Variation in HMA4 gene copy number and expression among Noccaea caerulescens populations presenting different levels of Cd tolerance and accumulation.

There is huge variability among populations of the hyperaccumulator Noccaea caerulescens (formerly Thlaspi caerulescens) in their capacity to tolerate and accumulate cadmium. To gain new insights into the mechanisms underlying this variability, we estimated cadmium fluxes and further characterized the N. caerulescens heavy metal ATPase 4 (NcHMA4) gene in three populations (two calamine, Saint-Félix-de-Pallières, France and Prayon, Belgium; one serpentine, Puente Basadre, Spain) presenting contrasting levels of tolerance and accumulation. Cadmium uptake and translocation varied among populations in the same way as accumulation; the population with the highest cadmium concentration in shoots (Saint Félix-de-Pallières) presented the highest capacity for uptake and translocation. We demonstrated that the four NcHMA4 copies identified in a previous study are not fixed at the species level, and that the copy truncated in the C-terminal part encodes a functional protein. NcHMA4 expression and gene copy number was lower in the serpentine population, which was the least efficient in cadmium translocation compared to the calamine populations. NcHMA4 expression was associated with the vascular tissue in all organs, with a maximum at the crown. Overall, our results indicate that differences in cadmium translocation ability of the studied populations appear to be controlled, at least partially, by NcHMA4, while the overexpression of NcHMA4 in the two calamine populations may result from convergent evolution.

GOLVEN secretory peptides regulate auxin carrier turnover during plant gravitropic responses.

Growth and development are coordinated by an array of intercellular communications. Known plant signaling molecules include phytohormones and hormone peptides. Although both classes can be implicated in the same developmental processes, little is known about the interplay between phytohormone action and peptide signaling within the cellular microenvironment. We show that genes coding for small secretory peptides, designated GOLVEN (GLV), modulate the distribution of the phytohormone auxin. The deregulation of the GLV function impairs the formation of auxin gradients and alters the reorientation of shoots and roots after a gravity stimulus. Specifically, the GLV signal modulates the trafficking dynamics of the auxin efflux carrier PIN-FORMED2 involved in root tropic responses and meristem organization. Our work links the local action of secretory peptides with phytohormone transport.

The AP-3 ß adaptin mediates the biogenesis and function of lytic vacuoles in Arabidopsis.

Plant vacuoles are essential multifunctional organelles largely distinct from similar organelles in other eukaryotes. Embryo protein storage vacuoles and the lytic vacuoles that perform a general degradation function are the best characterized, but little is known about the biogenesis and transition between these vacuolar types. Here, we designed a fluorescent marker-based forward genetic screen in Arabidopsis thaliana and identified a protein affected trafficking2 (pat2) mutant, whose lytic vacuoles display altered morphology and accumulation of proteins. Unlike other mutants affecting the vacuole, pat2 is specifically defective in the biogenesis, identity, and function of lytic vacuoles but shows normal sorting of proteins to storage vacuoles. PAT2 encodes a putative ß-subunit of adaptor protein complex 3 (AP-3) that can partially complement the corresponding yeast mutant. Manipulations of the putative AP-3 ß adaptin functions suggest a plant-specific role for the evolutionarily conserved AP-3 ß in mediating lytic vacuole performance and transition of storage into the lytic vacuoles independently of the main prevacuolar compartment-based trafficking route.

Systematic analysis of cell-cycle gene expression during Arabidopsis development.

The steady-state distribution of cell-cycle transcripts in Arabidopsis thaliana seedlings was studied in a broad in situ survey to provide a better understanding of the expression of cell-cycle genes during plant development. The 61 core cell-cycle genes analyzed were expressed at variable levels throughout the different plant tissues: 23 genes generally in dividing and young differentiating tissues, 34 genes mostly in both dividing and differentiated tissues and four gene transcripts primarily in differentiated tissues. Only 21 genes had a typical patchy expression pattern, indicating tight cell-cycle regulation. The increased expression of 27 cell-cycle genes in the root elongation zone hinted at their involvement in the switch from cell division to differentiation. The induction of 20 cell-cycle genes in differentiated cortical cells of etiolated hypocotyls pointed to their possible role in the process of endoreduplication. Of seven cyclin-dependent kinase inhibitor genes, five were upregulated in etiolated hypocotyls, suggesting a role in cell-cycle arrest. Nineteen genes were preferentially expressed in pericycle cells activated by auxin that give rise to lateral root primordia. Approximately 1800 images have been collected and can be queried via an online database. Our in situ analysis revealed that 70% of the cell-cycle genes, although expressed at different levels, show a large overlap in their localization. The lack of regulatory motifs in the upstream regions of the analyzed genes suggests the absence of a universal transcriptional control mechanism for all cell-cycle genes.

Fluorescence imaging-based screen identifies ARF GEF component of early endosomal trafficking.

Endocytic vesicle trafficking is crucial for regulating activity and localization of plasma membrane components, but the process is still poorly genetically defined in plants. Membrane proteins of the PIN-FORMED (PIN) family exhibit polar localization in plant cells and facilitate cellular efflux of the plant hormone auxin, thereby regulating multiple developmental processes. PIN proteins undergo constitutive endocytosis and GNOM ARF GEF-dependent recycling, and their localization is under extensive regulation by developmental and environmental cues. We designed a fluorescence imaging-based screen to identify Arabidopsis thaliana mutants defective in internalization of proteins including PINs from the plasma membrane. We identified three mutant loci, BFA-visualized endocytic trafficking defective1 (ben1) through ben3 that do not efficiently accumulate PIN1-GFP in intracellular compartments after inhibition of recycling and secretion by fungal toxin brefeldin A (BFA). Fine mapping revealed that BEN1 encodes an ARF GEF vesicle trafficking regulator from the functionally uncharacterized BIG class. ben1 mutant has been previously implicated in pathogen response and shows cell polarity, BFA sensitivity, and growth defects. BEN1 is involved in endocytosis of plasma membrane proteins and localizes to early endocytic compartments distinct from GNOM-positive endosomes. Our results identify BEN1 as the ARF GEF mediating early endosomal traffic.

Plant CLE peptides from two distinct functional classes synergistically induce division of vascular cells.

The Clavata3 (CLV3)/endosperm surrounding region (CLE) signaling peptides are encoded in large plant gene families. CLV3 and the other A-type CLE peptides promote cell differentiation in root and shoot apical meristems, whereas the B-type peptides (CLE41-CLE44) do not. Instead, CLE41 inhibits the differentiation of Zinnia elegans tracheary elements. To test whether CLE genes might code for antagonistic or synergistic functions, peptides from both types were combined through overexpression within or application onto Arabidopsis thaliana seedlings. The CLE41 peptide (CLE41p) promoted proliferation of vascular cells, although delaying differentiation into phloem and xylem cell lineages. Application of CLE41p or overexpression of CLE41 did not suppress the terminal differentiation of the root and shoot apices triggered by A-type CLE peptides. However, in combination, A-type peptides enhanced all of the phenotypes associated with CLE41 gain-of-function, leading to massive proliferation of vascular cells. This proliferation relied on auxin signaling because it was enhanced by exogenous application of a synthetic auxin, decreased by an auxin polar transport inhibitor, and abolished by a mutation in the Monopteros auxin response factor. These findings highlight that vascular patterning is a process controlled in time and space by different CLE peptides in conjunction with hormonal signaling.

The DP-E2F-like gene DEL1 controls the endocycle in Arabidopsis thaliana.

Endoreduplication or DNA replication without mitosis is widespread in nature. Well-known examples are fruit fly polytene chromosomes and cereal endosperm. Although endocycles are thought to be driven by the same regulators as those that control the G1-S transition of the mitotic cell cycle, the molecular mechanisms that differentiate mitotically dividing cells from endoreduplicating ones are largely unknown. A novel class of atypical E2F-like proteins has recently been identified and is designated E2F7 in mammals and DP-E2F-like (DEL) in Arabidopsis thaliana . We demonstrate that loss of DEL1 function resulted in increased ploidy levels, whereas ectopic expression of DEL1 reduced endoreduplication. Ploidy changes were correlated with altered expression of a subset of E2F target genes encoding proteins necessary for DNA replication. Because DEL1 proteins were postulated to antagonize the E2F pathway, we generated DEL1-E2Fa-DPa triple transgenics. DEL1 inhibited the endoreduplication phenotype, but not the ectopic cell divisions that resulted from the overexpression of both E2Fa and DPa, illustrating that DEL1 specifically represses the endocycle. Because DEL1 transcripts were detected exclusively in mitotically dividing cells, we conclude that DEL1 is an important novel inhibitor of the endocycle and preserves the mitotic state of proliferating cells by suppressing transcription of genes that are required for cells to enter the DNA endoreduplication cycle.

Dynamic cytoskeleton rearrangements in giant cells and syncytia of nematode-infected roots.

Giant cells induced by root knot nematodes and syncytia caused by cyst nematodes are large multinucleated feeding cells containing a dense cytoplasm generated during a complex host-parasite association in plant roots. To find out whether cytoskeleton changes occurred during feeding cell development, transcriptional activity of actin (ACT) and tubulin genes and organization of the ACT filaments and of the microtubules (MTs) were analyzed in situ. The importance of changes in the cytoskeleton architecture for the proper initiation and development of galls and syncytia was demonstrated by perturbing the cytoskeleton with chemical inhibitors. The expression levels of cytoskeletal components, such as tubulins and ACTs, are proposed to be upregulated to allow the assembly of a new cytoskeleton in expanding feeding cells. However, MTs and ACT filaments failed to properly organize and appeared partially depolymerized throughout feeding site development. Both the actin and tubulin cytoskeletons were strongly disrupted in syncytia and mitotic figures were never observed. In contrast, in giant cells, an ACT and cortical MT cytokeleton, although disturbed, was still visible. In addition, a functional mitotic apparatus was present that contained multiple large spindles and arrested phragmoplasts, but no pre-prophase bands. Chemical stabilization of the microtubular cytoskeleton with taxol blocked feeding site development. On the other hand, when the ACT or MT cytoskeleton of feeding cells was depolymerized by cytochalasin D or oryzalin, nematodes could complete their life cycle. Our data suggest that the cytoskeleton rearrangements and depolymerization induced by parasitic nematodes may be essential for a successful feeding process.

Analysis of the spatial expression pattern of seven Kip related proteins (KRPs) in the shoot apex of Arabidopsis thaliana.

BACKGROUND AND AIMS: Kip-related-proteins (KRPs), negative regulators of cell division, have recently been discovered in plants but their in planta function is as yet unclear. In this study the spatial expression of all seven KRP genes in shoot apices of Arabidopsis thaliana were compared. METHODS: In situ hybridization analyses were performed on longitudinal sections of shoot apices from 2-month-old Arabidopsis plants. KEY RESULTS: The study provides evidence for different expression pattern groups. KRP1 and KRP2 expression is restricted to the endoreduplicating tissues. In contrast, KRP4 and KRP5 expression is mainly restricted to mitotically dividing cells. KRP3, KRP6 and KRP7 can be found in both mitotically dividing and endoreduplicating cells. CONCLUSION: The results suggest differential roles for the distinct KRPs. KRP1 and KRP2 might specifically be involved in the establishment of polyploidy. In contrast, KRP4 and KRP5 might be involved in regulating the progression through the mitotic cell cycle. KRP3, KRP6 and KRP7 might have a function in both types of cell cycle.

An Arabidopsis thaliana pectin acetylesterase gene is upregulated in nematode feeding sites induced by root-knot and cyst nematodes.

By using differential display, gene expression was investigated in Arabidopsis thaliana roots shortly after nematode infection, and a putative pectin acetylesterase (PAE) homolog (DiDi 9C-12) was found to be upregulated. PAEs catalyze the deacetylation of pectin, a major compound of primary cell walls. mRNA in situ hybridization experiments showed that the expression of DiDi 9C-12 was enhanced very early after infection in initiating giant-cells and in cells surrounding the nematodes. Later on, the level of DiDi 9C-12 mRNA was lower in giant-cells and transcripts were mainly found in parenchyma, endodermis, and pericycle cells of the root gall. Twenty days after infection, DiDi 9C-12 transcripts could no longer be detected. DiDi 9C-12 transcripts were also found in young syncytia and in the cells surrounding the expanding syncytium. Our results suggest that plant parasitic nematodes can modulate the rapid growth of the feeding cells and the expansion of the root gall by triggering the expression of DiDi 9C-12. PAEs, which probably act together with a range of other pectin-degrading enzymes, could be involved in softening and loosening the primary cell wall in nematode-infected plant roots.