Michèle Crèvecoeur

Identification of a Ca(2+)-pectate binding site on an apoplastic peroxidase

An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca2+-induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus–insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca2+-pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca2+-pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.

Changing concepts in plant hormone action

SummaryA plant hormone is not, in the classic animal sense, a chemical synthesized in one organ, transported to a second organ to exert a chemical action to control a physiological event. Any phytohormone can be synthesized everywhere and can influence different growth and development processes at different places. The concept of physiological activity under hormonal control cannot be dissociated from changes in concentrations at the site of action, from spatial differences and changes in the tissues sensitivity to the compound, from its transport and its metabolism, from balances and interactions with the other phytohormones, or in their metabolic relationships, and in their signaling pathways as well. Secondary messengers are also involved. Hormonal involvement in physiological processes can appear through several distinct manifestations (as environmental sensors, homeostatic regulators and spatio-temporal synchronizers, resource allocators, biotime adjusters, etc.), dependent on or integrated with the primary biochemical pathways. The time has also passed for the hypothesized ‘specific’ developmental hormones, rhizocaline, canlocaline, and florigen: root, stem, and flower formation result from a sequential control of specific events at the right places through a coordinated control by electrical signals, the known phytohormones and nonspecific molecules of primary and secondary metabolism, and involve both cytoplasmic and apoplastic compartments. These contemporary views are examined in this review.

Expression of a peroxidase gene in zucchini in relation with hypocotyl growth

The expression of the gene encoding an anionic isoperoxidase from zucchini (Cucurbita pepo L., cv. Black Beauty) (APRX) has been analyzed by northern blot, in situ hybridization and isoelectric focusing (IEF). Hypocotyls and stems were the organs containing the highest amount of APRX transcripts. A detailed study of APRX expression in hypocotyls revealed that the mRNA accumulated mainly beneath the apical hook, in the outer half of the hook and in the root tip, three zones characterized by a relatively high elongation rate. Seedlings grown in darkness or under a 12-h photoperiod exhibited a maximum APRX expression 5 or 6 d after germination, preceding an acceleration of hypocotyl growth. The seedlings grown under continuous light had a shorter hypocotyl and a lower level of expression. Surprisingly, the variations of APRX transcript level observed in the different hypocotyl zones or in hypocotyls of different ages did not result in visible changes in the amounts of APRX activity, as determined after IEF separation. The distribution of lignin along the hypocotyl and in the hook was inversely related to the level of APRX transcript.

Long term in vitro-cultured plant cells show typical neoplastic features at the cytological level

Cells from a green normal (dependent on exogenous hormones) callus and from an achlorophyllous fully habituated (independent from exogenous hormones) callus, both generated from the same sugarbeet strain more than twenty years ago, were reexamined cytologically, ten years after the first comparative description. Cells from the habituated callus, already considered as neoplastic cells, because terminating a neoplastic progression where the organogenic totipotency was lost, still showed nuclear invaginations, polynucleolation, vacuolation of nucleoli and incomplete cell walls, nevertheless at a higher degree. The present study particularly shows that, compared to their previous description, normal cells have started to acquire some features (polynucleolation, nuclear invaginations…) that are typical of the neoplastic cells. This suggests that normal cells, on the long term, also entered a neoplastic progression, which should explain the known progressive loss of regeneration capacity of too long subcultured hormone‐dependent calli.

Long-Distance Transport of Thiamine (Vitamin B1) Is Concomitant with That of Polyamines

Long distance transport of thiamine is required for growth and development and is facilitated by PUT3, the polyamine transporter. Thiamine (vitamin B1) is ubiquitous and essential for cell energy supply in all organisms as a vital metabolic cofactor, known for over a century. In plants, it is established that biosynthesis de novo is taking place predominantly in green tissues and is furthermore limited to plastids. Therefore, transport mechanisms are required to mediate the movement of this polar metabolite from source to sink tissue to activate key enzymes in cellular energy generating pathways but are currently unknown. Similar to thiamine, polyamines are an essential set of charged molecules required for diverse aspects of growth and development, the homeostasis of which necessitates long-distance transport processes that have remained elusive. Here, a yeast-based screen allowed us to identify Arabidopsis (Arabidopsis thaliana) PUT3 as a thiamine transporter. A combination of biochemical, physiological, and genetic approaches permitted us to show that PUT3 mediates phloem transport of both thiamine and polyamines. Loss of function of PUT3 demonstrated that the tissue distribution of these metabolites is altered with growth and developmental consequences. The pivotal role of PUT3 mediated thiamine and polyamine homeostasis in plants, and its importance for plant fitness is revealed through these findings.

Identification and Expression of Nine Oak Aquaporin Genes in the Primary Root Axis of Two Oak Species, Quercus petraea and Quercus robur

Aquaporins (AQPs) belong to the Major Intrinsic Protein family that conducts water and other small solutes across biological membranes. This study aimed to identify and characterize AQP genes in the primary root axis of two oak species, Quercus petraea and Quercus robur. Nine putative AQP genes were cloned, and their expression was profiled in different developmental root zones by real-time PCR. A detailed examination of the predicted amino acid sequences and subsequent phylogenetic analysis showed that the isolated AQPs could be divided into two subfamilies, which included six plasma membrane intrinsic proteins (PIPs) and three tonoplast intrinsic proteins (TIPs). We characterized the anatomical features of the roots and defined three developmental root zones: the immature, transition and mature zones. Expression analysis of the AQPs was performed according to these root developmental stages. Our results showed that the expression of PIP2;3 and TIP1 was significantly higher in Quercus petraea compared with Quercus robur in the three root zones. However, PIP2;1 and TIP2;1 were found to be differentially expressed in the mature zone of the two oak species. Of the nine AQP genes identified and analyzed, we highlighted four genes that might facilitate a deeper understanding of how these two closely related tree species adapted to different environments.

A novel non-symbiotic hemoglobin from oak: Roles in root signalling and development?

The cellular and molecular adaptations of non-model woody species to environmental changes are still poorly understood. We have cloned and characterised a novel non-symbiotic hemoglobin from oak roots (QpHb1) which exhibits a specific cellular distribution in the root. The QpHb1 gene is strongly expressed in the protoderm and the protoxylem cells in two Quercus species (Q. petraea and Q. robur) with contrasting adaptive potential to drought and flooding. The constitutive expression of QpHb1 in both oak species in specific root tissues combined with the reported presence of nitric oxide in the same tissues and its potential for protein S-nitrosylation could support a role for non-symbiotic hemoglobins in signalling changes in the root environment and/or in controlling some aspects of root development. Addendum to: Parent C, Berger A, Folzer H, Dat J, Crèvecoeur M, Badot PM, Capelli N. A novel nonsymbiotic hemoglobin from oak: Cellular and tissue specificity of gene expression. New Phytol 2008; 177:142-54.

Glucose-6-phosphate dehydrogenase activity in spinach as measured by image analysis: A new approach for plant enzyme histochemistry

SummaryA relatively low-cost computer-assisted image analysis system is described. Software has been specifically written for the continuous monitoring of absorbance readings on cryostat sectons of plant tissues incubated in media to reveal enzyme activities. The equipment was tested by quantify ing glucose-6-phosphate dehydrogenase activity in cryostat sections from shoot apices of spinach plants. The reaction rate of the dehydrogenase activity was monitored at two incubation temperatures, 20°C and 30°C. Control incubations were performed in media lacking substrate. The specific test minus control reaction at 30°C was twice that at 20°C. Variation of the substrate concentration at 30°C yielded a Km value of 0.37 mM. These preliminary results show that our image analysis system can be used for kineti measurements of dehydrogenase activity in froizen tssue sections and constitute a new approach for enzyme histochemistry in the shoot apicla meristem.

Comparison of Floral Structure and Ontogeny in Monoecious and Dioecious Species of the Palm Tribe Chamaedoreeae (Arecaceae; Arecoideae)

Premise of research.u2003The sexuality of flowers is an important reproductive character in angiosperms. An insight into the evolutionary events that led to the appearance of monoecious and dioecious species can be gained by comparing closely related groups with contrasting characters. For this study, we focused on the tribe Chamaedoreeae, within which dioecy appears to have evolved twice from a monoecious ancestor. Methodology.u2003To improve our knowledge of flower structure and ontogeny in this group, SEM and anatomical sectioning were performed on inflorescences and flowers of the dioecious species Chamaedorea tepejilote and the monoecious species Hyophorbe lagenicaulis at different developmental stages. Pivotal results.u2003Our data highlighted that the higher degree of spatial sexual separation seen in the dioecious C. tepejilote, compared to the monoecious H. lagenicaulis, is accompanied by a more accentuated dimorphism between male and female flowers. More specifically, in the case of C. tepejilote, the vestigial reproductive organs (staminodes of the female flower and pistillode of the male flower) are more rudimentary structures, in terms of their developmental differentiation, than their homologs in H. lagenicaulis. Conclusions.u2003Our data suggest that the unisexual flowers already present in the monoecious ancestor of the Chamaedoreeae underwent further modifications either shortly before or since the appearance of dioecy in the genus Chamaedorea. These structural changes were presumably the result of genomic mutations causing earlier developmental arrest of the vestigial reproductive organs and are likely, in turn, to have conferred enhanced resource-allocation efficiency.