Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro.

We have established a detailed framework for the process of shoot regeneration from Arabidopsis root and hypocotyl explants grown in vitro. Using transgenic plant lines in which the GUS or GFP genes were fused to promoters of developmental genes (WUS, CLV1, CLV3, STM, CUC1, PLT1, RCH1, QC25), or to promoters of genes encoding indicators of the auxin response (DR5) or transport (PIN1), cytokinin (CK) response (ARR5) or synthesis (IPT5), or mitotic activity (CYCB1), we showed that regenerated shoots originated directly or indirectly from the pericycle cells adjacent to xylem poles. In addition, shoot regeneration appeared to be partly similar to the formation of lateral root meristems (LRMs). During pre-culture on a 2, 4-dichlorophenoxyacetic acid (2, 4-D)-rich callus-inducing medium (CIM), xylem pericycle reactivation established outgrowths that were not true calli but had many characteristics of LRMs. Transfer to a CK-rich shoot-inducing medium (SIM) resulted in early LRM-like primordia changing to shoot meristems. Direct origin of shoots from the xylem pericycle occurred upon direct culture on CK-containing media without prior growth on CIM. Thus, it appeared that the xylem pericycle is more pluripotent than previously thought. This pluripotency was accompanied by the ability of pericycle derivatives to retain diploidy, even after several rounds of cell division. In contrast, the phloem pericycle did not display such developmental plasticity, and responded to CKs with only periclinal divisions. Such observations reinforce the view that the pericycle is an 'extended meristem' that comprises two types of cell populations. They also suggest that the founder cells for LRM initiation are not initially fully specified for this developmental pathway.

Functional alterations of root meristematic cells of Arabidopsis thaliana induced by a simulated microgravity environment.

Environmental gravity modulates plant growth and development, and these processes are influenced by the balance between cell proliferation and differentiation in meristems. Meristematic cells are characterized by the coordination between cell proliferation and cell growth, that is, by the accurate regulation of cell cycle progression and the optimal production of biomass for the viability of daughter cells after division. Thus, cell growth is correlated with the rate of ribosome biogenesis and protein synthesis. We investigated the effects of simulated microgravity on cellular functions of the root meristem in a sequential study. Seedlings were grown in a clinostat, a device producing simulated microgravity, for periods between 3 and 10days. In a complementary study, seedlings were grown in a Random Positioning Machine (RPM) and sampled sequentially after similar periods of growth. Under these conditions, the cell proliferation rate and the regulation of cell cycle progression showed significant alterations, accompanied by a reduction of cell growth. However, the overall size of the root meristem did not change. Analysis of cell cycle phases by flow cytometry showed changes in their proportion and duration, and the expression of the cyclin B1 gene, a marker of entry in mitosis, was decreased, indicating altered cell cycle regulation. With respect to cell growth, the rate of ribosome biogenesis was reduced under simulated microgravity, as shown by morphological and morphometric nucleolar changes and variations in the levels of the nucleolar protein nucleolin. Furthermore, in a nucleolin mutant characterized by disorganized nucleolar structure, the microgravity treatment intensified disorganization. These results show that, regardless of the simulated microgravity device used, a great disruption of meristematic competence was the first response to the environmental alteration detected at early developmental stages. However, longer periods of exposure to simulated microgravity do not produce an intensification of the cellular damages or a detectable developmental alteration in seedlings analyzed at further stages of their growth. This suggests that the secondary response to the gravity alteration is a process of adaptation, whose mechanism is still unknown, which eventually results in viable adult plants.

Microgravity induces changes in microsome-associated proteins of Arabidopsis seedlings grown on board the international space station.

The "GENARA A" experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected.

Microsome-associated proteome modifications of Arabidopsis seedlings grown on board the International Space Station reveal the possible effect on plants of space stresses other than microgravity.

Growing plants in space for using them in bioregenerative life support systems during long-term human spaceflights needs improvement of our knowledge in how plants can adapt to space growth conditions. In a previous study performed on board the International Space Station (GENARA A experiment STS-132) we evaluate the global changes that microgravity can exert on the membrane proteome of Arabidopsis seedlings. Here we report additional data from this space experiment, taking advantage of the availability in the EMCS of a centrifuge to evaluate the effects of cues other than microgravity on the relative distribution of membrane proteins. Among the 1484 membrane proteins quantified, 227 proteins displayed no abundance differences between µ g and 1 g in space, while their abundances significantly differed between 1 g in space and 1 g on ground. A majority of these proteins (176) were over-represented in space samples and mainly belong to families corresponding to protein synthesis, degradation, transport, lipid metabolism, or ribosomal proteins. In the remaining set of 51 proteins that were under-represented in membranes, aquaporins and chloroplastic proteins are majority. These sets of proteins clearly appear as indicators of plant physiological processes affected in space by stressful factors others than microgravity.

Anatomical diversity and regressive evolution in trichomanoid filmy ferns (Hymenophyllaceae): a phylogenetic approach.

To infer the anatomical evolution of the Hymenophyllaceae (filmy ferns) and to test previously suggested scenarios of regressive evolution, we performed an exhaustive investigation of stem anatomy in the most variable lineage of the family, the trichomanoids, using a representative sampling of 50 species. The evolution of qualitative and quantitative anatomical characters and possibly related growth-forms was analyzed using a maximum likelihood approach. Potential correlations between selected characters were then statistically tested using a phylogenetic comparative method. Our investigations support the anatomical homogeneity of this family at the generic and sub-generic levels. Reduced and sub-collateral/collateral steles likely derived from an ancestral massive protostele, and sub-collateral/collateral types appear to be related to stem thickness reduction and root apparatus regression. These results corroborate the hypothesis of regressive evolution in the lineage, in terms of morphology as well as anatomy. In addition, a heterogeneous cortex, which is derived in the lineage, appears to be related to a colonial strategy and likely to a climbing phenotype. The evolutionary hypotheses proposed in this study lay the ground for further evolutionary analyses that take into account trichomanoid habitats and accurate ecological preferences.

Diarch symmetry of the vascular bundle in Arabidopsis root encompasses the pericycle and is reflected in distich lateral root initiation.

The outer tissues of dicotyledonous plant roots (i.e. epidermis, cortex, and endodermis) are clearly organized in distinct concentric layers in contrast to the diarch to polyarch vascular tissues of the central stele. Up to now, the outermost layer of the stele, the pericycle, has always been regarded, in accordance with the outer tissue layers, as one uniform concentric layer. However, considering its lateral root-forming competence, the pericycle is composed of two different cell types, with one subset of cells being associated with the xylem, showing strong competence to initiate cell division, whereas another group of cells, associated with the phloem, appears to remain quiescent. Here, we established, using detailed microscopy and specific Arabidopsis thaliana reporter lines, the existence of two distinct pericycle cell types. Analysis of two enhancer trap reporter lines further suggests that the specification between these two subsets takes place early during development, in relation with the determination of the vascular tissues. A genetic screen resulted in the isolation of mutants perturbed in pericycle differentiation. Detailed phenotypical analyses of two of these mutants, combined with observations made in known vascular mutants, revealed an intimate correlation between vascular organization, pericycle fate, and lateral root initiation potency, and illustrated the independence of pericycle differentiation and lateral root initiation from protoxylem differentiation. Taken together, our data show that the pericycle is a heterogeneous cell layer with two groups of cells set up in the root meristem by the same genetic pathway controlling the diarch organization of the vasculature.