Gibberellic acid (GA) promotes seed germination, elongation growth, and flowering time in plants. GA responses are repressed by DELLA proteins, which contain an N-terminal DELLA domain essential for GA-dependent proteasomal degradation of DELLA repressors. Mutations of or within the DELLA domain of DELLA repressors have been described for species including Arabidopsis thaliana, wheat (Triticum aestivum), maize (Zea mays), and barley (Hordeum vulgare), and we show that these mutations confer GA insensitivity when introduced into the Arabidopsis GA INSENSITIVE (GAI) DELLA repressor. We also demonstrate that Arabidopsis mutants lacking the three GA INSENSITIVE DWARF1 (GID1) GA receptor genes are GA insensitive with respect to GA-promoted growth responses, GA-promoted DELLA repressor degradation, and GA-regulated gene expression. Our genetic interaction studies indicate that GAI and its close homolog REPRESSOR OF ga1-3 are the major growth repressors in a GA receptor mutant background. We further demonstrate that the GA insensitivity of the GAI DELLA domain mutants is explained in all cases by the inability of the mutant proteins to interact with the GID1A GA receptor. Since we found that the GAI DELLA domain alone can mediate GA-dependent GID1A interactions, we propose that the DELLA domain functions as a receiver domain for activated GA receptors.

The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the—in many cells—asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.DOI: http://dx.doi.org/10.7554/eLife.02860.001

The phytohormone auxin is a major determinant of plant growth and differentiation. Directional auxin transport and auxin responses are required for proper embryogenesis, organ formation, vascular development, and tropisms. Members of several protein families, including the PIN auxin efflux facilitators, have been implicated in auxin transport; however, the regulation of auxin transport by signaling proteins remains largely unexplored. We have studied a family of four highly homologous AGC protein kinases, which we designated the D6 protein kinases (D6PKs). We found that d6pk mutants have defects in lateral root initiation, root gravitropism, and shoot differentiation in axillary shoots, and that these phenotypes correlate with a reduction in auxin transport. Interestingly, D6PK localizes to the basal (lower) membrane of Arabidopsis root cells, where it colocalizes with PIN1, PIN2 and PIN4. D6PK and PIN1 interact genetically, and D6PK phosphorylates PIN proteins in vitro and in vivo. Taken together, our data show that D6PK is required for efficient auxin transport and suggest that PIN proteins are D6PK phosphorylation targets.

Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.

Plants integrate different regulatory signals to control their growth and development. Although a number of physiological observations suggest that there is crosstalk between the phytohormone gibberellin (GA) and auxin, as well as with auxin transport, the molecular basis for this hormonal crosstalk remains largely unexplained. Here, we show that auxin transport is reduced in the inflorescences of Arabidopsis thaliana mutants deficient in GA biosynthesis and signaling. We further show that this reduced auxin transport correlates with a reduction in the abundance of PIN-FORMED (PIN) auxin efflux facilitators in GA-deficient plants and that PIN protein levels recover to wild-type levels following GA treatment. We also demonstrate that the regulation of PIN protein levels cannot be explained by a transcriptional regulation of the PIN genes but that GA deficiency promotes, at least in the case of PIN2, the targeting of PIN proteins for vacuolar degradation. In genetic studies, we reveal that the reduced auxin transport of GA mutants correlates with an impairment in two PINdependent growth processes, namely, cotyledon differentiation and root gravitropic responses. Our study thus presents evidence for a role of GA in these growth responses and for a GA-dependent modulation of PIN turnover that may be causative for these differential growth responses.