Developmental changes in the composition of brain microtubule-associated proteins have been studied in three species : the rat and the mouse, which are characterized by post-natal brain development, and the guinea-pig, whose brain is mature at birth.1. At an adult stage, and whatever the species, two major microtubule-associated proteins, which have been referred to MAP2 and z, have been identified by polyacrylamide gel electrophoresis. Rat z is composed of four closely spaced bands; mouse z contains only three components with one of them being present in higher proportion than the others; adult guinea-pig z is essentially present as a single band.2. Microtubule-associated proteins were also prepared at different stages of brain development. In the three species only two bands were seen in the z region at immature stages of development (fast z and slow z). However adult z factors progressively replace the young entities. In contrast, only small changes were seen in the proportion of MAP2.3. Peptide mapping analysis of the purified z entities confirmed that the four adult rat proteins are very similar. In contrast, peptide mapping of the two young rat z proteins were very different from each other and from those of the adult ones. Peptide mappings of young and adult MAP2 were only slightly different. 4. The activities of young z proteins and young MAP2 in promoting pure tubulin assembly were much lower than those of the adult ones. Young fast z and young slow z were purified and both show to be active in promoting pure tubulin polymerization.5 . These data demonstrate the existence of two types of heterogeneity of microtubule-associated proteins : plurality of protein species at every stage of brain development and changes in composition and activity dependent on development.

Germination of pollen grains is a crucial step in plant reproduction. However, the molecular mechanisms involved remain unclear. We investigated the role of PECTIN METHYLESTERASE48 (PME48), an enzyme implicated in the remodeling of pectins in Arabidopsis (Arabidopsis thaliana) pollen. A combination of functional genomics, gene expression, in vivo and in vitro pollen germination, immunolabeling, and biochemical analyses was used on wild-type and Atpme48 mutant plants. We showed that AtPME48 is specifically expressed in the male gametophyte and is the second most expressed PME in dry and imbibed pollen grains. Pollen grains from homozygous mutant lines displayed a significant delay in imbibition and germination in vitro and in vivo. Moreover, numerous pollen grains showed two tips emerging instead of one in the wild type. Immunolabeling and Fourier transform infrared analyses showed that the degree of methylesterification of the homogalacturonan was higher in pme482/2 pollen grains. In contrast, the PME activity was lower in pme482/2, partly due to a reduction of PME48 activity revealed by zymogram. Interestingly, the wild-type phenotype was restored in pme482/2 with the optimum germination medium supplemented with 2.5 mM calcium chloride, suggesting that in the wild-type pollen, the weakly methylesterified homogalacturonan is a source of Ca 2+ necessary for pollen germination. Although pollen-specific PMEs are traditionally associated with pollen tube elongation, this study provides strong evidence that PME48 impacts the mechanical properties of the intine wall during maturation of the pollen grain, which, in turn, influences pollen grain germination.

The onsert of neuronal differentiation is characterised by intensive neurite growth; because microtubule formation is strictly required during this process, in vitro assembly of the tubulin present in the rat brain has been studied at different stages of development: the rate of assembly is very slow in the early stages and increases progressively with age from birth until adulthood. Other data also suggested that the limiting factor in the young brain is the amount or activity of one or several of the minor components which co-polymerise into microtubules with tubulin. We show here that both the composition and the activity of the microtubule-associated proteins change during the time course of rat brain development.

Pectin methylesterases (PMEs) were detected in tobacco ( Nicotiana tabacum) pollen tubes grown in vitro. Seven PME isoforms exhibiting a wide isoelectric-point (pI) range (5.3-9.1) were found in crude extracts of pollen tubes. These isoforms were mainly retrieved in supernatants after low- and high-speed separation of the crude extract. Two isoforms, with pIs 5.5 and 7.3 and molecular weight about 158 kDa, were detected by immunoblotting with anti-flax PME antiserum. Localization of pectins and PME isoforms in pollen tubes was investigated by immunogold labelling with JIM5 monoclonal antibodies and anti-flax PME antiserum, respectively. In germinated pollen grains, two PME isoforms were mainly detected in the exine, Golgi apparatus and secretory vesicles. In pollen tubes the same two PME isoforms were distributed along the outer face of the plasma membrane in the vicinity of the inner layer of the cell wall, in the Golgi and around secretory vesicles. In pollen grains, PME isoforms were, in some cases, mixed with acidic pectins in proximity to the outer surface of the plasma membrane. In pollen tubes the presence of PMEs inside secretory vesicles carrying esterified pectins supports the hypothesis that, during pollen tube growth, PMEs could be transferred by secretory vesicles in a precursor form and be activated at the tip where exocytosis takes place.

Summary• Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development.• A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used.• We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia.• Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.

Background and Aims In Arabidopsis thaliana, the degree of methylesterification (DM) of homogalacturonans (HGs), the main pectic constituent of the cell wall, can be modified by pectin methylesterases (PMEs). In all organisms, two types of protein structure have been reported for PMEs: group 1 and group 2. In group 2 PMEs, the active part (PME domain, Pfam01095) is preceded by an N-terminal extension (PRO part), which shows similarities to PME inhibitors (PMEI domain, Pfam04043). This PRO part mediates retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating PME activity through a post-translational mechanism. This study investigated the roles of a subtilisin-type serine protease (SBT) in the processing of a PME isoform. † Methods Using a combination of functional genomics, biochemistry and proteomic approaches, the role of a specific SBT in the processing of a group 2 PME was assessed together with its consequences for plant development. † Key Results A group 2 PME, AtPME17 (At2g45220), was identified, which was highly co-expressed, both spatially and temporally, with AtSBT3.5 (At1g32940), a subtilisin-type serine protease (subtilase, SBT), during root development. PME activity was modified in roots of knockout mutants for both proteins with consequent effects on root growth. This suggested a role for SBT3.5 in the processing of PME17 in planta. Using transient expression in Nicotiana benthamiana, it was indeed shown that SBT3.5 can process PME17 at a specific single processing motif, releasing a mature isoform in the apoplasm. † Conclusions By revealing the potential role of SBT3.5 in the processing of PME17, this study brings new evidence of the complexity of the regulation of PMEs in plants, and highlights the need for identifying specific PME-SBT pairs.

Pectin methylesterases (PMEs) are ubiquitous enzymes present in the plant cell wall. They catalyse the demethylesterification of homogalacturonic acid units of pectins, which, in turn, can be associated with different physiological phenomena. In this study, different flax (Linum usitatissimum L.) PME isoforms were observed: neutral (pI 7.0 and 7.5, MW: 110 kDa), basic (pI 8.3 and 8.5, MW: 110 kDa) and very basic (pI>9.5, MW: 38 kDa). In an attempt to identify most of the expressed cell wall LuPME isoforms, polyclonal antibodies were raised against a conserved region of PME. These antibodies allowed the purification of the very basic PME isoform (pI 9.5, MW: 36 kDa) from flax cells, designated LuPME5. This isoform corresponds to the Lupme5 cDNA isolated, at the same time, from flax hypocotyls, by using the RACE-PCR technique. Semi-quantitative PCR experiments showed that the Lupme5 transcript was highly expressed in the hypocotyl zones where elongation is being achieved. Thus, this enzyme may be involved in cell wall stiffening.

During evolution of land plants, the first colonizing species presented leafy-dominant gametophytes, found in non-vascular plants (bryophytes). Today, bryophytes include liverworts, mosses, and hornworts. In the first seedless vascular plants (lycophytes), the sporophytic stage of life started to be predominant. In the seed producing plants, gymnosperms and angiosperms , the gametophytic stage is restricted to reproduction. In mosses and ferns, the haploid spores germinate and form a protonema, which develops into a leafy gametophyte producing rhizoids for anchorage, water and nutrient uptakes. The basal gymnosperms (cycads and Ginkgo ) reproduce by zooidogamy. Their pollen grains develop a multi-branched pollen tube that penetrates the nucellus and releases flagellated sperm cells that swim to the egg cell. The pollen grain of other gymnosperms (conifers and gnetophytes) as well as angiosperms germinates and produces a pollen tube that directly delivers the sperm cells to the ovule (siphonogamy). These different gametophytes, which are short or long-lived structures, share a common tip-growing mode of cell expansion. Tip-growth requires a massive cell wall deposition to promote cell elongation, but also a tight spatial and temporal control of the cell wall remodeling in order to modulate the mechanical properties of the cell wall. The growth rate of these cells is very variable depending on the structure and the species, ranging from very slow (protonemata, rhizoids, and some gymnosperm pollen tubes), to a slow to fast-growth in other gymnosperms and angiosperms. In addition, the structural diversity of the female counterparts in angiosperms (dry, semi-dry vs wet stigmas, short vs long, solid vs hollow styles) will impact the speed and efficiency of sperm delivery. As the evolution and diversity of the cell wall polysaccharides accompanied the diversification of cell wall structural proteins and remodeling enzymes, this review focuses on our current knowledge on the biochemistry, the distribution and remodeling of the main cell wall polymers (including cellulose, hemicelluloses, pectins, callose, arabinogalactan-proteins and extensins), during the tip-expansion of gametophytes from bryophytes, pteridophytes (lycophytes and monilophytes), gymnosperms and the monocot and eudicot angiosperms.