No abstract

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected.

Spontaneously arising channels that transport the phytohormone auxin provide positional cues for self-organizing aspects of plant development such as flexible vasculature regeneration or its patterning during leaf venation. The auxin canalization hypothesis proposes a feedback between auxin signaling and transport as the underlying mechanism, but molecular players await discovery. We identified part of the machinery that routes auxin transport. The auxin-regulated receptor CAMEL (Canalization-related Auxin-regulated Malectin-type RLK) together with CANAR (Canalization-related Receptor-like kinase) interact with and phosphorylate PIN auxin transporters. camel and canar mutants are impaired in PIN1 subcellular trafficking and auxin-mediated PIN polarization, which macroscopically manifests as defects in leaf venation and vasculature regeneration after wounding. The CAMEL-CANAR receptor complex is part of the auxin feedback that coordinates polarization of individual cells during auxin canalization.

Synchronized tissue polarization during regeneration or de novo vascular tissue formation is a plant-specific example of intercellular communication and coordinated development. According to the canalization hypothesis, the plant hormone auxin serves as polarizing signal that mediates directional channel formation underlying the spatio-temporal vasculature patterning. A necessary part of canalization is a positive feedback between auxin signaling and polarity of the intercellular auxin flow. The cellular and molecular mechanisms of this process are still poorly understood, not the least, because of a lack of a suitable model system. We show that the main genetic model plant, Arabidopsis (Arabidopsis thaliana) can be used to study the canalization during vascular cambium regeneration and new vasculature formation. We monitored localized auxin responses, directional auxin-transport channels formation, and establishment of new vascular cambium polarity during regenerative processes after stem wounding. The increased auxin response above and around the wound preceded the formation of PIN1 auxin transporter-marked channels from the primarily homogenous tissue and the transient, gradual changes in PIN1 localization preceded the polarity of newly formed vascular tissue. Thus, Arabidopsis is a useful model for studies of coordinated tissue polarization and vasculature formation after wounding allowing for genetic and mechanistic dissection of the canalization hypothesis.