Jean-Louis Hilbert

Review: Correlations between oxygen affinity and sequence classifications of plant hemoglobins

Plants express three phylogenetic classes of hemoglobins (Hb) based on sequence analyses. Class 1 and 2 Hbs are full‐length globins with the classical eight helix Mb‐like fold, whereas Class 3 plant Hbs resemble the truncated globins found in bacteria. With the exception of the specialized leghemoglobins, the physiological functions of these plant hemoglobins remain unknown. We have reviewed and, in some cases, measured new oxygen binding properties of a large number of Class 1 and 2 plant nonsymbiotic Hbs and leghemoglobins. We found that sequence classification correlates with distinct extents of hexacoordination with the distal histidine and markedly different overall oxygen affinities and association and dissociation rate constants. These results suggest strong selective pressure for the evolution of distinct physiological functions. The leghemoglobins evolved from the Class 2 globins and show no hexacoordination, very high rates of O2 binding (∼250 μM−1 s−1), moderately high rates of O2 dissociation (∼5–15 s−1), and high oxygen affinity (Kd or P50 ≈ 50 nM). These properties both facilitate O2 diffusion to respiring N2 fixing bacteria and reduce O2 tension in the root nodules of legumes. The Class 1 plant Hbs show weak hexacoordination (KHisE7 ≈ 2), moderate rates of O2 binding (∼25 μM−1 s−1), very small rates of O2 dissociation (∼0.16 s−1), and remarkably high O2 affinities (P50 ≈ 2 nM), suggesting a function involving O2 and nitric oxide (NO) scavenging. The Class 2 Hbs exhibit strong hexacoordination (KHisE7 ≈ 100), low rates of O2 binding (∼1 μM−1 s−1), moderately low O2 dissociation rate constants (∼1 s−1), and moderate, Mb‐like O2 affinities (P50 ≈ 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.

A proteomic approach to decipher chilling response from cold acclimation in pea (Pisum sativum L.).

Two pea lines (Pisum sativum L.) with contrasted behaviours towards chilling and subsequent frost were studied by a proteomic approach to better understand cold acclimation. Following a chilling period, the Champagne line becomes tolerant to frost whereas Terese remains sensitive. Variance analysis allowed to select 260 statistically variable spots with 68 identified proteins (35 in leaves, 18 in stems, and 15 in roots). These proteins were shared out in proteins related to chilling response or cold acclimation. The better adaptation of Champagne to chilling might be related to a higher content in proteins involved in photosynthesis and in defence mechanisms. Moreover Champagne might prevent freezing damage particularly thanks to a higher constitutive expression of housekeeping proteins related to Terese. After three days of subsequent frost, proteomes of previously chilled plants also showed significant differences compared to unchilled plants. Out of 112 statistically variable spots (44 in leaves, 38 in stems, and 30 in roots), 32 proteins were identified. These proteins were related to frost response or frost resistance. It seems that Champagne could resist to frost with the reorientation of the energy metabolism.

A glutathione S-transferase cDNA identified by mRNA differential display is upregulated during somatic embryogenesis in Cichorium.

CHI-GST1, a cDNA encoding a glutathione S-transferase, was isolated by differential display in leaf tissues of chicory, during the early stages of somatic embryogenesis. Expression analysis of the gene by Northern blot indicated that the transcript accumulation is specific of the leaf developing somatic embryogenesis and is not observed in leaf tissue of the non-embryogenic cultivar.

Association of sugar content QTL and PQL with physiological traits relevant to frost damage resistance in pea under field and controlled conditions

To increase yield in pea (Pisum sativum L.), autumn sowing would be preferable. Hence, frost tolerance of pea became a major trait of interest for breeders. In order to better understand the cold acclimation in pea, Champagne a frost tolerant line and Terese, a frost sensitive line, and their recombinant inbred lines (RIL) were studied. RIL frost tolerance was evaluated by a frost damage scale under field as well as controlled conditions. A quantitative trait loci (QTL) approach was used to identify chromosomal regions linked to frost tolerance. The detected QTL explained from 6.5 to 46.5% of the phenotypic variance. Amongst them, those located on linkage groups 5 and 6 were consistent with over all experiments, in field as well as in controlled environments. In order to improve the understanding of the frost tolerance mechanisms, several cold acclimation key characters such as concentration of sugars, electrolyte leakage, osmotic pressure, and activity of RuBisCO were assessed. Some of these physiological QTL colocalised with QTL for frost damage, in particular two raffinose QTL on LG5 and LG6 and one RuBisCO activity QTL on LG6, explaining 8.8 to 27.0% of the phenotypic variance. In addition, protein quantitative loci were mapped; some of them colocalised with frost damage and physiological QTL on LG5 and LG6, explaining 16.0–43.6% of the phenotypic variance. Raffinose metabolism and RuBisCO activity and its effect on photosynthesis might play a major role in cold acclimation of pea.

Characterization of expressed sequence tags obtained by SSH during somatic embryogenesis in Cichorium intybus L

BackgroundSomatic embryogenesis (SE) is an asexual propagation pathway requiring a somatic-to-embryonic transition of differentiated somatic cells toward embryogenic cells capable of producing embryos in a process resembling zygotic embryogenesis. In chicory, genetic variability with respect to the formation of somatic embryos was detected between plants from a population of Cichorium intybus L. landrace Koospol. Though all plants from this population were self incompatible, we managed by repeated selfing to obtain a few seeds from one highly embryogenic (E) plant, K59. Among the plants grown from these seeds, one plant, C15, was found to be non-embryogenic (NE) under our SE-inducing conditions. Being closely related, we decided to exploit the difference in SE capacity between K59 and its descendant C15 to study gene expression during the early stages of SE in chicory.ResultsCytological analysis indicated that in K59 leaf explants the first cell divisions leading to SE were observed at day 4 of culture. In contrast, in C15 explants no cell divisions were observed and SE development seemed arrested before cell reactivation. Using mRNAs isolated from leaf explants from both genotypes after 4 days of culture under SE-inducing conditions, an E and a NE cDNA-library were generated by SSH. A total of 3,348 ESTs from both libraries turned out to represent a maximum of 2,077 genes. In silico subtraction analysis sorted only 33 genes as differentially expressed in the E or NE genotype, indicating that SSH had resulted in an effective normalisation. Real-time RT-PCR was used to verify the expression levels of 48 genes represented by ESTs from either library. The results showed preferential expression of genes related to protein synthesis and cell division in the E genotype, and related to defence in the NE genotype.ConclusionIn accordance with the cytological observations, mRNA levels in explants from K59 and C15 collected at day 4 of SE culture reflected differential gene expression that presumably are related to processes accompanying early stages of direct SE. The E and NE library obtained thus represent important tools for subsequent detailed analysis of molecular mechanisms underlying this process in chicory, and its genetic control.

Inhibition of direct somatic embryogenesis by α-difluoromethylarginine in a Cichorium hybrid: effects on polyamine content and protein patterns

Direct somatic embryogenesis was induced in mesophyll cells of leaves of the hybrid Cichorium intybus L. var. sativum x C. endivia L. var. latifolia through a twostep procedure during 5 d in a M17 liquid medium supplemented with 330 mM glycerol. The lack of cell division during the induction step seems to be associated with a relatively stable low free-polyamine content. Synchronised divisions of competent cells occurred after transfer onto a glycerol-free M17 medium. Increased free-polyamine levels, especially putrescine, accompanied embryo differentiation. The addition of 0.1 to 10 mM α-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of polyamine biosynthesis in this Cichorium hybrid, to the medium caused a drastic decrease of polyamine content. Putrescine and spermidine rapidly decreased after 1 d incubation with DFMA and remained at ten times lower concentration than the controls. Embryogenesis was decreased (2 embryos per mm2 of leaf versus 12 embryos per mm2) in DFMA-incubated leaves. The addition of 5 mM putrescine to DFMA-treated cultures restored both the initial spermidine content and the embryogenesis. The effects of somatic-embryogenesis inhibition on protein biosynthesis were analysed using two-dimensional electrophoresis. In control samples, extracellular polypeptides were secreted into the culture medium. Two of these secreted polypeptides exhibited the same features as two tissue somatic-embryogenesis-related polypeptides. In the presence of 5 mM DFMA their contents in the medium were reduced, whereas they reached similar levels as in the control samples in the presence of DFMA and putrescine.

Embryogenesis-related protein synthesis and accumulation during early acquisition of somatic embryogenesis competence in Cichorium

Abstract Somatic embryogenesis in Cichorium intybus was achieved from leaves. In the presence of glycerol in a half-strength Murashige and Skoog medium, mesophyll cells were induced to undergo somatic embryogenesis. The first division occurred 3 h after transfer of the synchronised embryogenic cells to a medium lacking glycerol (expression medium). Protein accumulation and synthesis during early embryogenesis was analysed using two-dimensional PAGE. As demonstrated by silver-staining and autoradiography of [35S]methionine-labelled polypeptides, most of the changes in protein pattern occurred by the third day of induction, before the first cellular division and well before any overt signs of morphological changes. At least 15 embryogenesis-related proteins were detected during induction of cells for morphogenic competence. This suggests that acquisition of the embryogenic competence which involves reprogramming of differentiated cells occurred early during the induction step in Cichorium leaves. The role of the embryogenesis-related proteins in the embryogenesis process is still unknown but they may play different roles in the acquisition of the embryogenic competence.

Removal of the fibrillar network surrounding Cichorium somatic embryos using cytoskeleton inhibitors: analysis of proteic components.

Abstract In Cichorium ‘474’, the embryo globular stage was characterised by a fibrillar network linking peripheral neighbouring cells. To test a putative connection between this fibrillar network and the cytoskeleton (CTK), we have used CTK disrupting agents (cold; colchicine; cytochalasine B) on Cichorium somatic embryos. Scanning electron microscopy observations showed that these three treatments induced the disappearance of the fibrillar network and suggested that this network could take part of the CTK-plasma membrane (PM)-extracellular matrix (ECM) continuum. The treatment media containing the removal fibrillar network were used to analyse its proteic component by 2D-PAGE. Using a Sun SPARCstation computer, the comparison of the gels corresponding to the different treatments allowed us to detect a group of 25 common proteins recovered in the medium after each treatment and in somatic embryogenesis conditioned medium. During the short treatments applied on somatic embryos, a large amount of high molecular weight glycoproteins corresponding to arabinogalactan proteins (AGPs) have been quantified with β- d -glucosyl Yariv reagent and identified with monoclonal antibodies raised against AGP epitopes (JIM13, JIM16, LM2). The implication of the fibrillar network and AGPs in the continuum CTK-PM-ECM are discussed in attempt to elucidate their possible function during Cichorium somatic embryogenesis.

Immunolocalization of non-symbiotic hemoglobins during somatic embryogenesis in chicory.

Hemoglobins are ancient O2-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum × C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.

Phenolic Composition and Antioxidant and Antimicrobial Activities of Extracts Obtained from Crataegus azarolus L. var. aronia (Willd.) Batt. Ovaries Calli

Objective. Plant cell culture is an innovative technology to produce a variety of substances. Numerous plants synthesize among their secondary metabolites phenolic compounds which possess antioxidant and antimicrobial effects. Hawthorn (Crataegus) is one of these plants which has long been used in folk medicine and is widely utilized in pharmaceutical preparations mainly in neuro- and cardiosedative actions. Methods and Results. The production of polyphenol by fifty-two-week-old Crataegus azarolus var. aronia calli was studied in relation to growth variation and antioxidant and antimicrobial capacity within a subcultured period. The DPPH and ABTS