Strigolactones' ability to regulate root development may be executed by induction of the ethylene pathway

The newly defined phytohormones strigolactones (SLs) were recently shown to act as regulators of root development. Their positive effect on root-hair (RH) elongation enabled examination of their cross talk with auxin and ethylene. Analysis of wild-type plants and hormone-signaling mutants combined with hormonal treatments suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs. The SL and auxin hormonal pathways were suggested to converge for regulation of RH elongation; this convergence was suggested to be mediated via the ethylene pathway, and to include regulation of auxin transport.Key words: strigolactone, auxin, ethylene, root, root hair, lateral rootStrigolactones (SLs) are newly identified phytohormones that act as long-distance shoot-branching inhibitors (reviewed in ref. ¹). In Arabidopsis, SLs have been shown to be regulators of root development and architecture, by modulating primary root elongation and lateral root formation.^2,3 In addition, they were shown to have a positive effect on root-hair (RH) elongation.² All of these effects are mediated via the MAX2 F-box.^2,3In addition to SLs, two other plant hormones, auxin and ethylene, have been shown to affect root development, including lateral root formation and RH elongation.^4–6 Since all three phytohormones (SLs, auxin and ethylene) were shown to have a positive effect on RH elongation, we examined the epistatic relations between them by examining RH length.⁷ Our results led to the conclusion that SLs and ethylene are in the same pathway regulating RH elongation, where ethylene may be epistatic to SLs.⁷ Moreover, auxin signaling was shown to be needed to some extent for the RH response to SLs: the auxin-insensitive mutant tir1-1,⁸ was less sensitive to SLs than the wild type under low SL concentrations.⁷On the one hand, ethylene has been shown to induce the auxin response,^9–12 auxin synthesis in the root apex,^11,12 and acropetal and basipetal auxin transport in the root.^4,13 On the other, ethylene has been shown to be epistatic to SLs in the SL-induced RH-elongation response.⁷ Therefore, it might be that at least for RH elongation, SLs are in direct cross talk with ethylene, whereas the cross talk between SL and auxin pathways may converge through that of ethylene.⁷ The reduced response to SLs in tir1-1 may be derived from its reduced ethylene sensitivity;^7,14 this is in line with the notion of the ethylene pathway being a mediator in the cross talk between the SL and auxin pathways.The suggested ethylene-mediated convergence of auxin and SLs may be extended also to lateral root formation, and may involve regulation of auxin transport. In the root, SLs have been suggested to affect auxin efflux,^3,15 whereas ethylene has been shown to have a positive effect on auxin transport.^4,13 Hence, it might be that in the root, the SLs'' effect on auxin flux is mediated, at least in part, via the ethylene pathway. Ethylene''s ability to increase auxin transport in roots was associated with its negative effect on lateral root formation: ethylene was suggested to enhance polar IAA transport, leading to alterations in the quantity of auxin that unloads into the tissues to drive lateral root formation.⁴ Under conditions of sufficient phosphate, SL''s effect was similar to that of ethylene: SLs reduced the appearance of lateral roots; this was explained by their ability to change auxin flux.³ Taken together, one possibility is that the SLs'' ability to affect auxin flux and thereby lateral root formation in the roots is mediated by induction of ethylene synthesis.To conclude, root development may be regulated by a network of auxin, SL and ethylene cross talk.⁷ The possibility that similar networks exist elsewhere in the SLs'' regulation of plant development, including shoot architecture, cannot be excluded.     

Strigolactones as mediators of plant growth responses to environmental conditions

Strigolactones (SLs) have been recently identified as a new group of plant hormones or their derivatives thereof, shown to play a role in plant development. Evolutionary forces have driven the development of mechanisms in plants that allow adaptive adjustments to a variety of different habitats by employing plasticity in shoot and root growth and development. The ability of SLs to regulate both shoot and root development suggests a role in the plant''s response to its growth environment. To play this role, SL pathways need to be responsive to plant growth conditions, and affect plant growth toward increased adaptive adjustment. Here, the effects of SLs on shoot and root development are presented, and possible feedback loops between SLs and two environmental cues, light and nutrient status, are discussed; these might suggest a role for SLs in plants'' adaptive adjustment to growth conditions.Key words: strigolactones, light, nutrient status, root, shoot, branching, lateral roots, root hairsStrigolactones (SLs) are carotenoid-derived terpenoid lactones suggested to stem from the carotenoid pathway¹ via the activity of various oxygenases.^2,3 SLs production has been demonstrated in both monocotyledons and eudicotyledons (reviewed in ref. ⁴), suggesting their presence in many plant species.⁵ SLs are synthesized mainly in the roots and in some parts of the stem and then move towards the shoot apex (reviewed ref. ⁷).^6,8,9SLs were first characterized more than 40 years ago as germination stimulants of the parasitic plants Striga and Orobanche and later, as stimulants of arbuscular mycorrhiza hyphal branching as well (reviewed in ref. ^{4, 10–13}). Recently, SLs or derivatives thereof, have been identified as a new group of plant hormones, shown to play a role in inhibition of shoot branching,^2,3,8,9 thereby affecting shoot architecture; more recently they have also been shown to affect root growth by affecting auxin efflux.¹⁴Plants have developed mechanisms that allow adaptive adjustments to a variety of different habitats by employing plasticity in their growth and development.¹⁵ Shoot architecture is affected by environmental cues, such as light quality and quantity and nutrient status.^16–19 Root-system architecture and development are affected by environmental conditions such as nutrient availability (reviewed in ref. ^{20, 21}). At the same time, plant hormones are known to be involved in the regulation of plant growth, development and architecture (reviewed in ref. ^22–24) and to be mediators of the effects of environmental cues on plant development; one classic example is auxin''s role in the plant''s shade-avoidance response (reviewed in ref. ²⁵).The ability of SLs to regulate shoot and root development suggests that these phytohormones also have a role in the plant''s growth response to its environment. To play this putative role, SL pathways need to be responsive to plant growth conditions, and affect plant growth toward enhancing its adaptive adjustment. The present review examines the SLs'' possible role in adaptive adjustment of the plant''s response to growth conditions, by discussing their effect on plant development and the possible associations and feedback loops between SLs and two environmental cues: light and nutrient status.     

Auxin acts independently of DELLA proteins in regulating gibberellin levels

Shoot elongation is a vital process for plant development and productivity, in both ecological and economic contexts. Auxin and bioactive gibberellins (GAs), such as GA₁, play critical roles in the control of elongation,^1–3 along with environmental and endogenous factors, including other hormones such as the brassinosteroids.^4,5 The effect of auxins, such as indole-3-acetic acid (IAA), is at least in part mediated by its effect on GA metabolism,⁶ since auxin upregulates biosynthesis genes such as GA 3-oxidase and GA 20-oxidase and downregulates GA catabolism genes such as GA 2-oxidases, leading to elevated levels of bioactive GA₁.⁷ In our recent paper,¹ we have provided evidence that this action of IAA is largely independent of DELLA proteins, the negative regulators of GA action,^8,9 since the auxin effects are still present in the DELLA-deficient la cry-s genotype of pea. This was a crucial issue to resolve, since like auxin, the DELLAs also promote GA₁ synthesis and inhibit its deactivation. DELLAs are deactivated by GA, and thereby mediate a feedback system by which bioactive GA regulates its own level.¹⁰ However, our recent results,¹ in themselves, do not show the generality of the auxin-GA relationship across species and phylogenetic groups or across different tissue types and responses. Further, they do not touch on the ecological benefits of the auxin-GA interaction. These issues are discussed below as well as the need for the development of suitable experimental systems to allow this process to be examined.Key words: auxin, gibberellins, DELLA proteins, interactions, elongation     

Irrepressible,truncated Auxin Response Factors: Natural roles and applications in dissecting auxin gene regulation pathways

The molecularly well-characterized auxin signal transduction pathway involves two evolutionarily conserved families interacting through their C-terminal domains III and IV: the Auxin Response Factors (ARFs) and their repressors the Aux/IAAs, to control auxin-responsive genes, among them genes involved in auxin transport.^1,2 We have developed a new genetic tool to study ARF function. Using MONOPTEROS (MP)/ARF5, we have generated a truncated version of MP (MPΔ),³ which has lost the target domains for repression by Aux/IAA proteins. Besides exploring genetic interactions between MP and Aux/IAAs, we used this construct to trace MP’s role in vascular patterning, a previously characterized auxin dependent process.^4,5 Here we summarize examples of naturally occurring truncated ARFs and summarize potential applications of truncated ARFs as analytical tools.     

Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵     

Evaluating the function of putative hormone transporters

Hormones typically serve as long distance signaling molecules. To reach their site of action, hormones need to be transported from the sites of synthesis. Many plant hormones are mobile, thus requiring specific transport systems for the export from their source cells as well as subsequent import into target cells. Hormone transport in general is still poorly understood. Auxin is probably the most intensively studied plant hormone concerning transport in the moment. To advance our understanding of hormone transport we need two principal data sets: information on the properties of the transport systems including substrate specificity and kinetics, and we need to identify candidate genes for the respective transporters. Physiological transport data can provide an important basis for identifying and characterizing candidate transporters and to define their in vivo role. A recent publication in Plant Physiology highlights how kinetic and specificity studies may help to identify cytokinin transporters.¹Key words: kinetin, zeatin, adenine, phytohormone, transportBy definition, hormones are compounds that interact at low concentrations with cellular receptors to modulate signal transduction pathways. A comparison of the chemical structures of animal and plant hormones suggests potential common origins. Peptide hormones are found in both kingdoms and share common processing mechanisms (e.g., TRH, vasopressin and kinins in animals; systemins, phytosulfokines, self incompatibility peptides in plants).^2,3 Steroid hormones derived from cholesterol such as testosterone, cortisol and calcitriol regulate development in mammals; the steroid hormone brassinolide is essential for plant development.⁴ Glutamate can serve as metabolite and signal in both plants and animals.^5,6 Finally, lipid and phospholipid-derived signaling compounds such as linoleic acid and arachidonic acid also function in both plants and animals; with phospholipid-derived prostaglandins and eicosanoids bearing similarities to the plant defense compound jasmonic acid.⁷Other signaling compounds present in animals have yet to be shown to function in plants, e.g., glycoprotein hormones such as luteinizing hormone, follicle-stimulating hormone or thyroid-stimulating hormone have been not been described to exist in plants.⁸ Compounds structurally similar to animal amine-derived hormones derived from tyrosine and tryptophan (such as catecholamines and thyroxine) are also present in plants, but appear to function primarily in herbivore defense.⁹The best characterized, and arguably most important plant hormones, bear little similarity to animal hormones and are mechanistically distinct. These include auxins, cytokinins, gibberellins, abscisic acid, ethylene and an apparent carotenoid-derivative, the MAX-dependent regulator of auxin signaling.^10,11 Arguably, the stress response compound salicylic acid, which functions in stress, wounding and defense responses could also be considered a plant hormone.¹²Hormonal signaling mechanisms can be categorized as autocrine (acting at the site of biosynthesis), paracrine (acting in adjacent or proximal cells), and endocrine (acting in cells distal to the site of production). In both, plants and animals, paracrine and endocrine hormone action is mediated and influenced by multiple long distance delivery systems. Hormones move primarily through the circulatory system in animals, but, in plants, are mobilized by transpiration and source-sink flows, which can be directed by chemisomotically-driven cellular uptake and efflux. However, the mechanisms driving uptake and efflux at the cellular level, as well as the proteins that mediate this movement, are surprisingly similar in plants and animals, despite the dissimilarities of plant and animal cell structure (central vacuoles, cell walls and H⁺ versus K⁺/Na²⁺ in/out gradients).Surprisingly little is known about plant hormone transport. Most hormones have autocrine activity, but in order to act at a distance or to even act on adjacent cells they must be transported across membranes. The existence of cellular export and import mechanisms are suggested by the presence of multiple hormones in the phloem sap^13,14 and the well documented polar long distance movement of auxin.¹⁵ Brassinosteroid receptors have been demonstrated as integral plasma membrane proteins which receive the hormone signal from outside the cell.¹⁶ This suggests a need for the hormone to first move into the apoplasm after biosynthesis. However, until recently, only the cellular auxin transport mechanisms mediated by the AUX/LAX, PIN and AtABCB/PGP proteins has been well characterized (reviewed in ref. ¹⁷).The study of these transporters has benefited from the use of plant, yeast and animal expression systems to characterize the proteins involved. Analyses of auxin transport proteins have capitalized on earlier suppression cloning and radiotracer uptake studies used successfully to characterize ion and metabolite transporters in yeast.^18–21 In cases where yeast systems have proven intractable for analysis of auxin transport proteins, heterologous systems based on mammalian cell systems have proven to be highly effective for radiotracer uptake studies.^18–23 Xenopus oocyte expression has been successfully utilized to characterize the AUX/LAX family of auxin influx symporters.^24,25 Plant cell culture systems have also been used to characterize transport proteins. This can however be problematic when endogenous substrates are metabolized by the cells, as is the case with IAA in tobacco BY-2 and Arabidopsis cell cultures.¹⁹ It is also difficult to assess the function of plant proteins in undifferentiated cell cultures, which may differ from the native function in phloem or xylem parenchyma cells.A recent article describes the use of a heterologous expression system based on the fission yeast S. pombe to express and characterize the PIN1 auxin efflux protein after knock-out of the endogenous yeast PIN-like gene AEL1.²¹ Previously, PIN1 had only been functionally expressed in plant cell systems and was nonfunctional when expressed in baker''s yeast or mammalian cells.^19,22 This report suggests that PIN1, interacts synergistically with the AtABCB19/PGP19 auxin efflux transporter, but appears to also mediate auxin efflux on its own, consistent with the distant phylogenetic similarity of the auxin efflux transporter protein family to major facilitator proteins.Subsequent work in the Murphy lab has shown that S. pombe can be used for comparisons of all known auxin transporters in a single system in which all ABC transporters and a solitary AUX1-like gene had been knocked out (Yang and Murphy, unpublished). This system also allows for the more detailed analyses of substrate specificity, transport kinetics and coupling mechanisms (primary and secondary active transport, uniport, cotransport antiport) necessary for functional assignment of auxin transport proteins. This system may also provide an attractive alternative to baker''s yeast when functional expression of a plant protein in Saccharomyces cerevisiae proves unsuccessful.Similar efforts are required for characterizing the transport of all other plant hormones including cytokinin. Arabidopsis transporters mediating both trans-zeatin and adenine uptake had been identified using yeast as an expression system.²⁶ Recently, the Schulz and Frommer labs provided a reference data set for trans-zeatin uptake by characterizing radiolabeled trans-zeatin uptake in Arabidopsis cell cultures.¹ The data show that the uptake kinetics of trans-zeatin are multiphasic, indicating the presence of both low- and high-affinity transport systems. The protonophore CCCP is an effective inhibitor of cytokinin uptake, consistent with H⁺-mediated uptake. Other physiologically active cytokinins such as isopentenyladenine and benzylaminopurine are effective competitors of trans-zeatin uptake, whereas allantoin had no inhibitory effect. Adenine competes for zeatin uptake indicating that degradation products of cytokinin oxidases can be transported by the same systems. Comparison of adenine and trans-zeatin uptake in Arabidopsis seedlings reveals similar uptake kinetics. Kinetic properties as well as substrate specificity determined in cell cultures are compatible with the hypothesis that members of the plant-specific PUP transporter family may play a role in adenine transport to scavenge extracellular adenine. In addition, the findings are also compatible with the hypothesis that this class of transporters may be involved at least in low affinity (µM range) cytokinin uptake. PUPs are encoded by a large gene family of 21 members, so it is conceivable that other members of the family may be involved in high affinity transport. Systematic analyses of single knock outs in Arabidopsis and combinations thereof my help to shed more light on the role of PUPs in cytokinin transport.     

Redox regulation of auxin signaling and plant development in Arabidopsis

Thioredoxin (NTR/TRX) and glutathione (GSH/GRX) are the two major systems that play a key role in the maintenance of cellular redox homeostasis. They are essential for plant development, cell division or the response to environmental stresses. In a recent article,¹ we studied the interplay between the NADP-linked thioredoxin and glutathione systems in auxin signaling genetically, by associating TRX reductase (ntra ntrb) and glutathione biosynthesis (cad2) mutations. We show that these two thiol reduction pathways interfere with developmental processes. This occurs through modulation of auxin activity as shown by genetic analyses of loss of function mutations in a triple ntra ntrb cad2 mutant. The triple mutant develops almost normally at the rosette stage but fails to generate lateral organs from the inflorescence meristem, producing almost naked stems that are reminiscent of mutants affected in PAT (polar auxin transport) or biosynthesis. The triple mutant exhibits other defects in processes regulated by auxin, including a loss of apical dominance, vasculature defects and reduced secondary root production. Furthermore, it has lower auxin (IAA) levels and decreased capacity for PAT, suggesting that the NTR and glutathione pathways influence inflorescence meristem development through regulation of auxin transport and metabolism.Key words: arabidopsis, NTS pathway, NGS pathway, thioredoxin (TRX), glutaredoxine (GRX), polar auxin transport (PAT), auxin biosynthesis, pin-like phenotype, apical dominance, meristematic activityExposure of living organisms to environmental stresses triggers various defense and developmental responses. Redox signaling is involved in many aspects of these responses.^2–6 The key players in these responses are the NADPH-dependent glutathione/glutaredoxin system (NGS) and the NADPH-dependent thioredoxin system (NTS). TRX and GRX play key roles in the maintenance of cellular redox homeostasis.^7–10 Genetic approaches aiming to identify functions of TRX and GRX in knock-out plants have largely been limited by the absence of phenotypes of single mutants, presumably due to functional redundancies among members of the multigene families of TRX and GRX.¹¹ Interplay between NTS and NGS pathways have been studied in different organisms^12–17 and association of mutants involved in these two pathways have recently revealed new functions in several aspects of plant development.^4–6     

Strigolactones: Internal and external signals in plant symbioses?

As the newest plant hormone, strigolactone research is undergoing an exciting expansion. In less than five years, roles for strigolactones have been defined in shoot branching, secondary growth, root growth and nodulation, to add to the growing understanding of their role in arbuscular mycorrhizae and parasitic weed interactions.¹ Strigolactones are particularly fascinating as signaling molecules as they can act both inside the plant as an endogenous hormone and in the soil as a rhizosphere signal.^2-4 Our recent research has highlighted such a dual role for strigolactones, potentially acting as both an endogenous and exogenous signal for arbuscular mycorrhizal development.⁵ There is also significant interest in examining strigolactones as putative regulators of responses to environmental stimuli, especially the response to nutrient availability, given the strong regulation of strigolactone production by nitrate and phosphate observed in many species.^5,6 In particular, the potential for strigolactones to mediate the ecologically important response of mycorrhizal colonization to phosphate has been widely discussed. However, using a mutant approach we found that strigolactones are not essential for phosphate regulation of mycorrhizal colonization or nodulation.⁵ This is consistent with the relatively mild impairment of phosphate control of seedling root growth observed in Arabidopsis strigolactone mutants.⁷ This contrasts with the major role for strigolactones in phosphate control of shoot branching of rice and Arabidopsis^8,9 and indicates that the integration of strigolactones into our understanding of nutrient response will be complex. New data presented here, along with the recent discovery of phosphate specific CLE peptides,¹⁰ indicates a potential role for PsNARK, a component of the autoregulation of nodulation pathway, in phosphate control of nodulation.     

Staying in the fold: The SGT1/chaperone machinery in maintenance and evolution of leucine-rich repeat proteins

The conserved eukaryotic protein SGT1 (suppressor of G₂ allele of skp1) participates in diverse physiological processes such as cell cycle progression in yeast, plant immunity against pathogens and plant hormone signalling. Recent genetic and biochemical studies suggest that SGT1 functions as a novel co-chaperone for cytosolic/nuclear HSP90 and HSP70 molecular chaperones in the folding and maturation of substrate proteins. Since proteins containing the leucine-rich repeat (LRR) protein-protein interaction motif are overrepresented in SGT1-dependent phenomena, we consider whether LRR-containing proteins are preferential substrates of an SGT1/HSP70/HSP90 complex. Such a chaperone organisation is reminiscent of the HOP/HSP70/HSP90 machinery which controls maturation and activation of glucocorticoid receptors in animals. Drawing on this parallel, we discuss the possible contribution of an SGT1-chaperone complex in the folding and maturation of LRR-containing proteins and its evolutionary consequences for the emergence of novel LRR interaction surfaces.Key words: heat shock protein, SGT1, co-chaperone, HSP90, HSP70, leucine-rich repeat, LRR, resistance, SCF, ubiquitinThe proper folding and maturation of proteins is essential for cell viability during de novo protein synthesis, translocation, complex assembly or under denaturing stress conditions. A complex machinery composed of molecular chaperones (heat-shock proteins, HSPs) and their modulators known as co-chaperones, catalyzes these protein folding events.^1,2 In animals, defects in the chaperone machinery is implicated in an increasing number of diseases such as cancers, susceptibility to viruses, neurodegenerative disease and cystic fibrosis, and thus it has become a major pharmacological target.^3,4 In plants, molecular genetic studies have identified chaperones and co-chaperones as components of various physiological responses and are now starting to yield important information on how chaperones work. Notably, processes in plant innate immunity rely on the HSP70 and HSP90^5–7 chaperones as well as two recently characterised co-chaperones, RAR1 (required for Mla12 resistance) and SGT1 (suppressor of G₂ allele of skp1).^8–11SGT1 is a highly conserved and essential co-chaperone in eukaryotes and is organized into three structural domains: a tetratricopeptide repeat (TPR), a CHORD/SGT1 (CS) and an SGT1-specific (SGS) domain (Fig. 1A). SGT1 is involved in a number of apparently unrelated physiological responses ranging from cell cycle progression and adenylyl cyclase activity in yeast to plant immunity against pathogens, heat shock tolerance and plant hormone (auxin and jasmonic acid) signalling.^7–9,12,13 Because the SGT1 TPR domain is able to interact with Skp1, SGT1 was initially believed to be a component of SCF (Skp1/Cullin/F-box) E3 ubiquitin ligases that are important for auxin/JA signalling in plants and cell cycle progression in yeast.^13,14 However, mutagenesis of SGT1 revealed that the TPR domain is dispensable for plant immunity and auxin signalling.¹⁵ Also, SGT1-Skp1 interaction was not observed in Arabidopsis.¹³ More relevant to SGT1 functions appear to be the CS and SGS domains.¹⁶ The former is necessary and sufficient for RAR1 and HSP90 binding. The latter is the most conserved of all SGT1 domains and the site of numerous disabling mutations.^14,16,17Open in a separate windowFigure 1Model for SGT1/chaperone complex functions in the folding of LRR-containing proteins. (A) The structural domains of SGT1, their sites of action (above) and respective binding partners (below) are shown. N- and C-termini are indicated. TPR, tetratricopeptide repeat; CS, CHORD/SGT1; SGS, SGT1-specific. (B) Conceptual analogy between steroid receptor folding by the HOP/chaperone machinery and LRR protein folding by the SGT1/chaperone machinery. LRR motifs are overrepresented in processes requiring SGT1 such as plant immune receptor signalling, yeast adenylyl cyclase activity and plant or yeast SCF (Skp1/Cullin/F-box) E3 ubiquitin ligase activities. (C) Opposite forces drive LRR evolution. Structure of LRRs 16 to 18 of the F-box auxin receptor TIR1 is displayed as an illustration of the LRR folds.³⁰ Leucine/isoleucine residues (side chain displayed in yellow) are under strong purifying selection and build the hydrophobic LRR backbone (Left). By contrast, solvent-exposed residues of the β-strands define a polymorphic and hydrophilic binding surface conferring substrate specificity to the LRR (Right) and are often under diversifying selection.We recently demonstrated that Arabidopsis SGT1 interacts stably through its SGS domain with cytosolic/nuclear HSP70 chaperones.⁷ The SGS domain was both necessary and sufficient for HSP70 binding and mutations affecting SGT1-HSP70 interaction compromised JA/auxin signalling and immune responses. An independent in vitro study also found interaction between human SGT1 and HSP70.¹⁸ The finding that SGT1 protein interacts directly with two chaperones (HSP90/70) and one co-chaperone (RAR1) reinforces the notion that SGT1 behaves as a co-chaperone, nucleating a larger chaperone complex that is essential for eukaryotic physiology. A future challenge will be to dissect the chaperone network at the molecular and subcellular levels. In plant cells, SGT1 localization appears to be highly dynamic with conditional nuclear localization⁷ and its association with HSP90 was recently shown to be modulated in vitro by RAR1.¹⁶A co-chaperone function suits SGT1 diverse physiological roles better than a specific contribution to SCF ubiquitin E3 ligases. Because SGT1 does not affect HSP90 ATPase activity, SGT1 was proposed rather as a scaffold protein.^16,19 In the light of our findings and earlier studies,²⁰ SGT1 is reminiscent of HOP (Hsp70/Hsp90 organizing protein) which links HSP90 and HSP70 activities and mediates optimal substrate channelling between the two chaperones (Fig. 1B).²¹ While the contribution of the HSP70/HOP/HSP90 to the maturation of glucocorticoid receptors is well established,²¹ direct substrates of an HSP70/SGT1/HSP90 complex remain elusive.It is interesting that SGT1 appears to share a functional link with leucine-rich repeat- (LRR) containing proteins although LRR domains are not so widespread in eukaryotes. For example, plant SGT1 affects the activities of the SCF^TIR1 and SCF^COI1 E3 ligase complexes whose F-box proteins contain LRRs.¹³ Moreover, plant intracellular immune receptors comprise a large group of LRR proteins that recruit SGT1.^8,9 LRRs are also found in yeast adenylyl cyclase Cyr1p and the F-box protein Grr1p which is required for SGT1-dependent cyclin destruction during G₁/S transition.^12,14 Yeast 2-hybrid interaction assays also revealed that yeast and plant SGT1 tend to associate directly or indirectly with LRR proteins.^12,22,23 We speculate that SGT1 bridges the HSP90-HSC70 chaperone machinery with LRR proteins during complex maturation and/or activation. The only other structural motif linked to SGT1 are WD40 domains found in yeast Cdc4p F-box protein and SGT1 interactors identified in yeast two-hybrid screens.¹²What mechanisms underlie a preferential SGT1-LRR interaction? HSP70/SGT1/HSP90 may have co-evolved to assist specifically in folding and maturation of LRR proteins. Alternatively, LRR structures may have an intrinsically greater need for chaperoning activity to fold compared to other motifs. These two scenarios are not mutually exclusive. The LRR domain contains multiple 20 to 29 amino acid repeats, forming an α/β horseshoe fold.²⁴ Each repeat is rich in hydrophobic leucine/isoleucine residues which are buried inside the structure and form the structural backbone of the motif (Fig. 1C, left). Such residues are under strong purifying selection to preserve structure. These hydrophobic residues would render the LRR a possible HSP70 substrate.²⁵ By contrast, hydrophilic solvent- exposed residues of the β strands build a surface which confers ligand recognition specificity of the LRRs (Fig. 1C). In many plant immune receptors for instance, these residues are under diversifying selection that is likely to favour the emergence of novel pathogen recognition specificities in response to pathogen evolution.²⁶ The LRR domain of such a protein has to survive such antagonist selection forces and yet remain functional. Under strong selection pressure, LRR proteins might need to accommodate less stable LRRs because their recognition specificities are advantageous. This could be the point at which LRRs benefit most from a chaperoning machinery such as the HSP90/SGT1/HSP70 complex. This picture is reminiscent of the genetic buffering that HSP90 exerts on many traits to mask mutations that would normally be deleterious to protein folding and/or function, as revealed in Drosophila and Arabidopsis.²⁷ It will be interesting to test whether the HSP90/SGT1/HSP70 complex acts as a buffer for genetic variation, favouring the emergence of novel LRR recognition surfaces in, for example, highly co-evolved plant-pathogen interactions.^28,29     

The Twisted Dwarf's ABC: How Immunophilins Regulate Auxin Transport

There is increasing evidence that immunophilins function as key regulators of plant development. One of the best investigated members, the multi-domain FKBP TWISTED DWARF1 (TWD1)/FKBP42, has been shown to reside on both the vacuolar and plasma membranes where it interacts in mirror image with two pairs of ABC transporters, MRP1/ MRP2 and PGP1/PGP19(MDR1), respectively. Twisted dwarf1 and pgp1/pgp19 mutants display strongly overlapping phenotypes, including reduction and disorientation of growth, suggesting functional interaction.In a recent work using plant and heterologous expression systems, TWD1 has been demonstrated to modulate PGP-mediated export of the plant hormone auxin, which controls virtually all plant developmental processes. Here we summarize recent molecular models on TWD1 function in plant development and PGP-mediated auxin tranport and discuss open questions.Key Words: Twisted Dwarf1, plant development, auxin, immunophilin, P-glycoprotein, ABC transporterFK506-binding Proteins (FKBPs), together with unrelated cyclophilins, belong to the immunophilins, an ancient and ubiquitous protein family.^1,4,5 They were first described as receptors for immunosuppressive drugs in animal and human cells, FK506 and cyclosporin A, respectively.¹ All FKBP-type immunophilins share a characteristic peptidyl-prolyl cis-trans isomerase domain (PPIase domain or FKBD, Fig. 2A) making protein folding a key feature among immunophilins.² The best investigated example, the human cytosolic single-domain FKBP12, modulates Ca²⁺ release channels^6,7 and associates with the cell cycle regulator TGF-β.⁸ Furthermore, the human FKBP12/FK506 complex is known to bind and inhibit calcineurin activity,⁹ leading to immune response inhibition. However, not all single- and multiple-domain FKBPs own folding activity and, interestingly, many form distinct protein complexes with diverse functions.^3–5Open in a separate windowFigure 2Model of TWISTED DWARF 1 interacting proteins. (A) Domain structure of TWD1 and putative interacting proteins. FKBD, FK506-binding domain: TPR, tetratricopeptide repeat; CaM(-BD, calmodulin-binding domain; MA, membrane anchor. For details, see text. (B) Functional TWD1-ABC transporter complexes on both the vacuolar and plasma membrane. While for TWD1/PGP pairs, the positive regulatory role on auxin transport was demonstrated,¹⁸ the modulation of MRP-mediated vacuolar import of glutathion conjugates (GS-X) was established using mammalian test substrates¹⁷ because the in vivo substrates are unknown. Note that C-terminal nucleotide binding folds of MRP- and PGP-like ABC transporters interact with distinct functional domains of TWD1, the TPR and FKBD, respectively. The native auxin, IAAH, gets trapped by deprotonization upon uptake into the cell. Export is catalyzed by secondary active export via PIN-like efflux carriers¹⁵ and/or by primary active, ATP-driven P-glycoproteins (PGPs, right panel); loss-of TWD1 function abolishes PGP-mediated auxin export (left panel).     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴