Expanding the repertoire of secretory peptides controlling root development with comparative genome analysis and functional assays.

Plant genomes encode numerous small secretory peptides (SSPs) whose functions have yet to be explored. Based on structural features that characterize SSP families known to take part in postembryonic development, this comparative genome analysis resulted in the identification of genes coding for oligopeptides potentially involved in cell-to-cell communication. Because genome annotation based on short sequence homology is difficult, the criteria for the de novo identification and aggregation of conserved SSP sequences were first benchmarked across five reference plant species. The resulting gene families were then extended to 32 genome sequences, including major crops. The global phylogenetic pattern common to the functionally characterized SSP families suggests that their apparition and expansion coincide with that of the land plants. The SSP families can be searched online for members, sequences and consensus (http://bioinformatics.psb.ugent.be/webtools/PlantSSP/). Looking for putative regulators of root development, Arabidopsis thaliana SSP genes were further selected through transcriptome meta-analysis based on their expression at specific stages and in specific cell types in the course of the lateral root formation. As an additional indication that formerly uncharacterized SSPs may control development, this study showed that root growth and branching were altered by the application of synthetic peptides matching conserved SSP motifs, sometimes in very specific ways. The strategy used in the study, combining comparative genomics, transcriptome meta-analysis and peptide functional assays in planta, pinpoints factors potentially involved in non-cell-autonomous regulatory mechanisms. A similar approach can be implemented in different species for the study of a wide range of developmental programmes.

GOLVEN peptides as important regulatory signalling molecules of plant development.

The contribution of signalling peptides to plant development is increasingly evident as more new peptide families become identified. The recently discovered GLV/RGF/CLEL secreted peptide family comprises 11 members in Arabidopsis and has been shown by independent research groups to be involved in different plant developmental programmes such as root meristem maintenance, root hair development, and root and hypocotyl gravitropism. This short review summarizes our current knowledge on GLV/RGF/CLEL peptides and highlights future challenges to decipher their function.

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.

APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis.

Transcription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix-loop-helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators.

Systematic localization of the Arabidopsis core cell cycle proteins reveals novel cell division complexes.

Cell division depends on the correct localization of the cyclin-dependent kinases that are regulated by phosphorylation, cyclin proteolysis, and protein-protein interactions. Although immunological assays can define cell cycle protein abundance and localization, they are not suitable for detecting the dynamic rearrangements of molecular components during cell division. Here, we applied an in vivo approach to trace the subcellular localization of 60 Arabidopsis (Arabidopsis thaliana) core cell cycle proteins fused to green fluorescent proteins during cell division in tobacco (Nicotiana tabacum) and Arabidopsis. Several cell cycle proteins showed a dynamic association with mitotic structures, such as condensed chromosomes and the preprophase band in both species, suggesting a strong conservation of targeting mechanisms. Furthermore, colocalized proteins were shown to bind in vivo, strengthening their localization-function connection. Thus, we identified unknown spatiotemporal territories where functional cell cycle protein interactions are most likely to occur.

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.

Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco.

Although secondary metabolism in Nicotiana tabacum (L.) (tobacco) is rather well studied, many molecular aspects of the biosynthetic pathways and their regulation remain to be disclosed, even for prominent compounds such as nicotine and other pyridine alkaloids. To identify players in tobacco pyridine alkaloid biosynthesis a functional screen was performed, starting from a tobacco gene collection established previously by means of combined transcript profiling and metabolite analysis. First, full-length cDNA clones were isolated for 34 genes, corresponding to tobacco transcript tag sequences putatively associated with pyridine alkaloid metabolism. Full-length open reading frames were transferred to pCaMV35S-steered overexpression vectors. The effects of plant transformation with these expression cassettes on the accumulation of nicotine and other pyridine alkaloids were assessed in transgenic tobacco Bright-Yellow 2 (BY-2) cell suspensions and hairy root cultures. This screen identified potential catalysers of tobacco pyridine metabolism, amongst which a lysine decarboxylase-like gene and a GH3-like enzyme. Overexpression of the GH3-like enzyme, presumably involved in auxin homeostasis and designated NtNEG1 (Nicotiana tabacum Nicotine-Enhancing GH3 enzyme 1), increased nicotine levels in BY-2 hairy roots significantly. This study shows how functional genomics-based identification of genes potentially involved in biosynthetic pathways followed by systematic functional assays in plant cells can be used at large-scale to decipher plant metabolic networks at the molecular level.

Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells.

Although sequence information and genome annotation are improving at an impressive pace, functional ontology is still non-existent or rudimentary for most genes. In this regard, transient expression assays are very valuable for identification of short functional segments in particular pathways, because they can be performed rapidly and at a scale unattainable in stably transformed tissues. Vectors were constructed and protocols developed for systematic transient assays in plant protoplasts. To enhance throughput and reproducibility, protoplast treatments were performed entirely by a liquid-handling robot in multiwell plates, including polyethylene glycol/Ca2+ cell transfection with plasmid mixtures, washes and lysis. All transcriptional readouts were measured using a dual firefly/Renilla luciferase assay, in which the former was controlled by a reporter promoter and the latter by the 35S CaMV promoter, which served as internal normalization standard. The automated protocols were suitable for transient assays in protoplasts prepared from cell cultures of Nicotiana tabacum Bright Yellow-2 and Arabidopsis thaliana. They were implemented in a screen to discover potential regulators of genes coding for key enzymes in nicotine biosynthesis. Two novel tobacco transcription factors were found, NtORC1 and NtJAP1, that positively regulate the putrescine N-methyltransferase (PMT) promoter. In addition, combinatorial tests showed that these two factors act synergistically to induce PMT transcriptional activity. The development and use of high-throughput plant transient expression assays are discussed.