Peter Marhavý

Cytokinin Modulates Endocytic Trafficking of PIN1 Auxin Efflux Carrier to Control Plant Organogenesis

Cytokinin is an important regulator of plant growth and development. In Arabidopsis thaliana, the two-component phosphorelay mediated through a family of histidine kinases and response regulators is recognized as the principal cytokinin signal transduction mechanism activating the complex transcriptional response to control various developmental processes. Here, we identified an alternative mode of cytokinin action that uses endocytic trafficking as a means to direct plant organogenesis. This activity occurs downstream of known cytokinin receptors but through a branch of the cytokinin signaling pathway that does not involve transcriptional regulation. We show that cytokinin regulates endocytic recycling of the auxin efflux carrier PINFORMED1 (PIN1) by redirecting it for lytic degradation in vacuoles. Stimulation of the lytic PIN1 degradation is not a default effect for general downregulation of proteins from plasma membranes, but a specific mechanism to rapidly modulate the auxin distribution in cytokinin-mediated developmental processes.

Role of PIN-mediated auxin efflux in apical hook development of Arabidopsis thaliana.

The apical hook of dark-grown Arabidopsis seedlings is a simple structure that develops soon after germination to protect the meristem tissues during emergence through the soil and that opens upon exposure to light. Differential growth at the apical hook proceeds in three sequential steps that are regulated by multiple hormones, principally auxin and ethylene. We show that the progress of the apical hook through these developmental phases depends on the dynamic, asymmetric distribution of auxin, which is regulated by auxin efflux carriers of the PIN family. Several PIN proteins exhibited specific, partially overlapping spatial and temporal expression patterns, and their subcellular localization suggested auxin fluxes during hook development. Genetic manipulation of individual PIN activities interfered with different stages of hook development, implying that specific combinations of PIN genes are required for progress of the apical hook through the developmental phases. Furthermore, ethylene might modulate apical hook development by prolonging the formation phase and strongly suppressing the maintenance phase. This ethylene effect is in part mediated by regulation of PIN-dependent auxin efflux and auxin signaling.

Spatiotemporal Regulation of Lateral Root Organogenesis in Arabidopsis by Cytokinin

This work characterizes the role of cytokinins in lateral root organogenesis, finding that cytokinin activity is likely important to prevent lateral root initiation in close proximity to existing lateral root primordia. The results support the idea that stage-dependent effects of cytokinin are determined by the robustness of the auxin gradient, which might be enhanced in mature primordia. The architecture of a plant’s root system, established postembryonically, results from both coordinated root growth and lateral root branching. The plant hormones auxin and cytokinin are central endogenous signaling molecules that regulate lateral root organogenesis positively and negatively, respectively. Tight control and mutual balance of their antagonistic activities are particularly important during the early phases of lateral root organogenesis to ensure continuous lateral root initiation (LRI) and proper development of lateral root primordia (LRP). Here, we show that the early phases of lateral root organogenesis, including priming and initiation, take place in root zones with a repressed cytokinin response. Accordingly, ectopic overproduction of cytokinin in the root basal meristem most efficiently inhibits LRI. Enhanced cytokinin responses in pericycle cells between existing LRP might restrict LRI near existing LRP and, when compromised, ectopic LRI occurs. Furthermore, our results demonstrate that young LRP are more sensitive to perturbations in the cytokinin activity than are developmentally more advanced primordia. We hypothesize that the effect of cytokinin on the development of primordia possibly depends on the robustness and stability of the auxin gradient.

Cytokinin Controls Polarity of PIN1-Dependent Auxin Transport during Lateral Root Organogenesis

The plant hormones auxin and cytokinin mutually coordinate their activities to control various aspects of development [1-9], and their crosstalk occurs at multiple levels [10, 11]. Cytokinin-mediated modulation of auxin transport provides an efficient means to regulate auxin distribution in plant organs. Here, we demonstrate that cytokinin does not merely control the overall auxin flow capacity, but might also act as a polarizing cue and control the auxin stream directionality during plant organogenesis. Cytokinin enhances the PIN-FORMED1 (PIN1) auxin transporter depletion at specific polar domains, thus rearranging the cellular PIN polarities and directly regulating the auxin flow direction. This selective cytokinin sensitivity correlates with the PIN protein phosphorylation degree. PIN1 phosphomimicking mutations, as well as enhanced phosphorylation in plants with modulated activities of PIN-specific kinases and phosphatases, desensitize PIN1 to cytokinin. Our results reveal conceptually novel, cytokinin-driven polarization mechanism that operates in developmental processes involving rapid auxin stream redirection, such as lateral root organogenesis, in which a gradual PIN polarity switch defines the growth axis of the newly formed organ.

Auxin reflux between the endodermis and pericycle promotes lateral root initiation

Lateral root (LR) formation is initiated when pericycle cells accumulate auxin, thereby acquiring founder cell (FC) status and triggering asymmetric cell divisions, giving rise to a new primordium. How this auxin maximum in pericycle cells builds up and remains focused is not understood. We report that the endodermis plays an active role in the regulation of auxin accumulation and is instructive for FCs to progress during the LR initiation (LRI) phase. We describe the functional importance of a PIN3 (PIN‐formed) auxin efflux carrier‐dependent hormone reflux pathway between overlaying endodermal and pericycle FCs. Disrupting this reflux pathway causes dramatic defects in the progress of FCs towards the next initiation phase. Our data identify an unexpected regulatory function for the endodermis in LRI as part of the fine‐tuning mechanism that appears to act as a check point in LR organogenesis after FCs are specified.

The protein quality control system manages plant defence compound synthesis

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondary metabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Here we show that, in the legume Medicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membrane-anchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

An auxin transport mechanism restricts positive orthogravitropism in lateral roots

As soon as a seed germinates, plant growth relates to gravity to ensure that the root penetrates the soil and the shoot expands aerially. Whereas mechanisms of positive and negative orthogravitropism of primary roots and shoots are relatively well understood, lateral organs often show more complex growth behavior. Lateral roots (LRs) seemingly suppress positive gravitropic growth and show a defined gravitropic set-point angle (GSA) that allows radial expansion of the root system (plagiotropism). Despite its eminent importance for root architecture, it so far remains completely unknown how lateral organs partially suppress positive orthogravitropism. Here we show that the phytohormone auxin steers GSA formation and limits positive orthogravitropism in LR. Low and high auxin levels/signaling lead to radial or axial root systems, respectively. At a cellular level, it is the auxin transport-dependent regulation of asymmetric growth in the elongation zone that determines GSA. Our data suggest that strong repression of PIN4/PIN7 and transient PIN3 expression limit auxin redistribution in young LR columella cells. We conclude that PIN activity, by temporally limiting the asymmetric auxin fluxes in the tip of LRs, induces transient, differential growth responses in the elongation zone and, consequently, controls root architecture.

Cytokinin response factors regulate PIN-FORMED auxin transporters

Auxin and cytokinin are key endogenous regulators of plant development. Although cytokinin-mediated modulation of auxin distribution is a developmentally crucial hormonal interaction, its molecular basis is largely unknown. Here we show a direct regulatory link between cytokinin signalling and the auxin transport machinery uncovering a mechanistic framework for cytokinin-auxin cross-talk. We show that the CYTOKININ RESPONSE FACTORS (CRFs), transcription factors downstream of cytokinin perception, transcriptionally control genes encoding PIN-FORMED (PIN) auxin transporters at a specific PIN CYTOKININ RESPONSE ELEMENT (PCRE) domain. Removal of this cis-regulatory element effectively uncouples PIN transcription from the CRF-mediated cytokinin regulation and attenuates plant cytokinin sensitivity. We propose that CRFs represent a missing cross-talk component that fine-tunes auxin transport capacity downstream of cytokinin signalling to control plant development.

Genetic approach towards the identification of auxin-cytokinin crosstalk components involved in root development

Phytohormones are important plant growth regulators that control many developmental processes, such as cell division, cell differentiation, organogenesis and morphogenesis. They regulate a multitude of apparently unrelated physiological processes, often with overlapping roles, and they mutually modulate their effects. These features imply important synergistic and antagonistic interactions between the various plant hormones. Auxin and cytokinin are central hormones involved in the regulation of plant growth and development, including processes determining root architecture, such as root pole establishment during early embryogenesis, root meristem maintenance and lateral root organogenesis. Thus, to control root development both pathways put special demands on the mechanisms that balance their activities and mediate their interactions. Here, we summarize recent knowledge on the role of auxin and cytokinin in the regulation of root architecture with special focus on lateral root organogenesis, discuss the latest findings on the molecular mechanisms of their interactions, and present forward genetic screen as a tool to identify novel molecular components of the auxin and cytokinin crosstalk.

Targeted cell elimination reveals an auxin-guided biphasic mode of lateral root initiation

To sustain a lifelong ability to initiate organs, plants retain pools of undifferentiated cells with a preserved proliferation capacity. The root pericycle represents a unique tissue with conditional meristematic activity, and its tight control determines initiation of lateral organs. Here we show that the meristematic activity of the pericycle is constrained by the interaction with the adjacent endodermis. Release of these restraints by elimination of endodermal cells by single-cell ablation triggers the pericycle to re-enter the cell cycle. We found that endodermis removal substitutes for the phytohormone auxin-dependent initiation of the pericycle meristematic activity. However, auxin is indispensable to steer the cell division plane orientation of new organ-defining divisions. We propose a dual, spatiotemporally distinct role for auxin during lateral root initiation. In the endodermis, auxin releases constraints arising from cell-to-cell interactions that compromise the pericycle meristematic activity, whereas, in the pericycle, auxin defines the orientation of the cell division plane to initiate lateral roots.