Carlos S. Galvan-Ampudia

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.

Phosphorylation of Conserved PIN Motifs Directs Arabidopsis PIN1 Polarity and Auxin Transport

This work identifies the Ser residues located in three evolutionarily conserved TPRXS(N/S) motifs within the PIN1 auxin efflux carrier hydrophilic loop as substrates of the PINOID kinase. It shows that reversible phosphorylation of these Ser residues by PINOID and possibly other kinases is necessary and sufficient for proper PIN1 polar localization, auxin distribution, and regulated plant development. 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.

Halotropism is a response of plant roots to avoid a saline environment

Tropisms represent fascinating examples of how plants respond to environmental signals by adapting their growth and development. Here, a novel tropism is reported, halotropism, allowing plant seedlings to reduce their exposure to salinity by circumventing a saline environment. In response to a salt gradient, Arabidopsis, tomato, and sorghum roots were found to actively prioritize growth away from salinity above following the gravity axis. Directionality of this response is established by an active redistribution of the plant hormone auxin in the root tip, which is mediated by the PIN-FORMED 2 (PIN2) auxin efflux carrier. We show that salt-induced phospholipase D activity stimulates clathrin-mediated endocytosis of PIN2 at the side of the root facing the higher salt concentration. The intracellular relocalization of PIN2 allows for auxin redistribution and for the directional bending of the root away from the higher salt concentration. Our results thus identify a cellular pathway essential for the integration of environmental cues with auxin-regulated root growth that likely plays a key role in plant adaptative responses to salt stress.

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.

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.

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.

Phyllotaxis: from patterns of organogenesis at the meristem to shoot architecture

The primary architecture of the aerial part of plants is controlled by the shoot apical meristem, a specialized tissue containing a stem cell niche. The iterative generation of new aerial organs, (leaves, lateral inflorescences, and flowers) at the meristem follows regular patterns, called phyllotaxis. Phyllotaxis has long been proposed to self‐organize from the combined action of growth and of inhibitory fields blocking organogenesis in the vicinity of existing organs in the meristem. In this review, we will highlight how a combination of mathematical/computational modeling and experimental biology has demonstrated that the spatiotemporal distribution of the plant hormone auxin controls both organogenesis and the establishment of inhibitory fields. We will discuss recent advances showing that auxin likely acts through a combination of biochemical and mechanical regulatory mechanisms that control not only the pattern of organogenesis in the meristem but also postmeristematic growth, to shape the shoot. WIREs Dev Biol 2016, 5:460–473. doi: 10.1002/wdev.231

Signal integration by GSK3 kinases in the root.

Signal integration is central to the regulation of patterning during plant development. During lateral root initiation, a signalling pathway controlled by the phloem-secreted TDIF peptide is found to activate the auxin signalling pathway independently of auxin, through phosphorylation of ARF transcription factors by GSK3 (Shaggy-like) kinases.

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

Significance In most biological processes, genes have to be activated and/or repressed. In plants, the TOPLESS protein is essential for gene repression through its action as a corepressor bridging transcription factor with chromatin remodeling complexes. Here we combine biochemical and structural studies to describe the structure of TOPLESS, how it tetramerizes, and how it interacts with its protein partners. We show that both the tetramerization interface and the binding site for protein partners have been conserved since algae, highlighting the ancestrality of TOPLESS function. Comparison of this plant protein with one of its animal counterparts also shows how corepressors can use a common domain differently to achieve similar properties, illustrating the tinkering of evolution in 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.

3-d Tessellation of Plant Tissue - A Dual Optimization Approach to Cell-Level Meristem Reconstruction from Microscopy Images

The goal of this paper is the reconstruction of topologically accurate 3-dimensional triangular meshes representing a complex, multi-layered plant tissue structure. Based on time sequences of meristem images of the model plant Arabidopsis thaliana, displaying fluorescence markers on either cell membranes or cell nuclei under confocal laser scanning microscopy, we aim at obtaining faithful reconstructions of all the cell walls in the tissue. In the presented method, the problem is tackled under the angle of topology, and the shape of the cells is seen as the dual geometry of a 3-d simplicial complex accounting for their adjacency relationships. We present a method for optimizing such complexes using an energy minimization process, designed to make them fit to the actual adjacencies in the tissue. The resulting dual meshes constitute a light discrete representation of the cell surfaces that enables fast visualization, and quantitative analysis, and allows in silico physical and mechanical simulations on real-world data.