A network of transcriptional repressors modulates auxin responses.

The regulation of signalling capacity, combined with the spatiotemporal distribution of developmental signals themselves, is pivotal in setting developmental responses in both plants and animals1. The hormone auxin is a key signal for plant growth and development that acts through the AUXIN RESPONSE FACTOR (ARF) transcription factors2-4. A subset of these, the conserved class A ARFs5, are transcriptional activators of auxin-responsive target genes that are essential for regulating auxin signalling throughout the plant lifecycle2,3. Although class A ARFs have tissue-specific expression patterns, how their expression is regulated is unknown. Here we show, by investigating chromatin modifications and accessibility, that loci encoding these proteins are constitutively open for transcription. Through yeast one-hybrid screening, we identify the transcriptional regulators of the genes encoding class A ARFs from Arabidopsis thaliana and demonstrate that each gene is controlled by specific sets of transcriptional regulators. Transient transformation assays and expression analyses in mutants reveal that, in planta, the majority of these regulators repress the transcription of genes encoding class A ARFs. These observations support a scenario in which the default configuration of open chromatin enables a network of transcriptional repressors to regulate expression levels of class A ARF proteins and modulate auxin signalling output throughout development.

TPLATE complex-dependent endocytosis attenuates CLAVATA1 signaling for shoot apical meristem maintenance.

Endocytosis regulates the turnover of cell surface localized receptors, which are crucial for plants to rapidly respond to stimuli. The evolutionary ancient TPLATE complex (TPC) plays an essential role in endocytosis in Arabidopsis plants. Knockout or knockdown of single TPC subunits causes male sterility and seedling lethality phenotypes, complicating analysis of the roles of TPC during plant development. Partially functional alleles of TPC subunits however only cause mild developmental deviations. Here, we took advantage of the partially functional TPLATE allele, WDXM2, to investigate a role for TPC-dependent endocytosis in receptor-mediated signaling. We discovered that reduced TPC-dependent endocytosis confers a hypersensitivity to very low doses of CLAVATA3 peptide signaling. This hypersensitivity correlated with the abundance of the CLAVATA3 receptor protein kinase CLAVATA1 at the plasma membrane. Genetic and biochemical analysis as well as live-cell imaging revealed that TPC-dependent regulation of CLAVATA3-dependent internalization of CLAVATA1 from the plasma membrane is required for shoot stem cell homeostasis. Our findings provide evidence that TPC-mediated endocytosis and degradation of CLAVATA1 is a mechanism to dampen CLAVATA3-mediated signaling during plant development.

Low Blue Light Enhances Phototropism by Releasing Cryptochrome1-Mediated Inhibition of PIF4 Expression.

Shade-avoiding plants, including Arabidopsis (Arabidopsis thaliana), display a number of growth responses, such as elongation of stem-like structures and repositioning of leaves, elicited by shade cues, including a reduction in the blue and red portions of the solar spectrum and a low-red to far-red ratio. Shade also promotes phototropism of de-etiolated seedlings through repression of phytochrome B, presumably to enhance capture of unfiltered sunlight. Here we show that both low blue light and a low-red to far-red light ratio are required to rapidly enhance phototropism in Arabidopsis seedlings. However, prolonged low blue light treatments are sufficient to promote phototropism through reduced cryptochrome1 (cry1) activation. The enhanced phototropic response of cry1 mutants in the lab and in response to natural canopies depends on PHYTOCHROME INTERACTING FACTORs (PIFs). In favorable light conditions, cry1 limits the expression of PIF4, while in low blue light, PIF4 expression increases, which contributes to phototropic enhancement. The analysis of quantitative DII-Venus, an auxin signaling reporter, indicates that low blue light leads to enhanced auxin signaling in the hypocotyl and, upon phototropic stimulation, a steeper auxin signaling gradient across the hypocotyl. We conclude that phototropic enhancement by canopy shade results from the combined activities of phytochrome B and cry1 that converge on PIF regulation.

E2FB Interacts with RETINOBLASTOMA RELATED and Regulates Cell Proliferation during Leaf Development.

Cell cycle entry and quiescence are regulated by the E2F transcription factors in association with RETINOBLASTOMA-RELATED (RBR). E2FB is considered to be a transcriptional activator of cell cycle genes, but its function during development remains poorly understood. Here, by studying E2FB-RBR interaction, E2F target gene expression, and epidermal cell number and shape in e2fb mutant and overexpression lines during leaf development in Arabidopsis (Arabidopsis thaliana), we show that E2FB in association with RBR plays a role in the inhibition of cell proliferation to establish quiescence. In young leaves, both RBR and E2FB are abundant and form a repressor complex that is reinforced by an autoregulatory loop. Increased E2FB levels, either by expression driven by its own promoter or ectopically together with DIMERIZATION PARTNER A, further elevate the amount of this repressor complex, leading to reduced leaf cell number. Cell overproliferation in e2fb mutants and in plants overexpressing a truncated form of E2FB lacking the RBR binding domain strongly suggested that RBR repression specifically acts through E2FB. The increased number of small cells below the guard cells and of fully developed stomata indicated that meristemoids preferentially hyperproliferate. As leaf development progresses and cells differentiate, the amount of RBR and E2FB gradually declined. At this stage, elevation of E2FB level can overcome RBR repression, leading to reactivation of cell division in pavement cells. In summary, E2FB in association with RBR is central to regulating cell proliferation during organ development to determine final leaf cell number.

WUSCHEL in the shoot apical meristem: old player, new tricks.

The maintenance of the stem cell niche in the shoot apical meristem, the structure that generates all of the aerial organs of the plant, relies on a canonical feedback loop between WUSCHEL (WUS) and CLAVATA3 (CLV3). WUS is a homeodomain transcription factor expressed in the organizing centre that moves to the central zone to promote stem cell fate. CLV3 is a peptide whose expression is induced by WUS in the central zone and that can move back to the organizing centre to inhibit WUS expression. Within the past 20 years since the initial formulation of the CLV-WUS feedback loop, the mechanisms of stem cell maintenance have been intensively studied and the function of WUS has been redefined. In this review, we highlight the most recent advances in our comprehension of the molecular mechanisms of WUS function, of its interaction with other transcription factors and hormonal signals, and of its connection to environmental signals. Through this, we will show how WUS can integrate both internal and external cues to adapt meristem function to the plant environment.

Halotropism requires phospholipase Dζ1-mediated modulation of cellular polarity of auxin transport carriers.

Endocytosis and relocalization of auxin carriers represent important mechanisms for adaptive plant growth and developmental responses. Both root gravitropism and halotropism have been shown to be dependent on relocalization of auxin transporters. Following their homology to mammalian phospholipase Ds (PLDs), plant PLDζ-type enzymes are likely candidates to regulate auxin carrier endocytosis. We investigated root tropic responses for an Arabidopsis pldζ1-KO mutant and its effect on the dynamics of two auxin transporters during salt stress, that is, PIN2 and AUX1. We found altered root growth and halotropic and gravitropic responses in the absence of PLDζ1 and report a role for PLDζ1 in the polar localization of PIN2. Additionally, irrespective of the genetic background, salt stress induced changes in AUX1 polarity. Utilizing our previous computational model, we found that these novel salt-induced AUX1 changes contribute to halotropic auxin asymmetry. We also report the formation of "osmotic stress-induced membrane structures." These large membrane structures are formed at the plasma membrane shortly after NaCl or sorbitol treatment and have a prolonged presence in a pldζ1 mutant. Taken together, these results show a crucial role for PLDζ1 in both ionic and osmotic stress-induced auxin carrier dynamics during salt stress.

Structure of the Arabidopsis TOPLESS corepressor provides insight into the evolution of transcriptional repression.

Transcriptional repression involves a class of proteins called corepressors that link transcription factors to chromatin remodeling complexes. In plants such as Arabidopsis thaliana, the most prominent corepressor is TOPLESS (TPL), which plays a key role in hormone signaling and development. Here we present the crystallographic structure of the Arabidopsis TPL N-terminal region comprising the LisH and CTLH (C-terminal to LisH) domains and a newly identified third region, which corresponds to a CRA domain. Comparing the structure of TPL with the mammalian TBL1, which shares a similar domain structure and performs a parallel corepressor function, revealed that the plant TPLs have evolved a new tetramerization interface and unique and highly conserved surface for interaction with repressors. Using site-directed mutagenesis, we validated those surfaces in vitro and in vivo and showed that TPL tetramerization and repressor binding are interdependent. Our results illustrate how evolution used a common set of protein domains to create a diversity of corepressors, achieving similar properties with different molecular solutions.

Identification and functional characterization of the Arabidopsis†Snf1-related protein kinase SnRK2.4 phosphatidic acid-binding domain.

Phosphatidic acid (PA) is an important signalling lipid involved in various stress-induced signalling cascades. Two SnRK2 protein kinases (SnRK2.4 and SnRK2.10), previously identified as PA-binding proteins, are shown here to prefer binding to PA over other anionic phospholipids and to associate with cellular membranes in response to salt stress in Arabidopsis roots. A 42 amino acid sequence was identified as the primary PA-binding domain (PABD) of SnRK2.4. Unlike the full-length SnRK2.4, neither the PABD-YFP fusion protein nor the SnRK2.10 re-localized into punctate structures upon salt stress treatment, showing that additional domains of the SnRK2.4 protein are required for its re-localization during salt stress. Within the PABD, five basic amino acids, conserved in class 1 SnRK2s, were found to be necessary for PA binding. Remarkably, plants overexpressing the PABD, but not a non-PA-binding mutant version, showed a severe reduction in root growth. Together, this study biochemically characterizes the PA-SnRK2.4 interaction and shows that functionality of the SnRK2.4 PABD affects root development.

A regulated auxin minimum is required for seed dispersal in Arabidopsis.

Local hormone maxima are essential for the development of multicellular structures and organs. For example, steroid hormones accumulate in specific cell types of the animal fetus to induce sexual differentiation and concentration peaks of the plant hormone auxin direct organ initiation and mediate tissue patterning. Here we provide an example of a regulated local hormone minimum required during organogenesis. Our results demonstrate that formation of a local auxin minimum is necessary for specification of the valve margin separation layer where Arabidopsis fruit opening takes place. Consequently, ectopic production of auxin, specifically in valve margin cells, leads to a complete loss of proper cell fate determination. The valve margin identity factor INDEHISCENT (IND) is responsible for forming the auxin minimum by coordinating auxin efflux in separation-layer cells. We propose that the simplicity of formation and maintenance make local hormone minima particularly well suited to specify a small number of cells such as the stripes at the valve margins.

A quantitative gibberellin signaling biosensor reveals a role for gibberellins in internode specification at the shoot apical meristem.

Growth at the shoot apical meristem (SAM) is essential for shoot architecture construction. The phytohormones gibberellins (GA) play a pivotal role in coordinating plant growth, but their role in the SAM remains mostly unknown. Here, we developed a ratiometric GA signaling biosensor by engineering one of the DELLA proteins, to suppress its master regulatory function in GA transcriptional responses while preserving its degradation upon GA sensing. We demonstrate that this degradation-based biosensor accurately reports on cellular changes in GA levels and perception during development. We used this biosensor to map GA signaling activity in the SAM. We show that high GA signaling is found primarily in cells located between organ primordia that are the precursors of internodes. By gain- and loss-of-function approaches, we further demonstrate that GAs regulate cell division plane orientation to establish the typical cellular organization of internodes, thus contributing to internode specification in the SAM.

The Snf1-related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance of root system architecture during salt stress.

The sucrose non-fermenting-1-related protein kinase 2 (SnRK2) family represents a unique family of plant-specific protein kinases implicated in cellular signalling in response to osmotic stress. In our studies, we observed that two class 1 SnRK2 kinases, SnRK2.4 and SnRK2.10, are rapidly and transiently activated in Arabidopsis roots after exposure to salt. Under saline conditions, snrk2.4 knockout mutants had a reduced primary root length, while snrk2.10 mutants exhibited a reduction in the number of lateral roots. The reduced lateral root density was found to be a combinatory effect of a decrease in the number of lateral root primordia and an increase in the number of arrested lateral root primordia. The phenotypes were in agreement with the observed expression patterns of genomic yellow fluorescent protein (YFP) fusions of SnRK2.10 and -2.4, under control of their native promoter sequences. SnRK2.10 was found to be expressed in the vascular tissue at the base of a developing lateral root, whereas SnRK2.4 was expressed throughout the root, with higher expression in the vascular system. Salt stress triggered a rapid re-localization of SnRK2.4-YFP from the cytosol to punctate structures in root epidermal cells. Differential centrifugation experiments of isolated Arabidopsis root proteins confirmed recruitment of endogenous SnRK2.4/2.10 to membranes upon exposure to salt, supporting their observed binding affinity for the phospholipid phosphatidic acid. Together, our results reveal a role for SnRK2.4 and -2.10 in root growth and architecture in saline conditions.

Plasma membrane-bound AGC3 kinases phosphorylate PIN auxin carriers at TPRXS(N/S) motifs to direct apical PIN recycling.

Polar membrane cargo delivery is crucial for establishing cell polarity and for directional transport processes. In plants, polar trafficking mediates the dynamic asymmetric distribution of PIN FORMED (PIN) carriers, which drive polar cell-to-cell transport of the hormone auxin, thereby generating auxin maxima and minima that control development. The Arabidopsis PINOID (PID) protein kinase instructs apical PIN localization by phosphorylating PINs. Here, we identified the PID homologs WAG1 and WAG2 as new PIN polarity regulators. We show that the AGC3 kinases PID, WAG1 and WAG2, and not other plant AGC kinases, instruct recruitment of PINs into the apical recycling pathway by phosphorylating the middle serine in three conserved TPRXS(N/S) motifs within the PIN central hydrophilic loop. Our results put forward a model by which apolarly localized PID, WAG1 and WAG2 phosphorylate PINs at the plasma membrane after default non-polar PIN secretion, and trigger endocytosis-dependent apical PIN recycling. This phosphorylation-triggered apical PIN recycling competes with ARF-GEF GNOM-dependent basal recycling to promote apical PIN localization. In planta, expression domains of PID, WAG1 and WAG2 correlate with apical localization of PINs in those cell types, indicating the importance of these kinases for apical PIN localization. Our data show that by directing polar PIN localization and PIN-mediated polar auxin transport, the three AGC3 kinases redundantly regulate cotyledon development, root meristem size and gravitropic response, indicating their involvement in both programmed and adaptive plant development.

Phosphorylation of conserved PIN motifs directs Arabidopsis PIN1 polarity and auxin transport.

Polar cell-to-cell transport of auxin by plasma membrane-localized PIN-FORMED (PIN) auxin efflux carriers generates auxin gradients that provide positional information for various plant developmental processes. The apical-basal polar localization of the PIN proteins that determines the direction of auxin flow is controlled by reversible phosphorylation of the PIN hydrophilic loop (PINHL). Here, we identified three evolutionarily conserved TPRXS(N/S) motifs within the PIN1HL and proved that the central Ser residues were phosphorylated by the PINOID (PID) kinase. Loss-of-phosphorylation PIN1:green fluorescent protein (GFP) (Ser to Ala) induced inflorescence defects, correlating with their basal localization in the shoot apex, and induced internalization of PIN1:GFP during embryogenesis, leading to strong embryo defects. Conversely, phosphomimic PIN1:GFP (Ser to Glu) showed apical localization in the shoot apex but did not rescue pin1 inflorescence defects. Both loss-of-phosphorylation and phosphomimic PIN1:GFP proteins were insensitive to PID overexpression. The basal localization of loss-of-phosphorylation PIN1:GFP increased auxin accumulation in the root tips, partially rescuing PID overexpression-induced root collapse. Collectively, our data indicate that reversible phosphorylation of the conserved Ser residues in the PIN1HL by PID (and possibly by other AGC kinases) is required and sufficient for proper PIN1 localization and is thus essential for generating the differential auxin distribution that directs plant development.

Regulators of floral fragrance production and their target genes in petunia are not exclusively active in the epidermal cells of petals.

In which cells of the flower volatile biosynthesis takes place is unclear. In rose and snapdragon, some enzymes of the volatile phenylpropanoid/benzenoid pathway have been shown to be present in the epidermal cells of petals. It is therefore generally believed that the production of these compounds occurs in these cells. However, whether the entire pathway is active in these cells and whether it is exclusively active in these cells remains to be proven. Cell-specific transcription factors activating these genes will determine in which cells they are expressed. In petunia, the transcription factor EMISSION OF BENZENOIDS II (EOBII) activates the ODORANT1 (ODO1) promoter and the promoter of the biosynthetic gene isoeugenol synthase (IGS). The regulator ODO1 in turn activates the promoter of the shikimate gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Here the identification of a new target gene of ODO1, encoding an ABC transporter localized on the plasma membrane, PhABCG1, which is co-expressed with ODO1, is described. PhABCG1 expression is up-regulated in petals overexpressing ODO1 through activation of the PhABCG1 promoter. Interestingly, the ODO1, PhABCG1, and IGS promoters were active in petunia protoplasts originating from both epidermal and mesophyll cell layers of the petal, suggesting that the volatile phenylpropanoid/benzenoid pathway in petunia is active in these different cell types. Since volatile release occurs from epidermal cells, trafficking of (volatile) compounds between cell layers must be involved, but the exact function of PhABCG1 remains to be resolved.

Methods to Visualize Auxin and Cytokinin Signaling Activity in the Shoot Apical Meristem.

Visualizing the distribution of hormone signaling activity such as auxin and cytokinins is of key importance for understanding regulation of plant development and physiology. Live imaging and genetically encoded hormone biosensors and reporters allow monitoring the spatial and temporal distribution of these phytohormones. Here, we describe how to cultivate live shoot apical meristems after dissection for observation under the confocal microscope for up to 4 days. The shoot apical meristems are maintained on an appropriate medium allowing them to grow and initiate new organs at a frequency similar to plants grown on soil. Meristems expressing hormone biosensors and reporters allows following hormone signaling activity distribution at high spatiotemporal resolution without chemical fixation, an approach that that can also be applied to follow the dynamics of expression in vivo of any fluorescent marker.

Temporal integration of auxin information for the regulation of patterning.

Positional information is essential for coordinating the development of multicellular organisms. In plants, positional information provided by the hormone auxin regulates rhythmic organ production at the shoot apex, but the spatio-temporal dynamics of auxin gradients is unknown. We used quantitative imaging to demonstrate that auxin carries high-definition graded information not only in space but also in time. We show that, during organogenesis, temporal patterns of auxin arise from rhythmic centrifugal waves of high auxin travelling through the tissue faster than growth. We further demonstrate that temporal integration of auxin concentration is required to trigger the auxin-dependent transcription associated with organogenesis. This provides a mechanism to temporally differentiate sites of organ initiation and exemplifies how spatio-temporal positional information can be used to create rhythmicity.

Plants, like animals and many other multicellular organisms, control their body architecture by creating organized patterns of cells. These patterns are generally defined by signal molecules whose levels differ across the tissue and change over time. This tells the cells where they are located in the tissue and therefore helps them know what tasks to perform. A plant hormone called auxin is one such signal molecule and it controls when and where plants produce new leaves and flowers. Over time, this process gives rise to the dashing arrangements of spiraling organs exhibited by many plant species. The leaves and flowers form from a relatively small group of cells at the tip of a growing stem known as the shoot apical meristem. Auxin accumulates at precise locations within the shoot apical meristem before cells activate the genes required to make a new leaf or flower. However, the precise role of auxin in forming these new organs remained unclear because the tools to observe the process in enough detail were lacking. Galvan-Ampudia, Cerutti et al. have now developed new microscopy and computational approaches to observe auxin in a small plant known as Arabidopsis thaliana. This showed that dozens of shoot apical meristems exhibited very similar patterns of auxin. Images taken over a period of several hours showed that the locations where auxin accumulated were not fixed on a group of cells but instead shifted away from the center of the shoot apical meristems faster than the tissue grew. This suggested the cells experience rapidly changing levels of auxin. Further experiments revealed that the cells needed to be exposed to a high level of auxin over time to activate genes required to form an organ. This mechanism sheds a new light on how auxin regulates when and where plants make new leaves and flowers. The tools developed by Galvan-Ampudia, Cerutti et al. could be used to study the role of auxin in other plant tissues, and to investigate how plants regulate the response to other plant hormones.

Plant evolution: AGC kinases tell the auxin tale.

The signaling molecule auxin is a central regulator of plant development, which instructs tissue and organ patterning, and couples environmental stimuli to developmental responses. Here, we discuss the function of PINOID (PID) and the phototropins, members of the plant specific AGCVIII protein kinases, and their role in triggering and regulating development by controlling PIN-FORMED (PIN) auxin transporter-generated auxin gradients and maxima. We propose that the AGCVIII kinase gene family evolved from an ancestral phototropin gene, and that the co-evolution of PID-like and PIN gene families marks the transition of plants from water to land. We hypothesize that the PID-like kinases function in parallel to, or downstream of, the phototropins to orient plant development by establishing the direction of polar auxin transport.

WUSCHEL acts as an auxin response rheostat to maintain apical stem cells in Arabidopsis.

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.