Walid El Kayal

An Efficient Procedure to Stably Introduce Genes into an Economically Important Pulp Tree (Eucalyptus grandis × Eucalyptus urophylla)

Regeneration problems are one of the main limitations preventing the wider application of genetic engineering strategies to the genus Eucalyptus. Seedlings from Eucalyptus grandis × Eucalyptus urophylla were selected according to their regeneration (adventitious organogenesis) and transformation capacity. After in vitro cloning, the best genotype of 250 tested was transformed via Agrobacterium tumefaciens. A cinnamyl alcohol dehydrogenase (CAD) antisense cDNA from Eucalyptus gunnii was transferred, under the control of the 35S CaMV promoter with a double enhancer sequence, into a selected genotype. According to kanamycin resistance and PCR verification, 120 transformants were generated. 58% were significantly inhibited for CAD activity, and nine exhibited the highest down-regulation, ranging from 69 to 78% (22% residual activity). Southern blot hybridisation showed a low transgene copy number, ranging from 1 to 4, depending on the transgenic line. Northern analyses on the 5–16 and 3–23 lines (respectively one and two insertion sites) demonstrated the antisense origin of CAD gene inhibition. With respectively 26 and 22% of residual CAD activity, these two lines were considered as the most interesting and transferred to the greenhouse for further analyses.

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 seasons 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.

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 trees 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 trees adaptive response to overwintering.

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.

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.

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.

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)-β-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.

Transcript profiling combined with functional annotation of 2,662 ESTs provides a molecular picture of Eucalyptus gunnii cold acclimation

Plants respond to low temperatures through an intricately coordinated transcription network which results in cold acclimation, a phenomenon by which plants increase their freezing tolerance. To analyse the global transcriptome of Eucalyptus gunnii cold-acclimated leaves, 2,662 ESTs were classified by FunCat and the corresponding transcripts quantified throughout a cold acclimation programme using macro-array technique. This combined analysis resulted in the description of the temporal patterns of gene expression with regard to their identity and corresponding functional categories. This paper proposes a predictive hierarchical classification of the main protective mechanisms likely to participate in the increased cold tolerance of E. gunnii. Given the time course and level of the LEA/dehydrin accumulation, the cryoprotection through proteins may explain most of the hardening. This cryoprotection would be completed by sugar synthesis (raffinose and maltose). As a permanent response, red-ox regulation and protection of membranes or macromolecules against peroxidation look mainly associated with metallothioneins. A limited part of cold response seems to be dedicated to dehydration avoidance through osmoprotectants or to frost avoidance through antifreeze proteins and deposition of cuticle wax.

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.

Diverse chitinases are invoked during the activity-dormancy transition in spruce

North temperate tree species such as white spruce (Picea glauca [Moench] Voss) have evolved strategies to protect themselves against abiotic and biotic stresses that trees encounter during the inclement winter months. Chitinases not only play well-documented roles in plant defense but also function during physiological and developmental preparations for overwintering, including growth cessation, cold and desiccation acclimation, and dormancy acquisition. Phylogenetic analysis of 31 white spruce and 52 Norway spruce chitinases identified genes falling into each of the five clusters, which sometimes—but not always—separated the different biochemical classes of chitinases. Digital expression profiling of white spruce and Norway spruce chitinases across multiple conditions revealed a range of spatiotemporal expression patterns. Transcript abundance profiling in buds, needles, stems, and roots by quantitative RT-PCR suggested roles for eight white spruce chitinases during the growth-to-dormancy transition. In silico analyses of these eight sequences suggested that two cluster 2/class I chitinases function as chitinolytic enzymes in the tree’s constitutive defense arsenal during the winter months. A cluster 2/class I, cluster 2/class II, and cluster 1/class IV chitinase each exhibit hallmarks of antifreeze proteins. Additionally, two cluster 2/class I chitinases and a cluster 1/class IV chitinase may serve as vegetative storage proteins. One cluster 3/class II chitinase exhibited attributes suggesting that it is a chitinase-like gene functioning in cell wall synthesis. Taken together, our results imply that dormancy-associated chitinases act in concert to (1) confer protection against freezing injury, pests, and pathogens, (2) store nitrogen, and (3) promote cell maturation that precedes growth cessation.