Detoxification of monoterpenes by a family of plant glycosyltransferases.

Plant monoterpenes are challenging compounds, since they often act as solvents, and thus have both phytotoxic and antimicrobial properties. In this study an approach is developed to identify and characterize enzymes that can detoxify monoterpenoids, and thus would protect both plants and microbial production systems from these compounds. Plants respond to the presence of monoterpenes by expressing glycosyltransferases (UGTs), which conjugate the monoterpenoids into glycosides. By identifying these enzymes in a transcriptomics approach using Mentha × piperita, a family of UGTs was identified which is active on cyclic monoterpenoids such as menthol, and on acyclic monoterpenoids such as geranic acid. Other members of this family, from tomato, were also shown to be active on these monoterpenoids. In vitro and in vivo activity of different UGTs were tested with different substrates. We found that some glycosyltransferases significantly affect the toxicity of selected monoterpenoids in Escherichia coli, suggesting that glycosyltransferases can protect cells from monoterpenoid toxicity.

The power of seaweeds as plant biostimulants to boost crop production under abiotic stress.

Abiotic stresses like drought and salinity are major factors resulting in crop yield losses and soil degradation worldwide. To meet increasing food demands, we must improve crop productivity, especially under increasing abiotic stresses due to climate change. Recent studies suggest that seaweed-based biostimulants could be a solution to this problem. Here, we summarize the current findings of using these biostimulants and highlight current knowledge gaps. Seaweed extracts were shown to enhance nutrient uptake and improve growth performance in crops under stressed and normal conditions. Seaweed extracts contain several active compounds, for example, polysaccharides, polyphenols and phytohormones. Although some of these compounds have growth-promoting properties on plants, the molecular mechanisms that underly seaweed extract action remain understudied. In this paper, we review the role of these extracts and their bioactive compounds as plant biostimulants. The targeted application of seaweed extract to improve crop performance and protein accumulation is also discussed.

Root plasticity under abiotic stress.

Abiotic stresses increasingly threaten existing ecological and agricultural systems across the globe. Plant roots perceive these stresses in the soil and adapt their architecture accordingly. This review provides insights into recent discoveries showing the importance of root system architecture (RSA) and plasticity for the survival and development of plants under heat, cold, drought, salt, and flooding stress. In addition, we review the molecular regulation and hormonal pathways involved in controlling RSA plasticity, main root growth, branching and lateral root growth, root hair development, and formation of adventitious roots. Several stresses affect root anatomy by causing aerenchyma formation, lignin and suberin deposition, and Casparian strip modulation. Roots can also actively grow toward favorable soil conditions and avoid environments detrimental to their development. Recent advances in understanding the cellular mechanisms behind these different root tropisms are discussed. Understanding root plasticity will be instrumental for the development of crops that are resilient in the face of abiotic stress.

Several geranylgeranyl diphosphate synthase isoforms supply metabolic substrates for carotenoid biosynthesis in tomato.

Geranylgeranyl diphosphate (GGPP) produced by GGPP synthase (GGPPS) serves as a precursor for many plastidial isoprenoids, including carotenoids. Phytoene synthase (PSY) converts GGPP into phytoene, the first committed intermediate of the carotenoid pathway. Here we used biochemical, molecular, and genetic tools to characterise the plastidial members of the GGPPS family in tomato (Solanum lycopersicum) and their interaction with PSY isoforms. The three tomato GGPPS isoforms found to localise in plastids (SlG1, 2 and 3) exhibit similar kinetic parameters. Gene expression analyses showed a preferential association of individual GGPPS and PSY isoforms when carotenoid biosynthesis was induced during root mycorrhization, seedling de-etiolation and fruit ripening. SlG2, but not SlG3, physically interacts with PSY proteins. By contrast, CRISPR-Cas9 mutants defective in SlG3 showed a stronger impact on carotenoid levels and derived metabolic, physiological and developmental phenotypes compared with those impaired in SlG2. Double mutants defective in both genes could not be rescued. Our work demonstrates that the bulk of GGPP production in tomato chloroplasts and chromoplasts relies on two cooperating GGPPS paralogues, unlike other plant species such as Arabidopsis thaliana, rice or pepper, which produce their essential plastidial isoprenoids using a single GGPPS isoform.

Phenotyping Tomato Root Developmental Plasticity in Response to Salinity in Soil Rhizotrons.

Plants have developed multiple strategies to respond to salt stress. In order to identify new traits related to salt tolerance, with potential breeding application, the research focus has recently been shifted to include root system architecture (RSA) and root plasticity. Using a simple but effective root phenotyping system containing soil (rhizotrons), RSA of several tomato cultivars and their response to salinity was investigated. We observed a high level of root plasticity of tomato seedlings under salt stress. The general root architecture was substantially modified in response to salt, especially with respect to position of the lateral roots in the soil. At the soil surface, where salt accumulates, lateral root emergence was most strongly inhibited. Within the set of tomato cultivars, H1015 was the most tolerant to salinity in both developmental stages studied. A significant correlation between several root traits and aboveground growth parameters was observed, highlighting a possible role for regulation of both ion content and root architecture in salt stress resilience.

The application of a biostimulant based on tannins affects root architecture and improves tolerance to salinity in tomato plants.

Roots have important roles for plants to withstand adverse environmental conditions, including salt stress. Biostimulant application was shown to enhance plant resilience towards abiotic stresses. Here, we studied the effect of a tannin-based biostimulant on tomato (Solanum lycopersicum L.) grown under salt stress conditions. We investigated the related changes at both root architecture (via imaging and biometric analysis) and gene expression (RNA-Seq/qPCR) levels. Moreover, in order to identify the main compounds potentially involved in the observed effects, the chemical composition of the biostimulant was evaluated by UV/Vis and HPLC-ESI-Orbitrap analysis. Sixteen compounds, known to be involved in root development and having a potential antioxidant properties were identified. Significant increase of root weight (+ 24%) and length (+ 23%) was observed when the plants were grown under salt stress and treated with the biostimulant. Moreover, transcriptome analysis revealed that the application of the biostimulant upregulated 285 genes, most of which correlated to root development and salt stress tolerance. The 171 downregulated genes were mainly involved in nutrient uptake. These data demonstrated that the biostimulant is able not only to restore root growth in salty soils, but also to provide the adequate plant nourishment by regulating the expression of essential transcription factors and stress responsive genes.

MYB5-like and bHLH influence flavonoid composition in pomegranate.

The fruit of the pomegranate (Punica granatum L.) is an important nutraceutical food rich in polyphenolic compounds, including hydrolysable tannins, anthocyanins and flavonols. Their composition varies according to cultivar, tissue and fruit development stage and is probably regulated by a combination of MYB and bHLH type transcription factors (TFs). In this study, metabolomics analysis during fruit developmental stages in the main pomegranate cultivars, Wonderful and Valenciana with contrasting colour of their ripe fruits, showed that flavonols were mostly present in flowers while catechins were highest in unripe fruits and anthocyanins in late fruit maturation stages. A novel MYB TF, PgMYB5-like, was identified, which differs from previously isolated pomegranate TFs by unique C-terminal protein motifs and lack of the amino-acid residues conserved among anthocyanins promoting MYBs. In both pomegranate cultivars the expression of PgMYB5-like was high at flowering stage, while it decreased during fruit ripening. A previously identified bHLH-type TF, PgbHLH, also showed high transcript levels at flowering stage in both cultivars, while it showed a decrease in expression during fruit ripening in cv. Valenciana, but not in cv. Wonderful. Functional analysis of both TFs was performed by agro-infiltration into Nicotiana benthamiana leaves. Plants infiltrated with the PgMYB5-like+PgbHLH combined construct showed a specific and significant accumulation of intermediates of the flavonoid pathway, especially dihydroflavonols, while anthocyanins were not produced. Thus, we propose a role for PgMYB5-like and PgbHLH in the first steps of flavonoid production in flowers and in unripe fruits. The expression patterns of these two TFs may be key in determining the differential flavonoid composition in both flowers and fruits of the pomegranate varieties Wonderful and Valenciana.

A Biostimulant Seed Treatment Improved Heat Stress Tolerance During Cucumber Seed Germination by Acting on the Antioxidant System and Glyoxylate Cycle.

Seed enhancement technologies have the potential to improve germination and seedling growth under environmental stress. The effects of KIEM®, an innovative biostimulant based on lignin derivatives and containing plant-derived amino acids and molybdenum, were investigated on cucumber (Cucumis sativus L.) seed germination. To determine the metabolic targets of this product, biometric, transcriptional and biochemical analyses were carried out on both non-treated and KIEM®-treated seeds incubated for 24 and 48 h under standard (28°C) and heat stress (35°C) conditions. The application of the biostimulant as a seed treatment increased the percent germination (+6.54%) and fresh biomass (+13%) at 48 h, and decreased the content of H2O2 in treated seeds at 28°C (-70%) and at 35°C (-80%). These changes in biometric and biochemical properties were accompanied by changes in expression levels of the genes coding for ROS-producing (RBOH) and scavenging (SOD, CAT, GST) enzymes and their specific activity. In general, the treatment with KIEM® in heat-stress condition appeared to stimulate a higher accumulation of three scavenger gene transcripts: CuZnSOD (+1.78), MnSOD (+1.75), and CAT (+3.39), while the FeSOD isoform was dramatically downregulated (0.24). Moreover, the amount of non-protein thiols, important antioxidant molecules, was increased by the biostimulant after 48 h (+20%). Taken together these results suggest that KIEM® acts through mitigation of the effects of the oxidative stress. Moreover, after 48 h, the pre-sowing treatment with KIEM® increased the transcription levels (+1.5) and the activity of isocitrate lyase (+37%), a key enzyme of the glyoxylate cycle, suggesting a potential effect of this product in speeding up the germination process. Finally, the chemical characterization of KIEM® identified five essential and three non-essential amino acids, and others bioactive compounds, including five organic and inorganic acids that might be potentially involved in its activity. Based on these data, insights on the potential mechanism of action of the biostimulant, suggested that there are broader applications as a product able to increase seed tolerance to different abiotic stress typical of adverse environmental conditions.

Transcription Factor-Mediated Control of Anthocyanin Biosynthesis in Vegetative Tissues.

Plants accumulate secondary metabolites to adapt to environmental conditions. These compounds, here exemplified by the purple-colored anthocyanins, are accumulated upon high temperatures, UV-light, drought, and nutrient deficiencies, and may contribute to tolerance to these stresses. Producing compounds is often part of a more broad response of the plant to changes in the environment. Here we investigate how a transcription-factor-mediated program for controlling anthocyanin biosynthesis also has effects on formation of specialized cell structures and changes in the plant root architecture. A systems biology approach was developed in tomato (Solanum lycopersicum) for coordinated induction of biosynthesis of anthocyanins, in a tissue- and development-independent manner. A transcription factor couple from Antirrhinum that is known to control anthocyanin biosynthesis was introduced in tomato under control of a dexamethasone-inducible promoter. By application of dexamethasone, anthocyanin formation was induced within 24 h in vegetative tissues and in undifferentiated cells. Profiles of metabolites and gene expression were analyzed in several tomato tissues. Changes in concentration of anthocyanins and other phenolic compounds were observed in all tested tissues, accompanied by induction of the biosynthetic pathways leading from Glc to anthocyanins. A number of pathways that are not known to be involved in anthocyanin biosynthesis were observed to be regulated. Anthocyanin-producing plants displayed profound physiological and architectural changes, depending on the tissue, including root branching, root epithelial cell morphology, seed germination, and leaf conductance. The inducible anthocyanin-production system reveals a range of phenomena that accompanies anthocyanin biosynthesis in tomato, including adaptions of the plants architecture and physiology.

Transcriptional control of fleshy fruit development and ripening.

Fleshy fruits have evolved to be attractive to frugivores in order to enhance seed dispersal, and have become an indispensable part of the human diet. Here we review the recent advances in the understanding of transcriptional regulation of fleshy fruit development and ripening with a focus on tomato. While aspects of fruit development are probably conserved throughout the angiosperms, including the model plant Arabidopsis thaliana, it is shown that the likely orthologues of Arabidopsis genes have distinct functions in fleshy fruits. The model for the study of fleshy fruit development is tomato, because of the availability of single gene mutants and transgenic knock-down lines. In other species, our knowledge is often incomplete or absent. Tomato fruit size and shape are co-determined by transcription factors acting during formation of the ovary. Other transcription factors play a role in fruit chloroplast formation, and upon ripening impact quality aspects such as secondary metabolite content. In tomato, the transcription factors NON-RIPENING (NOR), COLORLESS NON-RIPENING (CNR), and RIPENING INHIBITOR (MADS-RIN) in concert with ethylene signalling regulate ripening, possibly in response to a developmental switch. Additional components include TOMATO AGAMOUS-LIKE1 (TAGL1), APETALA2a (AP2a), and FRUITFULL (FUL1 and FUL2). The links between this highly connected regulatory network and downstream effectors modulating colour, texture, and flavour are still relatively poorly understood. Intertwined with this network is post-transcriptional regulation by fruit-expressed microRNAs targeting several of these transcription factors. This important developmental process is also governed by changes in DNA methylation levels and possibly chromatin remodelling.

Identification, cloning and characterization of the tomato TCP transcription factor family.

BACKGROUND: TCP proteins are plant-specific transcription factors, which are known to have a wide range of functions in different plant species such as in leaf development, flower symmetry, shoot branching, and senescence. Only a small number of TCP genes has been characterised from tomato (Solanum lycopersicum). Here we report several functional features of the members of the entire family present in the tomato genome. RESULTS: We have identified 30 Solanum lycopersicum SlTCP genes, most of which have not been described before. Phylogenetic analysis clearly distinguishes two homology classes of the SlTCP transcription factor family - class I and class II. Class II differentiates in two subclasses, the CIN-TCP subclass and the CYC/TB1 subclass, involved in leaf development and axillary shoots formation, respectively. The expression patterns of all members were determined by quantitative PCR. Several SlTCP genes, like SlTCP12, SlTCP15 and SlTCP18 are preferentially expressed in the tomato fruit, suggesting a role during fruit development or ripening. These genes are regulated by RIN (RIPENING INHIBITOR), CNR (COLORLESS NON-RIPENING) and SlAP2a (APETALA2a) proteins, which are transcription factors with key roles in ripening. With a yeast one-hybrid assay we demonstrated that RIN binds the promoter fragments of SlTCP12, SlTCP15 and SlTCP18, and that CNR binds the SlTCP18 promoter. This data strongly suggests that these class I SlTCP proteins are involved in ripening. Furthermore, we demonstrate that SlTCPs bind the promoter fragments of members of their own family, indicating that they regulate each other. Additional yeast one-hybrid studies performed with Arabidopsis transcription factors revealed binding of the promoter fragments by proteins involved in the ethylene signal transduction pathway, contributing to the idea that these SlTCP genes are involved in the ripening process. Yeast two-hybrid data shows that SlTCP proteins can form homo and heterodimers, suggesting that they act together in order to form functional protein complexes and together regulate developmental processes in tomato. CONCLUSIONS: The comprehensive analysis we performed, like phylogenetic analysis, expression studies, identification of the upstream regulators and the dimerization specificity of the tomato TCP transcription factor family provides the basis for functional studies to reveal the role of this family in tomato development.

Identification of microRNA targets in tomato fruit development using high-throughput sequencing and degradome analysis.

MicroRNAs (miRNAs) play important roles in plant development through regulation of gene expression by mRNA degradation or translational inhibition. Despite the fact that tomato (Solanum lycopersicum) is the model system for studying fleshy fruit development and ripening, only a few experimentally proven miRNA targets are known, and the role of miRNA action in these processes remains largely unknown. Here, by using parallel analysis of RNA ends (PARE) for global identification of miRNA targets and comparing four different stages of tomato fruit development, a total of 119 target genes of miRNAs were identified. Of these, 106 appeared to be new targets. A large part of the identified targets (56) coded for transcription factors. Auxin response factors, as well as two known ripening regulators, colorless non-ripening (CNR) and APETALA2a (SlAP2a), with developmentally regulated degradation patterns were identified. The levels of the intact messenger of both CNR and AP2a are actively modulated during ripening, by miR156/157 and miR172, respectively. Additionally, two TAS3-mRNA loci were identified as targets of miR390. Other targets such as Argonaute 1 (AGO1), shown to be involved in miRNA biogenesis in other plant species, were identified, which suggests a feedback loop regulation of this process. In this study, it is shown that miRNA-guided cleavage of mRNAs is likely to play an important role in tomato fruit development and ripening.

The tomato FRUITFULL homologs TDR4/FUL1 and MBP7/FUL2 regulate ethylene-independent aspects of fruit ripening.

Tomato (Solanum lycopersicum) contains two close homologs of the Arabidopsis thaliana MADS domain transcription factor FRUITFULL (FUL), FUL1 (previously called TDR4) and FUL2 (previously MBP7). Both proteins interact with the ripening regulator RIPENING INHIBITOR (RIN) and are expressed during fruit ripening. To elucidate their function in tomato, we characterized single and double FUL1 and FUL2 knockdown lines. Whereas the single lines only showed very mild alterations in fruit pigmentation, the double silenced lines exhibited an orange-ripe fruit phenotype due to highly reduced lycopene levels, suggesting that FUL1 and FUL2 have a redundant function in fruit ripening. More detailed analyses of the phenotype, transcriptome, and metabolome of the fruits silenced for both FUL1 and FUL2 suggest that the genes are involved in cell wall modification, the production of cuticle components and volatiles, and glutamic acid (Glu) accumulation. Glu is responsible for the characteristic umami taste of the present-day cultivated tomato fruit. In contrast with previously identified ripening regulators, FUL1 and FUL2 do not regulate ethylene biosynthesis but influence ripening in an ethylene-independent manner. Our data combined with those of others suggest that FUL1/2 and TOMATO AGAMOUS-LIKE1 regulate different subsets of the known RIN targets, probably in a protein complex with the latter.

Cell plate restricted association of DRP1A and PIN proteins is required for cell polarity establishment in Arabidopsis.

The polarized transport of the phytohormone auxin [1], which is crucial for the regulation of different stages of plant development [2, 3], depends on the asymmetric plasma membrane distribution of the PIN-FORMED (PIN) auxin efflux carriers [4, 5]. The PIN polar localization results from clathrin-mediated endocytosis (CME) from the plasma membrane and subsequent polar recycling [6]. The Arabidopsis genome encodes two groups of dynamin-related proteins (DRPs) that show homology to mammalian dynamin-a protein required for fission of endocytic vesicles during CME [7, 8]. Here we show by coimmunoprecipitation (coIP), bimolecular fluorescence complementation (BiFC), and Förster resonance energy transfer (FRET) that members of the DRP1 group closely associate with PIN proteins at the cell plate. Localization and phenotypic analysis of novel drp1 mutants revealed a requirement for DRP1 function in correct PIN distribution and in auxin-mediated development. We propose that rapid and specific internalization of PIN proteins mediated by the DRP1 proteins and the associated CME machinery from the cell plate membranes during cytokinesis is an important mechanism for proper polar PIN positioning in interphase cells.

Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening.

Fruit ripening in tomato (Solanum lycopersicum) requires the coordination of both developmental cues as well as the plant hormone ethylene. Although the role of ethylene in mediating climacteric ripening has been established, knowledge regarding the developmental regulators that modulate the involvement of ethylene in tomato fruit ripening is still lacking. Here, we show that the tomato APETALA2a (AP2a) transcription factor regulates fruit ripening via regulation of ethylene biosynthesis and signaling. RNA interference (RNAi)-mediated repression of AP2a resulted in alterations in fruit shape, orange ripe fruits, and altered carotenoid accumulation. Microarray expression analyses of the ripe AP2 RNAi fruits showed altered expression of genes involved in various metabolic pathways, such as the phenylpropanoid and carotenoid pathways, as well as in hormone synthesis and perception. Genes involved in chromoplast differentiation and other ripening-associated processes were also differentially expressed, but softening and ethylene biosynthesis occurred in the transgenic plants. Ripening regulators RIPENING-INHIBITOR, NON-RIPENING, and COLORLESS NON-RIPENING (CNR) function upstream of AP2a and positively regulate its expression. In the pericarp of AP2 RNAi fruits, mRNA levels of CNR were elevated, indicating that AP2a and CNR are part of a negative feedback loop in the regulation of ripening. Moreover, we demonstrated that CNR binds to the promoter of AP2a in vitro.

Proteomics insights into plant signaling and development.

Mass spectrometry-based proteomics is used to gain insight into the abundance and subcellular localization of cellular signaling components, the composition of molecular complexes and the regulation of signaling pathways. Multicellular organisms have evolved signaling networks and fast responses to stimuli that can be discovered and monitored by the use of advanced proteomics techniques in combination with traditional functional analysis. Plants are multicellular organisms and products of tightly regulated developmental programmes that respond to environmental conditions and internal cues. Plant development is orchestrated by inter- and intracellular signaling molecules, receptors and transcriptional regulators, which act in a temporal and spatially coordinated manner. Here we review recent advances in proteomics applications used to understand complex cellular signaling processes in plants.

Identification of in vitro phosphorylation sites in the Arabidopsis thaliana somatic embryogenesis receptor-like kinases.

The Arabidopsis thaliana somatic embryogenesis receptor-like kinase (SERK) family consists of five leucine-rich repeat receptor-like kinases (LRR-RLKs) with diverse functions such as brassinosteroid insensitive 1 (BRI1)-mediated brassinosteroid perception, development and innate immunity. The autophosphorylation activity of the kinase domains of the five SERK proteins was compared and the phosphorylated residues were identified by LC-MS/MS. Differences in autophosphorylation that ranged from high activity of SERK1, intermediate activities for SERK2 and SERK3 to low activity for SERK5 were noted. In the SERK1 kinase the C-terminally located residue Ser-562 controls full autophosphorylation activity. Activation loop phosphorylation, including that of residue Thr-462 previously shown to be required for SERK1 kinase activity, was not affected. In vivo SERK1 phosphorylation was induced by brassinosteroids. Immunoprecipitation of CFP-tagged SERK1 from plant extracts followed by MS/MS identified Ser-303, Thr-337, Thr-459, Thr-462, Thr-463, Thr-468, and Ser-612 or Thr-613 or Tyr-614 as in vivo phosphorylation sites of SERK1. Transphosphorylation of SERK1 by the kinase domain of the main brassinosteroid receptor BRI1 occurred only on Ser-299 and Thr-462. This suggests both intra- and intermolecular control of SERK1 kinase activity. Conversely, BRI1 was transphosphorylated by the kinase domain of SERK1 on Ser-887. BRI1 kinase activity was not required for interaction with the SERK1 receptor in a pull down assay.

Solubilization, activation, and insecticidal activity of Bacillus thuringiensis serovar thompsoni HD542 crystal proteins.

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40-kDa accompanying protein, is one of a small group of nontypical, less well-studied members of the Cry family of insecticidal proteins and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this paper, we describe the characterization of the Cry15Aa and 40-kDa protein's biochemical and insecticidal properties and the mode of action. Both proteins were solubilized above pH 10 in vitro. Incubation of solubilized crystal proteins with trypsin or insect midgut extracts rapidly processed the 40-kDa protein to fragments too small to be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the Cry15 protein yielded a stable product of approximately 30 kDa. Protein N-terminal sequencing showed that Cry15 processing occurs exclusively at the C-terminal end. Cry15 protein showed in vitro hemolytic activity, which was greatly enhanced by preincubation with trypsin or insect gut extract. Larvae of the lepidopteran insects Manduca sexta, Cydia pomonella, and Pieris rapae were susceptible to crystals, and presolubilization of the crystals enhanced activity to P. rapae. Activity for all three species was enhanced by preincubation with trypsin. Larvae of Helicoverpa armigera and Spodoptera exigua were relatively insensitive to crystals, and activity against these insects was not enhanced by prior solubilization or trypsin treatment. The 40-kDa crystal protein showed no activity in the insects tested, nor did its addition or coexpression in Escherichia coli increase the activity of Cry15 in insecticidal and hemolytic assays.

In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy.

The Arabidopsis (Arabidopsis thaliana) AAA ATPase CDC48A was fused to cerulean fluorescent protein and yellow fluorescent protein. AAA ATPases like CDC48 are only active in hexameric form. Förster resonance energy transfer-based fluorescence lifetime imaging microscopy using CDC48A-cerulean fluorescent protein and CDC48A-yellow fluorescent protein showed interaction between two adjacent protomers, demonstrating homo-oligomerization occurs in living plant cells. Interaction between CDC48A and the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) transmembrane receptor occurs in very restricted domains at the plasma membrane. In these domains the predominant form of the fluorescently tagged CDC48A protein is a hexamer, suggesting that SERK1 is associated with the active form of CDC48A in vivo. SERK1 trans-phosphorylates CDC48A on Ser-41. Förster resonance energy transfer-fluorescence lifetime imaging microscopy was used to show that in vivo the C-terminal domains of CDC48A stay in close proximity. Employing fluorescence correlation spectroscopy, it was shown that CDC48A hexamers are part of larger complexes.

Advances in understanding brassinosteroid signaling.

Brassinosteroids (BRs) function as signaling molecules in plants and are involved in processes such as stem elongation, vascular differentiation, male fertility, timing of senescence and flowering, leaf development, and resistance to biotic and abiotic stresses. Unlike animal steroids that are perceived by nuclear receptors, BRs are perceived by transmembrane receptor kinase complexes that initiate a phosphorylation-mediated signaling cascade to transduce the steroid signal. BR binding to the extracellular domain of the receptor BRI1 induces kinase activation and hetero-oligomerization with the second transmembrane kinase BAK1. Activated BRI1 then dissociates from the BRI1-interacting protein BKI1, a newly identified negative regulator of BR signaling. In the presence of BR, the kinase BIN2, which is the Arabidopsis homolog of GSK3 (glycogen synthase kinase 3), is inhibited by an unknown mechanism, leading to dephosphorylation of BES1 and BZR1 inside the nucleus. This allows BES1 and BZR1 to homodimerize or combine with other transcription factors to bind to promoters of BR-responsive genes. These studies of BR signaling in plants have revealed signaling pathways that are distinctly different from related ones operating in animal cells.