PIP) expression in colonized plants. Mycorrhization increased shoot water potential, transpiration, net photosynthetic rates, root hydraulic conductivity and root cortical cell hydraulic conductivity in seedlings. At 20°C, OE plants had higher root hydraulic conductivity compared with WT plants and the increases were accompanied by higher expression of P. glauca PIP GQ03401_M18.1 in roots. In contrast to WT L. bicolor, the effects of OE fungi on root and root cortical cell hydraulic conductivities were abolished at 10 and 5°C in the absence of major changes in the examined transcript levels of P. glauca root PIPs. The results provide evidence for the importance of fungal aquaporins in root water transport of mycorrhizal plants. They also demonstrate links between hyphal water transport, root aquaporin expression and root water transport in ECM plants.

Influence of water deficit on the molecular responses of Pinus contorta†×†Pinus banksiana mature trees to infection by the mountain pine beetle fungal associate, Grosmannia clavigera.

Conifers exhibit a number of constitutive and induced mechanisms to defend against attack by pests and pathogens such as mountain pine beetle (Dendroctonus ponderosae Hopkins) and their fungal associates. Ecological studies have demonstrated that stressed trees are more susceptible to attack by mountain pine beetle than their healthy counterparts. In this study, we tested the hypothesis that water deficit affects constitutive and induced responses of mature lodgepole pine × jack pine hybrids (Pinus contorta Dougl. ex Loud. var. latifolia Engelm. ex S. Wats. × Pinus banksiana Lamb.) to inoculation with the mountain pine beetle fungal associate Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield. The degree of stress induced by the imposed water-deficit treatment was sufficient to reduce photosynthesis. Grosmannia clavigera-induced lesions exhibited significantly reduced dimensions in water-deficit trees relative to well-watered trees at 5 weeks after inoculation. Treatment-associated cellular-level changes in secondary phloem were also observed. Quantitative RT-PCR was used to analyze transcript abundance profiles of 18 genes belonging to four families classically associated with biotic and abiotic stress responses: aquaporins (AQPs), dehydration-responsive element binding (DREB), terpene synthases (TPSs) and chitinases (CHIs). Transcript abundance profiles of a TIP2 AQP and a TINY-like DREB decreased significantly in fungus-inoculated trees, but not in response to water deficit. One TPS, Pcb(+)-3-carene synthase, and the Class II CHIs PcbCHI2.1 and PcbCHI2.2 showed increased expression under water-deficit conditions in the absence of fungal inoculation, while another TPS, Pcb(E)-ß-farnesene synthase-like, and two CHIs, PcbCHI1.1 and PcbCHI4.1, showed attenuated expression under water-deficit conditions in the presence of fungal inoculation. The effects were observed both locally and systemically. These results demonstrate that both constitutive and induced carbon- and nitrogen-based defenses are affected by water deficit, suggesting potential consequences for mountain pine beetle dynamics, particularly in novel environments.

Characterization of gibberellin-signalling elements during plum fruit ontogeny defines the essentiality of gibberellin in fruit development.

Fruit growth is a coordinated, complex interaction of cell division, differentiation and expansion. Gibberellin (GA) involvement in the reproductive events is an important aspect of GA effects. Perennial fruit-trees such as plum (Prunus salicina L.) have distinct features that are economically important and provide opportunities to dissect specific GA mechanisms. Currently, very little is known on the molecular mechanism(s) mediating GA effects on fruit development. Determination of bioactive GA content during plum fruit ontogeny revealed that GA1 and GA4 are critical for fruit growth and development. Further, characterization of several genes involved in GA-signalling showed that their transcriptional regulation are generally GA-dependent, confirming their involvement in GA-signalling. Based on these results, a model is presented elucidating how the potential association between GA and other hormones may contribute to fruit development. PslGID1 proteins structure, Y2H and BiFC assays indicated that plum GA-receptors can form a complex with AtDELLA-repressors in a GA-dependent manner. Moreover, phenotypical-, molecular- and GA-analyses of various Arabidopsis backgrounds ectopically expressing PslGID1 sequences provide evidence on their role as active GA-signalling components that mediate GA-responsiveness. Our findings support the critical contribution of GA alone or in association with other hormones in mediating plum fruit growth and development.

Integrated transcriptomic and proteomic profiling of white spruce stems during the transition from active growth to dormancy.

In the autumn, stems of woody perennials such as forest trees undergo a transition from active growth to dormancy. We used microarray transcriptomic profiling in combination with a proteomics analysis to elucidate processes that occur during this growth-to-dormancy transition in a conifer, white spruce (Picea glauca[Moench] Voss). Several differentially expressed genes were likely associated with the developmental transition that occurs during growth cessation in the cambial zone and the concomitant completion of cell maturation in vascular tissues. Genes encoding for cell wall and membrane biosynthetic enzymes showed transcript abundance patterns consistent with completion of cell maturation, and also of cell wall and membrane modifications potentially enabling cells to withstand the harsh conditions of winter. Several differentially expressed genes were identified that encoded putative regulators of cambial activity, cell development and of the photoperiodic pathway. Reconfiguration of carbon allocation figured centrally in the tree's overwintering preparations. For example, genes associated with carbon-based defences such as terpenoids were down-regulated, while many genes associated with protein-based defences and other stress mitigation mechanisms were up-regulated. Several of these correspond to proteins that were accumulated during the growth-to-dormancy transition, emphasizing the importance of stress protection in the tree's adaptive response to overwintering.

Molecular events of apical bud formation in white spruce, Picea glauca.

Bud formation is an adaptive trait that temperate forest trees have acquired to facilitate seasonal synchronization. We have characterized transcriptome-level changes that occur during bud formation of white spruce [Picea glauca (Moench) Voss], a primarily determinate species in which preformed stem units contained within the apical bud constitute most of next season's growth. Microarray analysis identified 4460 differentially expressed sequences in shoot tips during short day-induced bud formation. Cluster analysis revealed distinct temporal patterns of expression, and functional classification of genes in these clusters implied molecular processes that coincide with anatomical changes occurring in the developing bud. Comparing expression profiles in developing buds under long day and short day conditions identified possible photoperiod-responsive genes that may not be essential for bud development. Several genes putatively associated with hormone signalling were identified, and hormone quantification revealed distinct profiles for abscisic acid (ABA), cytokinins, auxin and their metabolites that can be related to morphological changes to the bud. Comparison of gene expression profiles during bud formation in different tissues revealed 108 genes that are differentially expressed only in developing buds and show greater transcript abundance in developing buds than other tissues. These findings provide a temporal roadmap of bud formation in white spruce.

Two EguCBF1 genes overexpressed in Eucalyptus display a different impact on stress tolerance and plant development.

Two C-repeat binding factor genes (EguCBF1a/b), isolated from E. gunnii and differentially cold-regulated, were constitutively overexpressed in a cold-sensitive Eucalyptus hybrid. In addition to the expected improvement on freezing tolerance, some resulting transgenic lines (EguCBF1a-OE and EguCBF1b-OE) exhibited a decrease in stomata density and an over-accumulation of anthocyanins also observed to a lesser extent in a cold-acclimated control plant. Given that the induction of five putative CBF target genes was observed in CBF-overexpressing lines as well as in the cold-acclimated control line, these phenotypes might be related to cold acclimation. In comparison with the control plant, the most altered transgenic line (EguCBF1a-OE A1 line), exhibited reduced growth and better water retention capacity. This modified phenotype includes reduced leaf area and thickness associated with a decrease in cell size, as well as a higher oil gland density and a wax deposition on the cuticle. Surprisingly, the EguCBF1b-OE B9 line, with a level of transgene expression equivalent to the A1 line, showed a less marked phenotype, suggesting a difference in transactivation efficiency between EguCBF1A and B factors. The features of these transgenic lines provide the first signs of adaptive mechanisms controlled by CBF transcription factors in an evergreen broad-leaved tree. These data also open new prospects towards genetic improvement on Eucalyptus for freezing tolerance.

Transcriptional profiling of sunflower plants growing under low temperatures reveals an extensive down-regulation of gene expression associated with chilling sensitivity.

Being able to sow early to maximize the growing season and to escape drought stress has increased the importance of low-temperature tolerance in sunflower. Yet knowledge about the molecular basis of sunflower response to low temperature is still lacking. To address this issue, nylon microarrays containing >8000 putative unigenes were developed and used. Early- and late-flowering genotypes were sown at 15 degrees C and grown until the two-leaf stage when they were subjected to 7 degrees C until the four-leaf stage. The transcriptional profiles of low temperature-grown plants (15 degrees C and 7 degrees C) were compared with those grown under standard conditions (25 degrees C). Two-step ANOVA normalization and analysis models were used to identify the differentially expressed genes. A total of 108 cDNA clones having a P-value <10(-3) were found to be differentially expressed between the low temperature-grown plants (15 degrees C and 7 degrees C) and their corresponding two-leaf- and four-leaf-stage controls across the two genotypes. About 90% of these genes were down-regulated. This includes genes potentially involved in the metabolism of carbohydrate and energy, protein synthesis, signal transduction, and transport function. Comparing gene expression profiles at 15 degrees C and 7 degrees C revealed that only four genes can be considered as differentially expressed, in both genotypes, suggesting that similar genetic programmes underlie the response of sunflower plants to these temperature regimes. The analysis also revealed that early- and late-flowering genotypes respond similarly to low-temperature tolerance as justified by the low number of genes showing a significant genotype x treatment interaction effect. It seems likely that the down-regulation and/or non-induction of genes having a critical role in low-temperature tolerance may be responsible for the sensitivity of sunflower plants to low-temperature tolerance. The results reported provide an initial characterization of the transcriptome activity of sunflower, as a chilling-sensitive plant under suboptimal temperatures, and could be of importance to reveal the potential differences between chilling-sensitive and chilling-tolerant species.