Arabidopsis thaliana SHOOT MERISTEMLESS Substitutes for Medicago truncatula SINGLE LEAFLET1 to Form Complex Leaves and Petals.

LEAFY plant-specific transcription factors, which are key regulators of flower meristem identity and floral patterning, also contribute to meristem activity. Notably, in some legumes, LFY orthologs such as Medicago truncatula SINGLE LEAFLET (SGL1) are essential in maintaining an undifferentiated and proliferating fate required for leaflet formation. This function contrasts with most other species, in which leaf dissection depends on the reactivation of KNOTTED-like class I homeobox genes (KNOXI). KNOXI and SGL1 genes appear to induce leaf complexity through conserved downstream genes such as the meristematic and boundary CUP-SHAPED COTYLEDON genes. Here, we compare in M. truncatula the function of SGL1 with that of the Arabidopsis thaliana KNOXI gene, SHOOT MERISTEMLESS (AtSTM). Our data show that AtSTM can substitute for SGL1 to form complex leaves when ectopically expressed in M. truncatula. The shared function between AtSTM and SGL1 extended to the major contribution of SGL1 during floral development as ectopic AtSTM expression could promote floral organ identity gene expression in sgl1 flowers and restore sepal shape and petal formation. Together, our work reveals a function for AtSTM in floral organ identity and a higher level of interchangeability between meristematic and floral identity functions for the AtSTM and SGL1 transcription factors than previously thought.

Increased expression of a phloem membrane protein encoded by NHL26 alters phloem export and sugar partitioning in Arabidopsis.

The complex process of phloem sugar transport involves symplasmic and apoplasmic events. We characterized Arabidopsis thaliana lines ectopically expressing a phloem-specific gene encoding NDR1/HIN1-like26 (NHL26), a putative membrane protein. NHL26 overexpressor plants grew more slowly than wild-type plants, accumulated high levels of carbohydrates in mature leaves, and had a higher shoot biomass, contrasting with slower root growth and a lower seed yield. Similar effects were observed when NHL26 was overexpressed in companion cells, under the control of a companion cell-specific promoter. The soluble sugar content of the phloem sap and sink organs was lower than that in the wild type, providing evidence of a sugar export defect. This was confirmed in a phloem-export assay with the symplastic tracer carboxyfluorescein diacetate. Leaf sugar accumulation was accompanied by higher organic acid, amino acid, and protein contents, whereas analysis of the metabolite profile of phloem sap exudate revealed no change in amino acid or organic acid content, indicating a specific effect on sugar export. NHL26 was found to be located in the phloem plasmodesmata and the endoplasmic reticulum. These findings reveal that NHL26 accumulation affects either the permeability of plasmodesmata or sugar signaling in companion cells, with a specific effect on sugar export.

Soluble and filamentous proteins in Arabidopsis sieve elements.

Phloem sieve elements are highly differentiated cells involved in the long-distance transport of photoassimilates. These cells contain both aggregated phloem-proteins (P-proteins) and soluble proteins, which are also translocated by mass flow. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to carry out a proteomic survey of the phloem exudate of Arabidopsis thaliana, collected by the ethylenediaminetetraacetic acid (EDTA)-facilitated method. We identified 287 proteins, a large proportion of which were enzymes involved in the metabolic precursor generation and amino acid synthesis, suggesting that sieve tubes display high levels of metabolic activity. RNA-binding proteins, defence proteins and lectins were also found. No putative P-proteins were detected in the EDTA-exudate fraction, indicating a lack of long-distance translocation of such proteins in Arabidopsis. In parallel, we investigated the organization of P-proteins, by high-resolution transmission electron microscopy, and the localization of the phloem lectin PP2, a putative P-protein component, by immunolocalization with antibodies against PP2-A1. Transmission electron microscopy observations of P-proteins revealed bundles of filaments resembling strings of beads. PP2-A1 was found weakly associated with these structures in the sieve elements and bound to plastids. These observations suggest that PP2-A1 is anchored to P-proteins and organelles rather than being a structural component of P-proteins.

S phase of the cell cycle: a key phase for the regulation of thermodormancy in barley grain.

The aim of the present work was to investigate the occurrence of the cell cycle during germination as related to thermodormancy in barley (Hordeum vulgare L., cv. Pewter) grains in relation with abscisic acid (ABA) by: (i) flow cytometry to determine the progression of the cell cycle; and (ii) reverse transcription-PCR to characterize the expression of some important genes involved in cell-cycle regulation. In dry embryos, cells are mostly (82%) arrested in G1 phase of the cell cycle, the remaining cells being in the G2 (17%) or S phase (0.9%). Germination at 20 °C was associated with an increase in the nuclei population in G2 and S (up to 32.5-44.5 and 9.2-11.3%, respectively, after 18-24h). At 30 °C, partial reactivation of the cell cycle occurred in embryos of dormant grains that did not germinate. Incubation with 50mM hydroxyurea suggests that thermodormancy resulted in a blocking of the nuclei in the S phase. In dry dormant grains, transcripts of CDKA1, CYCA3, KRP4, and WEE1 were present, while those of CDKB1, CDKD1, CYCB1, and CYCD4 were not detected. Incubation at 30 °C resulted in a strong reduction of CDKB1, CYCB1, and CYCD4 expression and overexpression of CDK1 and KRP4. ABA had a similar effect as incubation at 30 °C on the expression of CDKB1, CYCB1, and CYCD4, but did not increase that of CDK1 and KRP4. Patterns of gene expression are discussed with regard to thermodormancy expression and ABA.

Live-Cell Imaging of Fluorescently Tagged Phloem Proteins with Confocal Microscopy.

Confocal laser scanning microscopy can enable observation of phloem cells in living tissues. Here we describe live imaging of phloem cells in the leaves and roots of Arabidopsis thaliana using fluorescently tagged proteins, either expressed in the vasculature using phloem specific promoters or constitutively expressed reference marker lines. Now, the majority of phloem cell types can be identified, allowing a precise cellular and subcellular localization of phloem proteins.

Live imaging of companion cells and sieve elements in Arabidopsis leaves.

The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.