Highlighting type A RRs as potential regulators of the dkHK1 multi-step phosphorelay pathway in Populus.

In previous studies, we highlighted a multistep phosphorelay (MSP) system in poplars composed of two hybrid-type Histidine aspartate Kinases, dkHK1a and dkHK1b, which interact with three Histidine Phosphotransfer proteins, dkHPt2, 7, and 9, which in turn interact with six type B Response Regulators. These interactions correspond to the dkHK1a-b/dkHPts/dkRRBs MSP. This MSP is putatively involved in an osmosensing pathway, as dkHK1a-b are orthologous to the Arabidopsis osmosensor AHK1, and able to complement a mutant yeast deleted for its osmosensors. Since type A RRs have been characterized as negative regulators in cytokinin MSP signaling due to their interaction with HPt proteins, we decided in this study to characterize poplar type A RRs and their implication in the MSP. For a global view of this MSP, we isolated 10 poplar type A RR cDNAs, and determined their subcellular localization to check the in silico prediction experimentally. For most of them, the in planta subcellular localization was as predicted, except for three RRAs, for which this experimental approach gave a more precise localization. Interaction studies using yeast two-hybrid and in planta BiFC assays, together with transcript expression analysis in poplar organs led to eight dkRRAs being singled out as partners which could interfere the dkHK1a-b/dkHPts/dkRRBs MSP identified in previous studies. Consequently, the results obtained in this study now provide an exhaustive view of dkHK1a-b partners belonging to a poplar MSP.

Genetic characterization of Libyan date palm resources by microsatellite markers.

Molecular typing of 377 female date palm trees belonging to 18 Libyan cultivars and representing common genotypes in the central Libyan oasis of Al Jufrah was performed using 16 highly polymorphic microsatellite or SSR loci. A total of 110 alleles with an average of 6.88 alleles per locus were scored indicating the high level of polymorphism existing among the cultivars thus allowing their genetic fingerprinting. Moreover 28 alleles out of 110 were fixed. All the cultivars were characterized by negative values of the Fixation Index (F) due to an excess of heterozygotes with respect to HW equilibrium. The pattern of genetic diversity among cultivars was estimated by codominant genetic distances and presented by principal coordinates analysis (PCoA). The observed pattern evidences the genetic diversity existing among cultivars that allow distinguishing them easily. The average dissimilarity internal to each cultivar ranged from 0 to 21. Seven cultivars showed value zero indicating no genetic difference within cultivar in agreement with their Fixation Index (F = 1). A varietal identification key was also built using multiloci genotyping with only three microsatellite loci that identified 23 alleles in total. The possibility to attribute the unknown male plant to a cultivar was also considered and male parentage analysis was performed. Fifty-five male plants out of 63 were assigned to a definite cultivar with high confidence level. The positive result obtained in identifying males confirmed the suitability of SSR for clone fingerprinting and cultivar identification, thus opening new prospects for date palm breeding.

Transcript analysis of stress defence genes in a white poplar clone inoculated with the arbuscular mycorrhizal fungus Glomus mosseae and grown on a polluted soil.

In this study we investigated if the symbiosis with the arbuscular mycorrhizal fungus Glomus mosseae, which contributes to alleviate heavy metal stress in plants, may affect the transcription of genes involved in the stress defence in the white poplar clone 'AL35' grown on a multimetal (Cu and Zn) contaminated soil. The results obtained showed that the symbiosis with G. mosseae reduced transcript abundance of genes involved in antioxidant defence in leaves and roots of 'AL35' plants grown on the heavy metal-polluted soil. Moreover, the interaction between this poplar clone and the arbuscular mycorrhizal fungus induced the gene coding for phytochelatin synthase in leaves, whereas the expression of genes involved in heavy metal homeostasis did not change in roots. The present results suggest that, in presence of high levels of heavy metals, inoculation with G. mosseae may confer to 'AL35' a more efficient control of the oxidant level. Moreover, in mycorrhizal plants heavy metal chelation pathways appear involved in the defence strategies in leaves, whereas in roots they do not seem to contribute to increase the plant tolerance of heavy metals.

Transcriptome changes in the cambial region of poplar (Populus alba L.) in response to water deficit.

A transcriptome analysis of the Populus alba cambial region was performed with the aim of elucidating the gene network underlying the response to water deficit within the cambium and differentiating derivative cambial cells. Water stress was induced in 1-year-old P. alba plants by withholding water for 9 days. At that time, leaf predawn water potential fell to -0.8 MPa, resulting in a significant reduction in stomatal conductance, CO(2) assimilation and a consistent increment of stem shrinkage. These effects were almost fully reversed by re-hydration. The water deficit resulted in changes in gene expression that affected several functional categories, such as protein metabolism, cell wall metabolism, stress response, transporters and transcriptional regulation. The function of up- and down-regulated genes is discussed considering the physiological response of the plants to water deficit.

Molecular cloning, characterisation and expression of a manganese superoxide dismutase gene from peach (Prunus persica [L.] Batsch).

Two cDNA clones encoding mitochondrial manganese superoxide dismutases (MnSODs) from peach ( Prunus persica [L.] Batsch) were identified, which show homologies to several plant MnSODs. The amino acid sequence predicted from one full-length clone ( MnSOD1) showed the highest homology to an MnSOD from Nicotiana plumbaginifolia (94%) and included a 24-amino acid transit peptide typical of those used to target proteins to the mitochondria. A second, partial clone ( MnSOD2) showed divergence from MnSOD1 in the 3' untranslated region. It could therefore derive from a second gene or from an allele of MnSOD1. Southern hybridisation analysis suggests the existence of two MnSOD genes in peach. SOD isoenzyme profiles, MnSOD1 expression and protein levels were studied in aerial vegetative tissues derived from plants of different ages and in adult plants during the seasonal cycle. Zymograms revealed at least two isoforms of MnSODs in pre-shooting vegetative buds and in developing fruits. Levels of MnSODs were lower in leaves derived from apical shoots of adult plants than in leaves derived from seedlings, basal shoots or in vitro propagated juvenile plants, which are considered as juvenile-like structures. The MnSOD1 transcript and protein followed the same pattern. The results suggest that the steady-state levels of MnSOD1 mRNA in leaves vary with both the ontogenetic stage and the growth rate of the tissues examined.

Differential activity of catalase and superoxide dismutase in seedlings and in vitro micropropagated oak (Quercus robur L.).

Catalase (CAT) and superoxide dismutase (SOD) activity profiles were examined by native polyacrylamide gel electrophoresis in different tissues of seedlings and microcuttings of oak (Quercus robur L.) initiated from crown material (NL100A) and from basal epicormic shoots (NL100R), which differ in rooting ability. Two CAT isoforms were differentially active in seedlings and microcuttings; in particular, CAT-2 was activated in the basal callus of rooted microshoots. SOD isoenzymes, Mn-SOD and at least four Cu/Zn-SODs were found to be present, with Mn-SODs particularly active in microcuttings. No differences were found between the electrophoretic profiles of the two lines despite their different ontogenetic origin. The strong activity of CAT-2 in rooted microshoots indicates that this isoform is a protein specifically related to rooting.

Anthocyanin and proteins as biochemical markers in maize endosperm cultures.

Endosperm maize cultures derived from a strain homozygous for all genes required for anthocyanin synthesis develop an intense pigmentation. Pigmenting ability is generally maintained in successive subcultures, altough colourless areas are frequently observed in pigmented cultures. The isolated colourless cell clusters show a growth rate higher than the coloured ones. These calli nevertheless do not lose the ability to synthesize anthocyanins, and in successive subcultures turn red again.The different growth rates associated with the ability of cells to accumulate pigments suggest the existence of different physiological states of the culture. To investigate this possibility we analyzed the polypeptide patterns of coloured and colourless cultures. SDS gel electrophoresis has demonstrated differences in soluble protein fractions, among which a 26 kD peptide, characteristic of pigmented tissues, has been evidenced. Zein, the major storage protein of maize endosperm is present, although at very low levels, both in pigmented and in unpigmented cultures, confirming that its synthesis occurs continuously in vitro.

Effect of the genes B and Pl on anthocyanin synthesis in maize endosperm culture.

B and Pl are two genes involved in anthocyanin biosynthesis in maize (Zea mays) plant tissues. In this work the effect of B and Pl on pigment accumulation is analyzed in endosperm tissues, either cultured in vitro or scraped off from the seed. The results obtained indicate that the two genes play a different role in callus pigmentation: B exerts a qualitative change in pigment composition, while Pl controls the rate of pigment accumulation in the callus. Anthocyanin synthesis in all strains analyzed appears to be light independent. Two cases of instability in pigment production arisen in the endosperm cultures are described and discussed in relation to epigenetic variation in secondary metabolite content in plant tissue culture.