AUX/LAX Genes Encode a Family of Auxin Influx Transporters That Perform Distinct Functions during Arabidopsis Development

Auxin transport, which is mediated by specialized influx and efflux carriers, plays a major role in many aspects of plant growth and development. AUXIN1 (AUX1) has been demonstrated to encode a high-affinity auxin influx carrier. In Arabidopsis thaliana, AUX1 belongs to a small multigene family comprising four highly conserved genes (i.e., AUX1 and LIKE AUX1 [LAX] genes LAX1, LAX2, and LAX3). We report that all four members of this AUX/LAX family display auxin uptake functions. Despite the conservation of their biochemical function, AUX1, LAX1, and LAX3 have been described to regulate distinct auxin-dependent developmental processes. Here, we report that LAX2 regulates vascular patterning in cotyledons. We also describe how regulatory and coding sequences of AUX/LAX genes have undergone subfunctionalization based on their distinct patterns of spatial expression and the inability of LAX sequences to rescue aux1 mutant phenotypes, respectively. Despite their high sequence similarity at the protein level, transgenic studies reveal that LAX proteins are not correctly targeted in the AUX1 expression domain. Domain swapping studies suggest that the N-terminal half of AUX1 is essential for correct LAX localization. We conclude that Arabidopsis AUX/LAX genes encode a family of auxin influx transporters that perform distinct developmental functions and have evolved distinct regulatory mechanisms.     

A small molecule inhibitor partitions two distinct pathways for trafficking of tonoplast intrinsic proteins in Arabidopsis

Tonoplast intrinsic proteins (TIPs) facilitate the membrane transport of water and other small molecules across the plant vacuolar membrane, and members of this family are expressed in specific developmental stages and tissue types. Delivery of TIP proteins to the tonoplast is thought to occur by vesicle-mediated traffic from the endoplasmic reticulum to the vacuole, and at least two pathways have been proposed, one that is Golgi-dependent and another that is Golgi-independent. However, the mechanisms for trafficking of vacuolar membrane proteins to the tonoplast remain poorly understood. Here we describe a chemical genetic approach to unravel the mechanisms of TIP protein targeting to the vacuole in Arabidopsis seedlings. We show that members of the TIP family are targeted to the vacuole via at least two distinct pathways, and we characterize the bioactivity of a novel inhibitor that can differentiate between them. We demonstrate that, unlike for TIP1;1, trafficking of markers for TIP3;1 and TIP2;1 is insensitive to Brefeldin A in Arabidopsis hypocotyls. Using a chemical inhibitor that may target this BFA-insensitive pathway for membrane proteins, we show that inhibition of this pathway results in impaired root hair growth and enhanced vacuolar targeting of the auxin efflux carrier PIN2 in the dark. Our results indicate that the vacuolar targeting of PIN2 and the BFA-insensitive pathway for tonoplast proteins may be mediated in part by common mechanisms.     

Isoform-specific subcellular localization among 14-3-3 proteins in Arabidopsis seems to be driven by client interactions

In most higher eukaryotes, the predominantly phosphoprotein-binding 14-3-3 proteins are the products of a multigene family, with many organisms having 10 or more family members. However, current models for 14-3-3/phosphopeptide interactions suggest that there is little specificity among 14-3-3s for diverse phosphopeptide clients. Therefore, the existence of sequence diversity among 14-3-3s within a single organism begs questions regarding the in vivo specificities of the interactions between the various 14-3-3s and their clients. Chief among those questions is, Do the different 14-3-3 isoforms interact with different clients within the same cell? Although the members of the Arabidopsis 14-3-3 family of proteins typically contain highly conserved regions of sequence, they also display distinctive variability with deep evolutionary roots. In the current study, a survey of several Arabidopsis 14-3-3/GFP fusions revealed that 14-3-3s demonstrate distinct and differential patterns of subcellular distribution, by using trichomes and stomate guard cells as in vivo experimental cellular contexts. The effects of client interaction on 14-3-3 localization were further analyzed by disrupting the partnering with peptide and chemical agents. Results indicate that 14-3-3 localization is both isoform specific and highly dependent upon interaction with cellular clients.     

Comprehensive analysis of CLE polypeptide signaling gene expression and overexpression activity in Arabidopsis

Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis (Arabidopsis thaliana), only a few families of extracellular signaling molecules have been discovered, and their biological roles are largely unknown. To expand our insight into the developmental processes potentially regulated by ligand-mediated signal transduction pathways, we undertook a systematic expression analysis of the members of the Arabidopsis CLAVATA3/ESR-RELATED (CLE) small signaling polypeptide family. Using reporter constructs, we show that the CLE genes have distinct and specific patterns of promoter activity. We find that each Arabidopsis tissue expresses at least one CLE gene, indicating that CLE-mediated signaling pathways are likely to play roles in many biological processes during the plant life cycle. Some CLE genes that are closely related in sequence have dissimilar expression profiles, yet in many tissues multiple CLE genes have overlapping patterns of promoter-driven reporter activity. This observation, plus the general absence of detectable morphological phenotypes in cle null mutants, suggest that a high degree of functional redundancy exists among CLE gene family members. Our work establishes a community resource of CLE-related biological materials and provides a platform for understanding and ultimately manipulating many different plant signaling systems.     

Activation of calcium/calmodulin regulated kinases.

Among numerous protein kinases found in mammalian cell systems there is a distinct subfamily of serine/threonine kinases that are regulated by calmodulin or other related activators in a calcium concentration dependent manner. Members of this family are involved in various cellular processes like cell proliferation and death, cell motility and metabolic pathways. In this contribution we shall review the available structural biology data on five members of this kinase family (calcium/calmodulin dependent kinase, twitchin kinase, titin kinase, phosphorylase kinase, myosin light chain kinase). As a common element, all these kinases contain a regulatory tail, which is C-terminal to their catalytic domain. The available 3D structures of two members, the serine/threonine kinases of the giant muscle proteins twitchin and titin in the autoinhibited conformation, show how this regulatory tail blocks their active sites. The structures suggest that activation of these kinases requires unblocking the active site from the C-terminal extension and conformational rearrangement of the active site loops. Small angle scattering data for myosin light chain kinase indicate a complete release of the C-terminal extension upon calcium/calmodulin binding. In addition, members of this family are regulated by diverse add-on mechanisms, including phosphorylation of residues within the activation segment or the P+1 loop as well as by additional regulatory subunits. The available structural data lead to the hypothesis of two different activation mechanisms upon binding to calcium sensitive proteins. In one model, the regulatory tail is entirely released ("fall-apart"). The alternative model ("looping-out") proposes a two-anchored release mechanism.     

Expression Profiles of Arabidopsis thaliana VQ Gene Family in Defense Related Hormones Treatments

Several VQ proteins are recently identified as WRKY factors interacting partners in Arabidopsis and involved in regulating physiological processes. Searching of genomic databases found that VQ gene family is specific to land plants and these VQ genes encode proteins characteristic of a conserved VQ motif. It consists of 34 representatives in Arabidopsis and can be divided into two groups based on the similarity of the amino acid sequences. To understand the functions of Arabidopsis VQ proteins, we examined the expression profiles of AtVQ genes in various defense related hormones treatments. qRT PCR analysis revealed that a majority of them were differently regulated in response to salicylic acid (SA), methyl jasmonate (MeJA), or 1 aminocyclopropane 1 carboxylate (ACC). And some members are induced by two of these three hormones. Moreover, four members (AtVQ3, AtVQ18, AtVQ23 and AtVQ24) are induced by SA, MeJA and ACC simultaneity. However, there is only one AtVQ gene (AtVQ27) is up regulated after spraying of abscisic acid (ABA). These results suggest that Arabidopsis VQ genes may be involved in plant defense responses.     

TGA1 and G-box binding factors: two distinct classes of Arabidopsis leucine zipper proteins compete for the G-box-like element TGACGTGG.

Regulatory elements containing the sequence ACGT are found in several plant promoters and are recognized by various basic/leucine zipper (bZIP) proteins. The Arabidopsis G-box binding factor 1 (GBF1), initially identified by its ability to bind to the palindromic G-box (CCACGTGG), also interacts with the TGACGT motif if this hexamer sequence is followed by either the dinucleotide GG--as found in the Hex motif of the wheat histone 3 promoter--or GT. Here we describe the isolation of an Arabidopsis bZIP protein, denoted TGA1, that also recognizes ACGT-containing sequences. However, TGA1 differs from members of the GBF family in the spectrum of base pair permutations flanking the ACGT sequence that are required for DNA binding. TGA1 primarily requires a TGACG motif and preferentially binds to those pentamers that are followed by a T residue. We show that although both TGA1 and GBF1 bind to the Hex motif (TGACGTGG), this binding can be distinguished on the basis of their specific DNA-protein contacts. Furthermore, TGA1 also differs from members of the GBF family in that it apparently does not form heterodimers with any member of this family.     

Molecular characterization of a novel gene family encoding ACT domain repeat proteins in Arabidopsis

In bacteria, the regulatory ACT domains serve as amino acid-binding sites in some feedback-regulated amino acid metabolic enzymes. We have identified a novel type of ACT domain-containing protein family in Arabidopsis whose members contain ACT domain repeats (the "ACR" protein family). There are at least eight ACR genes located on each of the five chromosomes in the Arabidopsis genome. Gene structure comparisons indicate that the ACR gene family may have arisen by gene duplications. Northern-blot analysis indicates that each member of the ACR gene family has a distinct expression pattern in various organs from 6-week-old Arabidopsis. Moreover, analyses of an ACR3 promoter-beta-glucuronidase (GUS) fusion in transgenic Arabidopsis revealed that the GUS activity formed a gradient in the developing leaves and sepals, whereas low or no GUS activity was detected in the basal regions. In 2-week-old Arabidopsis seedlings grown in tissue culture, the expression of the ACR gene family is differentially regulated by plant hormones, salt stress, cold stress, and light/dark treatment. The steady-state levels of ACR8 mRNA are dramatically increased by treatment with abscisic acid or salt. Levels of ACR3 and ACR4 mRNA are increased by treatment with benzyladenine. The amino acid sequences of Arabidopsis ACR proteins are most similar in the ACT domains to the bacterial sensor protein GlnD. The ACR proteins may function as novel regulatory or sensor proteins in plants.     

The Mathematical Model of the Bcl-2 Family Mediated MOMP Regulation Can Perform a Non-Trivial Pattern Recognition

Interactions between individual members of the B-cell lymphoma 2 (Bcl-2) family of proteins form a regulatory network governing mitochondrial outer membrane permeabilization (MOMP). Bcl-2 family initiated MOMP causes release of the inter-membrane pro-apoptotic proteins to cytosol and creates a cytosolic environment suitable for the executionary phase of apoptosis. We designed the mathematical model of this regulatory network where the synthesis rates of the Bcl-2 family members served as the independent inputs. Using computational simulations, we have then analyzed the response of the model to up-/downregulation of the Bcl-2 proteins. Under several assumptions, and using estimated reaction parameters, a non-linear stimulus-response emerged, whose characteristics are associated with bistability and switch-like behavior. Interestingly, using the principal component analysis (PCA) we have shown that the given model of the Bcl-2 family interactions classifies the random combinations of inputs into two distinct classes, and responds to these by one of the two qualitatively distinct outputs. As we showed, the emergence of this behavior requires specific organization of the interactions between particular Bcl-2 proteins.     

A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.

The mammalian olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants presumably results from the association of odorous ligands with specific receptors on olfactory sensory neurons. To address the problem of olfactory perception at a molecular level, we have cloned and characterized 18 different members of an extremely large multigene family that encodes seven transmembrane domain proteins whose expression is restricted to the olfactory epithelium. The members of this novel gene family are likely to encode a diverse family of odorant receptors.     

Comprehensive approach to genes involved in cell wall modifications in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The plant cell wall is of supermolecular architecture, and is composed of various types of heterogeneous polymers. A few thousand enzymes and structural proteins are directly involved in the construction processes, and in the functional aspects of the dynamic architecture in Arabidopsis thaliana. Most of these proteins are encoded by multigene families, and most members within each family share significant similarities in structural features, but often exhibit differing expression profiles and physiological functions. Thus, for the molecular dissection of cell wall dynamics, it is necessary to distinguish individual members within a family of proteins. As a first step towards characterizing the processes involved in cell wall dynamics, we have manufactured a gene-specific 70-mer oligo microarray that consists of 765 genes classified into 30 putative families of proteins that are implicated in the cell wall dynamics of Arabidopsis. By using this array system, we identified several sets of genes that exhibit organ preferential expression profiles. We also identified gene sets that are expressed differentially at certain specific growth stages of the Arabidopsis inflorescence stem. Our results indicate that there is a division of roles among family members within each of the putative cell wall-related gene families.