Cytokinin Targets Auxin Transport to Promote Shoot Branching.

Cytokinin promotes shoot branching by activating axillary buds, but its mechanism of action in Arabidopsis (Arabidopsis thaliana) in this process is unclear. We have shown previously that a hextuple mutant lacking a clade of type-A Arabidopsis Response Regulators (ARRs) known to act in cytokinin signaling has reduced shoot branching compared with the wild type. Since these proteins typically act as negative regulators of cytokinin signaling, this is an unexpected result. To explore this paradox more deeply, we characterized the effects of loss of function of the type-B ARR, ARR1, which positively regulates cytokinin-induced gene expression. The arr1 mutant has increased branching, consistent with a role antagonistic to the type-A ARRs but in apparent conflict with the known positive role for cytokinin in bud activation. We show that the arr branching phenotypes correlate with increases in stem auxin transport and steady-state levels of the auxin export proteins PIN3 and PIN7 on the plasma membrane of xylem-associated cells in the main stem. Cytokinin treatment results in an increased accumulation of PIN3, PIN7, and the closely related PIN4 within several hours, and loss of PIN3, PIN4, and PIN7 can partially rescue the arr1 branching phenotype. This suggests that there are multiple signaling pathways for cytokinin in bud outgrowth; one of these pathways regulates PIN proteins in shoots, independently of the canonical signaling function of the ARR genes tested here. A hypothesis consistent with the arr shoot phenotypes is that feedback control of biosynthesis leads to altered cytokinin accumulation, driving cytokinin signaling via this pathway.European Research Council Gatsby Charitable Foundatio

Cytokinin is required for escape but not release from auxin mediated apical dominance.

Auxin produced by an active primary shoot apex is transported down the main stem and inhibits the growth of the axillary buds below it, contributing to apical dominance. Here we use Arabidopsis thaliana cytokinin (CK) biosynthetic and signalling mutants to probe the role of CK in this process. It is well established that bud outgrowth is promoted by CK, and that CK synthesis is inhibited by auxin, leading to the hypothesis that release from apical dominance relies on an increased supply of CK to buds. Our data confirm that decapitation induces the expression of at least one ISOPENTENYLTRANSFERASE (IPT) CK biosynthetic gene in the stem. We further show that transcript abundance of a clade of the CK-responsive type-A Arabidopsis response regulator (ARR) genes increases in buds following CK supply, and that, contrary to their typical action as inhibitors of CK signalling, these genes are required for CK-mediated bud activation. However, analysis of the relevant arr and ipt multiple mutants demonstrates that defects in bud CK response do not affect auxin-mediated bud inhibition, and increased IPT transcript levels are not needed for bud release following decapitation. Instead, our data suggest that CK acts to overcome auxin-mediated bud inhibition, allowing buds to escape apical dominance under favourable conditions, such as high nitrate availability

The pea branching RMS2 gene encodes the PsAFB4/5 auxin receptor and is involved in an auxin-strigolactone regulation loop

Strigolactones (SLs) are well known for their role in repressing shoot branching. In pea, increased transcript levels of SL biosynthesis genes are observed in stems of highly branched SL deficient (ramosus1 (rms1) and rms5) and SL response (rms3 and rms4) mutants indicative of negative feedback control. In contrast, the highly branched rms2 mutant has reduced transcript levels of SL biosynthesis genes. Grafting studies and hormone quantification led to a model where RMS2 mediates a shoot-to-root feedback signal that regulates both SL biosynthesis gene transcript levels and xylem sap levels of cytokinin exported from roots. Here we cloned RMS2 using synteny with Medicago truncatula and demonstrated that it encodes a putative auxin receptor of the AFB4/5 clade. Phenotypes similar to rms2 were found in Arabidopsis afb4/5 mutants, including increased shoot branching, low expression of SL biosynthesis genes and high auxin levels in stems. Moreover, afb4/5 and rms2 display a specific resistance to the herbicide picloram. Yeast-two-hybrid experiments supported the hypothesis that the RMS2 protein functions as an auxin receptor. SL root feeding using hydroponics repressed auxin levels in stems and down-regulated transcript levels of auxin biosynthesis genes within one hour. This auxin down-regulation was also observed in plants treated with the polar auxin transport inhibitor NPA. Together these data suggest a homeostatic feedback loop in which auxin up-regulates SL synthesis in an RMS2-dependent manner and SL down-regulates auxin synthesis in an RMS3 and RMS4- dependent manner

Axillary bud outgrowth in herbaceous shoots: How do strigolactones fit into the picture?

Strigolactones have recently been identified as the long sought-after signal required to inhibit shoot branching (Gomez-Roldan et al. 2008; Umehara et al. 2008; reviewed in Dun et al. 2009). Here we briefly describe the evidence for strigolactone inhibition of shoot branching and, more extensively, the broader context of this action. We address the central question of why strigolactone mutants exhibit a varied branching phenotype across a wide range of experimental conditions. Where knowledge is available, we highlight the role of other hormones in dictating these phenotypes and describe those instances where our knowledge of known plant hormones and their interactions falls considerably short of explaining the phenotypes. This review will focus on bud outgrowth in herbaceous species because knowledge on the role of strigolactones in shoot branching to date barely extends beyond this group of plants. © Springe

Cytokinin is required for escape but not release from auxin mediated apical dominance.

Auxin produced by an active primary shoot apex is transported down the main stem and inhibits the growth of the axillary buds below it, contributing to apical dominance. Here we use Arabidopsis thaliana cytokinin (CK) biosynthetic and signalling mutants to probe the role of CK in this process. It is well established that bud outgrowth is promoted by CK, and that CK synthesis is inhibited by auxin, leading to the hypothesis that release from apical dominance relies on an increased supply of CK to buds. Our data confirm that decapitation induces the expression of at least one ISOPENTENYLTRANSFERASE (IPT) CK biosynthetic gene in the stem. We further show that transcript abundance of a clade of the CK-responsive type-A Arabidopsis response regulator (ARR) genes increases in buds following CK supply, and that, contrary to their typical action as inhibitors of CK signalling, these genes are required for CK-mediated bud activation. However, analysis of the relevant arr and ipt multiple mutants demonstrates that defects in bud CK response do not affect auxin-mediated bud inhibition, and increased IPT transcript levels are not needed for bud release following decapitation. Instead, our data suggest that CK acts to overcome auxin-mediated bud inhibition, allowing buds to escape apical dominance under favourable conditions, such as high nitrate availability

Targeting antigen to diverse APCs inactivates memory CD8+ T cells without eliciting tissue-destructive effector function

Memory T cells develop early during the preclinical stages of autoimmune diseases and have traditionally been considered resistant to tolerance induction. As such, they may represent a potent barrier to the successful immunotherapy of established autoimmune diseases. It was recently shown that memory CD8+ T cell responses are terminated when Ag is genetically targeted to steady-state dendritic cells. However, under these conditions, inactivation of memory CD8+ T cells is slow, allowing transiently expanded memory CD8+ T cells to exert tissue-destructive effector function. In this study, we compared different Ag-targeting strategies and show, using an MHC class II promoter to drive Ag expression in a diverse range of APCs, that CD8+ memory T cells can be rapidly inactivated by MHC class II+ hematopoietic APCs through a mechanism that involves a rapid and sustained downregulation of TCR, in which the effector response of CD8+ memory cells is rapidly truncated and Ag-expressing target tissue destruction is prevented. Our data provide the first demonstration that genetically targeting Ag to a broad range of MHC class II+ APC types is a highly efficient way to terminate memory CD8+ T cell responses to prevent tissue-destructive effector function and potentially established autoimmune diseases. Copyright © 2010 by The American Association of Immunologists, Inc

Research data supporting 'Connective auxin transport in the shoot facilitates communication between shoot apices'.

Full datasets underlying graphs presented in the above named paper.This research data supports 'Connective auxin transport in the shoot facilitates communication between shoot apices', which will be published in the 'PLoS Biology' journal. This record will be updated with publication details.10.1371/journal.pbio.1002446This work was supported by the ERC [grant number N° 294514 – EnCoDe] and the Swedish Governmental Agency for Innovation Systems (VINNOVA) and the Swedish Research Council (VR) [grant number GAT3272C

Multi-channel models of auxin transport capture observed stem dynamics.

<p><b>A)</b> The three types of grid structure used to represent tissue organization in the single and multi-channel models. In all three models, cells have dimensions 0.1 mm x 0.01 mm; auxin diffusion rate: D = 16e-3 mm²/min. <b>B)</b> Computer simulations compared to empirical data of the drainage of auxin in an excised stem segment; parameter values are as in C and E, with additional synthesis rates: 3×10⁻⁵ auxin units /min^-1 (single channel model), 3.8×10⁻⁶ auxin units/min^-1 (three-channel model). The empirical data are as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002446#pbio.1002446.g003" target="_blank">Fig 3A</a>. <b>C–E)</b> Computer simulations compared to empirical data of the auxin pulse assay in WT using the single channel model (C), the two-channel model (D), and the three-channel model (E). For the measured data, experiments were as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002446#pbio.1002446.g003" target="_blank">Fig 3B and 3C</a>. Error bars show the s.e.m., <i>n</i> = 8 per time point. <b>F)</b> Same simulation as in E, but for the <i>pin1</i> mutant and later time points. For the measured data, experiments were as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002446#pbio.1002446.g003" target="_blank">Fig 3B and 3C</a>, but using <i>pin1-613</i>. Error bars show the s.e.m., <i>n</i> = 8 per time point. Parameter values <u>(mm/min)</u>: <b>C.</b> <i>p</i> = 16, q as shown on graph; <b>D.</b> <u>high conductance polar channel:</u> p₁ = 6, q₁ = 0.1; q₁₂ = 0.; <u>non-polar channel:</u> q₃ = 0.01, q₃₂ = 0.01; <b>E.</b> <u>high conductance polar channel:</u> p₁ = 2, q₁ = 0.2; q₁₂ = 9 10⁻⁴; <u>low conductance polar channel:</u> p₂ = 0.3, q₂ = 0.7, q₂₃ = q₂₂ = q₂₁ = 0.01; <u>non-polar channel:</u> q₃ = 0.3, q₃₂ = 2.5 10⁻⁴; <b>F.</b> <u>high conductance polar channel:</u> p₁ = 0.5, q₁ = 0.2; q₁₂ = 9 10⁻⁴; <u>low conductance polar:</u> p₂ = 0.075, q₂ = 0.7, q₂₃ = q₂₂ = q₂₁ = 0.01; <u>non-polar channel:</u> q₃ = 0.3, q₃₂ = 2.5 10⁻⁴.</p

PIN1 dynamics in the stem are nonlinear.

<p>PIN1-GFP expression in xylem parenchyma cells (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002446#pbio.1002446.g001" target="_blank">Fig 1</a>) in longitudinally hand sectioned ~2 cm basal inflorescence stem segments of 6-wk-old <i>PIN1pro</i>:<i>PIN1-GFP</i> plants. “Apical” and “basal” (at the left) refer to which end of the segment is being imaged. Numbers in the right hand corner indicate the number of segments/the number examined in which basal, polar PIN1 localization was seen in this treatment. Green signal indicates PIN1-GFP, red signal is chloroplast autofluorescence. <b>A)</b> Freshly harvested stem segments. <b>B,D)</b> Stem segments held vertically in Petri dishes between 2 agar blocks with 1 μM NAA supplied in the apical block for 1, 3, or 6 d, respectively. <b>E–L)</b> Stem segments held vertically in Petri dishes between 2 agar blocks with no hormone treatment (‘—‘) for 4 hours (E, I), 1 d (F, J), 3 d (G, K) or 6 d (H, L) at the apical end (E–H) or basal end (I–L) of the segment.</p