SummaryControlled plant growth requires regulation through a variety of signaling molecules, including steroids, peptides, radicals of oxygen and nitrogen, as well as the 'classical' phytohormone groups. Auxin is critical for the control of plant growth and also orchestrates many developmental processes, such as the formation of new roots. It modulates root architecture both slowly, through actions at the transcriptional level and, more rapidly, by mechanisms targeting primarily plasma membrane sensory systems and intracellular signaling pathways. The latter reactions use several second messengers, including Ca 2+ , nitric oxide (NO) and reactive oxygen species (ROS). Here, we investigated the different roles of two auxins, the major auxin indole-3-acetic acid (IAA) and another endogenous auxin indole-3-butyric acid (IBA), in the lateral root formation process of Arabidopsis and maize. This was mainly analyzed by different types of fluorescence microscopy and inhibitors of NO production.This study revealed that peroxisomal IBA to IAA conversion is followed by peroxisomal NO, which is important for IBA-induced lateral root formation.We conclude that peroxisomal NO emerges as a new player in auxin-induced root organogenesis. In particular, the spatially and temporally coordinated release of NO and IAA from peroxisomes is behind the strong promotion of lateral root formation via IBA.

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BackgroundHydroxyproline rich glycoproteins (HRGPs) are implicated to have a role in many aspects of plant growth and development but there is limited knowledge about their localization and function during somatic embryogenesis of higher plants. In this study, the localization and function of hydroxyproline rich glycoproteins in embryogenic cells (ECs) and somatic embryos of banana were investigated by using immunobloting and immunocytochemistry with monoclonal JIM11 and JIM20 antibodies as well as by treatment with 3,4-dehydro-L-proline (3,4-DHP, an inhibitor of extensin biosynthesis), and by immunomodulation with the JIM11 antibody.ResultsImmunofluorescence labelling of JIM11 and JIM20 hydroxyproline rich glycoprotein epitopes was relatively weak in non-embryogenic cells (NECs), mainly on the edge of small cell aggregates. On the other hand, hydroxyproline rich glycoprotein epitopes were found to be enriched in early embryogenic cells as well as in various developmental stages of somatic embryos. Embryogenic cells (ECs), proembryos and globular embryos showed strong labelling of hydroxyproline rich glycoprotein epitopes, especially in their cell walls and outer surface layer, so-called extracellular matrix (ECM). This hydroxyproline rich glycoprotein signal at embryo surfaces decreased and/or fully disappeared during later developmental stages (e.g. pear-shaped and cotyledonary stages) of embryos. In these later developmental embryogenic stages, however, new prominent hydroxyproline rich glycoprotein labelling appeared in tri-cellular junctions among parenchymatic cells inside these embryos. Overall immunofluorescence labelling of late stage embryos with JIM20 antibody was weaker than that of JIM11. Western blot analysis supported the above immunolocalization data. The treatment with 3,4-DHP inhibited the development of embryogenic cells and decreased the rate of embryo germination. Embryo-like structures, which developed after 3,4-DHP treatment showed aberrant non-compact epidermis with discontinuous ECM at the outer surface as well as much less immunolabelling with the JIM11 antibody. This treatment also decreased the plant regeneration capacity in embryogenic banana cultures. Finally, immunomodulation of surface hydroxyproline rich glycoproteins by co-culture of embryos with the JIM11 antibody resulted in a much lower germination capacity of these embryos.ConclusionsThese results suggest that hydroxyproline rich glycoproteins play an important developmental role, especially in the process of regeneration and germination of embryos during plant regeneration via somatic embryogenesis. Proper content and localization of hydroxyproline rich glycoproteins seem to be essential for the formation and regeneration of banana somatic embryos.

Plant lipid transfer proteins (LTPs) constitute a family of small proteins recognized as being extracellular. In agreement with this notion, several lines of evidence have shown the apoplastic localization of HaAP10, a LTP from Helianthus annuus dry seeds. However, HaAP10 was recently detected intracellularly in imbibing seeds. To clarify its distribution, immunolocalization experiments were performed during the course of germination and confirmed its intracellular localization upon early seed imbibition. Further assays using a hydrophobic dye, FM4-64, inhibitors of vesicular traffic, and immunolocalization of the pectin rhamnogalacturonan-II, allowed the conclusion that endocytosis is activated as soon as seed imbibition starts. Furthermore, this study demonstrated that HaAP10 is endocytosed throughout imbibition. Biochemical and cellular approaches indicate that the intracellular fraction of this LTP appears associated with oil bodies and some evidence also suggest its presence in glyoxysomes. So, HaAP10 is apoplastic in dry seeds and upon imbibition is rapidly internalized and relocalized to organelles involved in lipid metabolism. The results suggest that HaAP10 may be acting as a fatty acid shuttle between the oil body and the glyoxysome during seed germination. This concept is consistent with the initial proposition that LTPs participate in the intracellular transfer of lipids which was further denied based on their apparent extracellular localization. This report reveals for the first time the relocalization of a lipid transfer protein and opens new perspectives on its role.

Abbreviations: BFA, brefeldin A; DMSO, dimethylsulphoxide; FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-diethylaminophenylhexatrienyl)pyridinium dibromide; RG-II, rhamnogalacturonan-II During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4-64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunolocalization of rhamnogalacturonan-II (RG-II) complexed with boron showed that whereas this pectin is localized only in the cell walls of dry seed embryos, it starts to be intracellular once the imbibition started. Brefeldin A (BFA) exposure resulted in recruitment of the intracellular RG-II pectin complexes into the endocytic BFA-induced compartments, confirming the endocytic origin of the RG-II signal detected intracellularly. Finally, germination was significantly delayed when Arabidopsis seeds were germinated in the presence of inhibitors of endocytic pathways, suggesting that trafficking of extracellular molecules might play an important role in the overcome of germination. This work constitutes the first demonstration of endocytic processes during germination and opens new perspectives about the role of the extracellular matrix and membrane components in seed germination.