Potassium flux in the pollen tubes was essential in plant sexual reproduction

Potassium channels are controlling K⁺ transport across plasma membrane and thus playing a central role in all aspects of osmolarity as well as numerous other functions in plants, including in sexual reproduction. We have used whole-cell and single-channel patch-clamp recording techniques investigated the regulation of intracellular free Ca²⁺-activated outward K⁺ channels in Pyrus pyrifolia pollen tube protoplasts. We have also showed the channels could be inhibited by heme and activated carbon monoxide (CO). In the presence of oxygen and NADPH, hemoxygenases catalyzes heme degradation, producing biliverdin, iron and CO. Considered the oxygen concentration approaching zero in the ovary, the heme will inhibit the K⁺ outward flux from the intracellular of pollen tube, increasing the pollen tubes osmolarity, inducing pollen tube burst. Here we discuss the putative role of K⁺ channels in plant sexual reproduction.Key words: pear, pollen, K⁺ channels, heme, carbon monoxideIon channels in the pollen tube play critical roles in mediation pollen germination and pollen tube growth.^1–3 Early studies were focus on the plasma membrane calcium channel regulation and cytosolic free calcium concentration variation in the pollen tube reason by which was one of the most important second messengers in plants.^3–7 However, reports have also showed that the potassium channels in the pollen tubes were also involved in several important steps of plant sexual reproduction.^8–19 Recently, more reports further demonstrated this phenomena.^20–24 In the report by Lu et al. they demonstrated that two cation/proton exchangers (CHX), CHX21 and CHX23, are essential for pollen tube growth guidance in Arabidopsis.²² chx21 chx23 double mutant induces the fertility impaired, but which is unchanged in both single chx21 or chx23 mutants. They have also found that the double mutant pollen grains germination and pollen tube growth in the transmitting tract were not difference with the wild-type, however, the double mutant pollen tubes fail to turn toward ovules.²² Protein localization experiments show CHX23 is expressed in the endoplasmic reticulum of pollen tubes; functional analysis results showed that CHX23 as a K⁺ transporter mediates K⁺ uptake in a pH-dependent manner. So, these protein affect the signal transduction pathway of pollen tube growth toward to the ovule by controlling the cation balance and pH in the pollen tube.²² Amien et al. identified a signaling ligand of defensin-like (DEFL) protein, ZmES4, which expressed in maize synergid. ZmES4 activates the maize pollen tube tip plasma membrane K⁺ Shaker channel KZM1.²⁰ This finding is also very interesting. Pollen tube bursting suggested to be based on the osmotic stress; the influx of K⁺ mediated by ZmES4-activated KZM1 will trigger rapid plasma membrane depolarization, which induced the pollen tube tip burst.²⁰ Furthermore, the osmotic increasing induced by too much K⁺ in the cytosolic of pollen tube was not only resulted by inward K⁺ channel activation, but also resulted by outward K⁺ channel inhibition in the pollen tube plasma membrane. In our report, we find a intracellular Ca²⁺-sensitive outward K⁺ channel in pear pollen tube plasma membrane, which could be inhibited by heme and activated by heme oxidative production, carbon monoxide (CO), may play a functional role in the pollen tube brusting.²³In the presence of oxygen and NADPH, hemoxygenases catalyzes heme degradation, producing biliverdin, iron and CO.²⁵ Early reports showed that oxygen plays an important role in plant sexual reproduction. Pollen tubes grow through the style toward the ovary with high speed, a process that consumes tremendous amounts of energy and requires rapid oxygen uptake by pollen tubes.²⁶ Pollen grains have roughly 20 times the level of mitochondria and respire 10 times faster than vegetative tissue.^12,27–29 Furthermore, oxygen has been proposed as a possible cue for pollen-tube guidance.³⁰ Indeed, the existence of an oxygen gradient in the unpollinated style has been shown in some species such as Hipeastrum hybridum. Oxygen pressure is high in the stigma and style but suddenly decreases at the base of the style, approaching zero in the ovary. Moreover, pollen-tube growth itself creates hypoxic regions within the style.³¹ Therefore, we suggest that the outward K⁺ channel inhibited by heme is dominant compared with which activated by CO when pollen tubes reach the ovary, based on where the hypoxic condition (Fig. 1). However, the gene encode the outward K⁺ channel in the pear pollen tube remains to be determined in the further study.Open in a separate windowFigure 1Reciprocal regulation of heme and carbon monoxide in putative Ca²⁺-activated outward K⁺ channel. Under normal condition, in the presence of NADPH, heme is metabolized by hemeoxygenase to generate carbon monoxide (CO), which activates outward K⁺ channel. However, without the oxygen, heme cannot be metabolized. The accumulated heme acts as an inhibitor of outward K⁺ channel, even in the presence of NADPH. The accumulated K⁺ in the cytosolic of pollen will induced the pollen tube depolarized, then burst.     

Why have chloroplasts developed a unique motility system?

Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.^1,2 Actin inhibitors effectively retard the movements of mitochondria,^3–6 peroxisomes,^5,7–11 Golgi stacks,^12,13 endoplasmic reticulum (ER),^14,15 and nuclei.^16–18 These organelles are co-aligned and associated with actin filaments.^{5,7,8,10–12,15,18} Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.¹⁹Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.^20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).²² The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.^23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,²⁶ whereas phot2 alone is essential for the avoidance response.^27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.^29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.²⁹ CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.^29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,^22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.     

Prions: En route from structural models to structures

The prion hypothesis^1–3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.^4,5 Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.^6,7 Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and—most probably—a partially ordered prion domain.⁶ For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.Key words: prion, NMR, solid-state NMR, MAS, structure, Ure2p, HET-sDespite the large interest in the basic mechanisms of fibril formation and prion propagation, little is known about the molecular structure of prions at atomic resolution and the mechanism of propagation. Prions with related properties to the ones responsible for mammalian diseases were also discovered in yeast and funghi^8,9 which provide convenient model system for their studies. Prion proteins described include the mammalian prion protein PrP, Ure2p,¹⁰ Rnq1p,¹¹ Sup35,¹² Swi1,¹³ and Cyc8,¹⁴ from bakers yeast (S. cervisiae) and HET-s from the filamentous fungus P. anserina. The soluble non-prion form of the proteins characterized in vitro is a globular protein with an unfolded, dynamically disordered N- or C-terminal tail.^15–18 In the prion form, the proteins form fibrillar aggregates, in which the tail adopts a different conformation and is thought to be the dominant structural element for fibril formation.Fibrills are difficult to structurally characterize at atomic resolution, as X-ray diffraction and liquid-state NMR cannot be applied because of the non-crystallinity and the mass of the fibrils. Solid-state NMR, in contrast, is nowadays well suited for this purpose. The size of the monomer, between 230 and 685 amino-acid residues for the prions of Figure 1, and therefore the number of resonances in the spectrum—that used to be large for structure determination—is now becoming tractable by this method.Open in a separate windowFigure 1Prions identified today and characterized as consisting of a prion domain (blue) and a globular domain (red).Prion proteins characterized so far were found to be usually constituted of two domains, namely the prion domain and the globular domain (see Fig. 1). This architecture suggests a divide-and-conquer approach to structure determination, in which the globular and prion domain are investigated separately. In isolation, the latter, or fragments thereof, were found to form β-sheet rich structures (e.g., Ure2p(1-89),^6,19 Rnq1p(153-405)²⁰ and HET-s(218-289)²¹). The same conclusion was reached by investigating Sup35(1-254).²² All these fragements have been characterized as amyloids, which we define in the sense that a significant part of the protein is involved in a cross-beta motif.²³ An atomic resolution structure however is available presently only for the HET-s prion domain, and was obtained from solid-state NMR²⁴ (vide infra). It contains mainly β-sheets, which form a triangular hydrophobic core. While this cross-beta structure can be classified as an amyloid, its triangular shape does deviate significantly from amyloid-like structures of smaller peptides.²³Regarding the globular domains, structures have been determined by x-ray crystallography (Ure2p^25,26 and HET-s²⁷), as well as NMR (mammal prions^15,28–30). All reveal a protein fold rich in α-helices, and dimeric structures for the Ure2 and HET-s proteins. The Ure2p fold resembles that of the β-class glutathione S-transferases (GST), but lacks GST activity.²⁵It is a central question for the structural biology of prions if the divide-and-conquer approach imposed by limitations in current structural approaches is valid. Or in other words: can the assembly of full-length prions simply be derived from the sum of the two folds observed for the isolated domains?     

Mannan synthase activity in the CSLD family

Cellulose Synthase Like (CSL) proteins are a group of plant glycosyltransferases that are predicted to synthesize β-1,4-linked polysaccharide backbones. CSLC, CSLF and CSLH families have been confirmed to synthesize xyloglucan and mixed linkage β-glucan, while CSLA family proteins have been shown to synthesize mannans. The polysaccharide products of the five remaining CSL families have not been determined. Five CSLD genes have been identified in Arabidopsis thaliana and a role in cell wall biosynthesis has been demonstrated by reverse genetics. We have extended past research by producing a series of double and triple Arabidopsis mutants and gathered evidence that CSLD2, CSLD3 and CSLD5 are involved in mannan synthesis and that their products are necessary for the transition between early developmental stages in Arabidopsis. Moreover, our data revealed a complex interaction between the three glycosyltransferases and brought new evidence regarding the formation of non-cellulosic polysaccharides through multimeric complexes.Key words: mannan, mannose, plant cell wall, glycosyltransferase, cellulose synthase like, CSL, biosynthesis, hemicelluloseThe plant cell wall is mainly composed of polysaccharides, which are often grouped into cellulose, hemicelluloses and pectin. Since the discovery of the first cellulose synthase (CESA) genes in cotton fibers,¹ the synthesis of cellulose has been extensively studied.² In contrast, the glycosyltransferases responsible for synthesizing hemicelluloses and pectin are still largely unidentified.^3,4,5 The CESA genes are members of a superfamily that includes genes with a high sequence similarity with CESA genes and are named Cellulose Synthase Like (CSL).⁶ The CSL genes have themselves been grouped into nine families designated CSLA, -B, -C, -D, -E, -F, -G, -H and -J (Figure 1A).^5,6 Mannan and glucomannan synthase activity has been demonstrated in the CSLA family,^7,8,9 while members of the CSLC family have been implicated in synthesis of the xyloglucan backbone.¹⁰ CSLF and CSLH, which are found only in grasses, are involved in synthesis of mixed linkage glucan.^11,12 The function of the remaining CSL families has not been determined. We have reported our research on the CSLD family in a recent publication.¹³ Of all the CSL families, CSLD possesses the most ancient intron/exon structure and is the most similar to the CESA family.⁶ CSLD genes are found in all sequenced genomes of terrestrial plants including Physcomitrella and Selaginella suggesting a highly conserved function throughout the plant kingdom (Figure 1A). Five genes (CSLD1 to CSLD5) and one apparent pseudogene (CSLD6) have been identified in Arabidopsis thaliana.¹⁴ Bernal et al.^14,15 studied knock-out mutants of the individual genes and presented evidence for a role in cell wall biosynthesis for each Arabidopsis CSLD. To elucidate the activity of the CSLD proteins and obtain further understanding of their biological role, we generated double mutants csld2/csld3, csld2/csld5, csld3/csld5 and the triple mutant csld2/csld3/csld5. Immunochemical, biochemical and complementation assays brought evidence that CSLD5 or CSLD2 in concomitance with CSLD3 act as mannan synthases.Open in a separate windowFigure 1(A) Schematic representation of the CESA superfamily phylogeny. The inset on the right is a detailed phylogenetic tree of CSLDs from Selaginella moellendorffii, Arabidopsis thaliana and Oryza sativa. The figure is modified from Ulvskov and Scheller.⁵ (B) Comparison of csld2, csld3, csld5 with Col-0 20 days after germination. The inflorescences of csld2 and csld3 were similar to Col-0 whereas csld5 had a delayed growth. Scale bar: 1 cm. (C) Col-0 and csld2/csld3/csld5 (triple mutant, TM) 40 days after germination. After 40 days, the triple mutant was barely developed and, as shown in the magnified inset, displayed purple coloration indicating accumulation of anthocyanins, a typical stress response. Scale bar: 2 mm.     

Leaf carbohydrate metabolism during defense: Intracellular sucrose-cleaving enzymes do not compensate repression of cell wall invertase

The significance of cell wall invertase (cwINV) for plant defense was investigated by comparing wild type (wt) tobacco Nicotiana tabacum L. Samsun NN (SNN) with plants with RNA interference-mediated repression of cwINV (SNN::cwINV) during the interaction with the oomycetic phytopathogen Phytophthora nicotianae. We have previously shown that the transgenic plants developed normally under standard growth conditions, but exhibited weaker defense reactions in infected source leaves and were less tolerant to the pathogen. Here, we show that repression of cwINV was not accompanied by any compensatory activities of intracellular sucrose-cleaving enzymes such as vacuolar and alkaline/neutral invertases or sucrose synthase (SUSY), neither in uninfected controls nor during infection. In wt source leaves vacuolar invertase did not respond to infection, and the activity of alkaline/neutral invertases increased only slightly. SUSY however, was distinctly stimulated, in parallel to enhanced cwINV. In SNN::cwINV SUSY-activation was largely repressed upon infection. SUSY may serve to allocate sucrose into callose deposition and other carbohydrate-consuming defense reactions. Its activity, however, seems to be directly affected by cwINV and the related reflux of carbohydrates from the apoplast into the mesophyll cells.Key words: cell wall invertase, apoplastic invertase, alkaline invertase, neutral invertase, sucrose synthase, plant defense, Nicotiana tabacum, Phytophthora nicotianaePlant defense against pathogens is costly in terms of energy and carbohydrates.^1,2 Sucrose (Suc) and its cleavage products glucose and fructose are central molecules for metabolism and sensing in higher plants (reviewed in refs. ³ and ⁴). Rapid mobilization of these carbohydrates seems to be an important factor determining the outcome of plant-pathogen interactions. In particular in source cells reprogramming of the carbon flow from Suc to hexoses may be a crucial process during defense.^1,2There are two alternative routes of sucrolytic carbohydrate mobilization. One route is reversible and involves an uridine 5′-diphosphate (UDP)-dependent cleavage catalyzed by sucrose synthase (SUSY). Its activity is limited by the concentrations of Suc and UDP in the cytosol, as the affinity of the enzyme to its substrate is relatively low (K_m for Suc 40–200 mM). The other route is the irreversible, hydrolytic cleavage by invertases (INVs), which exhibit high affinity to Suc (K_m 7–15 mM).⁵Plants possess three different types of INV isoenzymes, which can be distinguished by their solubility, subcellular localization, pH-optima and isoelectric point. Usually, they are subdivided into cell wall (cwINV), vacuolar (vacINV), and alkaline/neutral (a/nINVs) INVs.cwINV, also referred to as extracellular or apoplastic INV, is characterized by a low pH-optimum (pH 3.5–5.0) and usually ionically bound to the cell wall. It is the key enzyme of the apoplastic phloem unloading pathway and plays a crucial role in the regulation of source/sink relations (reviewed in refs. ^{3, 6–8}). A specific role during plant defense has been suggested, based on observations that cwINV is often induced during various plant-pathogen interactions, and the finding that overexpression of a yeast INV in the apoplast increases plant resistance.^6,8–10 It was shown, that a rapid induction of cwINV is, indeed, one of the early defense-related reactions in resistant tobacco source leaves after infection with Phytophthora nicotianae (P. nicotianae).¹¹ Finally, the whole infection area in wt leaves was covered with hypersensitive lesions, indicating that all cells had undergone hypersensitive cell death (Fig. 1A).^1,11 When the activity of cwINV was repressed by an RNAi construct, defense-related processes were impaired, and the infection site exhibited only small spots of hypersensitive lesions. Finally, the pathogen was able to sporulate, indicating a reduced resistance of these transgenic plants (Fig. 1A).¹Open in a separate windowFigure 1Defense-induced changes in the activity of intracellular sucrose-cleaving enzymes and their contribution to defense. (A) The repression of cwINV in source leaves of tobacco leads to impaired pathogen resistance and can not be compensated by other sucrose-cleaving enzymes. The intensity of defense reactions is amongst others indicated by the extent of hypersensitive lesions. (B and C) Absolute activity of vacuolar (B) and alkaline/neutral (C) INVs at the infection site (white symbols, control; black symbols, infection site). (D) Increase in SUSY activity at the infection site. All data points taken from noninfected control parts of the plants in each individual experiment and each point along the time scale of an experiment are set as 0%. At least three independent infections are averaged and their means are presented as percentage changes ± SE (circles, SNN; triangles, SNN::cwINV). Insets show the means of the absolute amount of activities (white symbols, control; black symbols, infection site). Material and methods according to Essmann, et al.¹vacINV, also labeled as soluble acidic INV, is characterized by a pH optimum between pH 5.0–5.5. Among others it determines the level of Suc stored in the vacuole and generates hexose-based sugar signals (reviewed in refs. ³ and ¹²). Yet, no specific role of vacINV during pathogen response has been reported. Although vacINV and cwINV are glycoproteins with similar enzymatic and biochemical properties and share a high degree of overall sequence homology and two conserved amino acid motifs,⁴ the activity of vacINV in tobacco source leaves was not changed due to the repression of the cwINV (Fig. 1B).¹ After infection with P. nicotianae the activity of vacINV in wt SNN did not respond under conditions where cwINV was stimulated.¹ There was also no significant change in the transgenic SNN::cwINV (Fig. 1B). This suggests that during biotic stress, there is no crosstalk between the regulation of cwINV and vacINV.a/nINVs exhibit activity maxima between pH 6.5 and 8.0, are not glycosylated and thought to be exclusively localized in the cytosol. But recent reports also point to a subcellular location in mitochondria and chloroplasts.^13,14 Only a few a/nINVs have been cloned and characterized, and not much is known about their physiological functions (reviewed in refs. ^{4, 14} and ¹⁵). Among other things they seem to be involved in osmotic or low-temperature stress response.^14,15 During the interaction between tobacco and P. nicotianae the activity of a/nINVs rose on average 17% in the resistant wt SNN between 1 to 9 hours post infection (Fig. 1C). By contrast, in SNN::cwINV the a/nINVs activities remained unchanged in control leaves and even after infection (Fig. 1C). This suggests that the defense related stimulation in a/nINVs activities is rather a secondary phenomenon, possibly in response to the enhanced cwINV activity and the related carbohydrate availability in the cytosol.SUSY can be found as a soluble enzyme in the cytosol, bound to the inner side of the plasma membrane or the outer membrane of mitochondria, depending on the phosphorylation status. It channels hexoses into polysaccharide biosynthesis (i.e., starch, cellulose and callose) and respiration.^12,16 There is also evidence that SUSY improves the metabolic performance at low internal oxygen levels¹⁷ but little is known about its role during plant defense. Callose formation is presumably one of the strongest sink reactions in plant cells.^1,18 Defense-related SUSY activity may serve to allocate Suc into callose deposition and other carbohydrate-consuming defense reactions. In fact, in the resistant wt the activity of SUSY increased upon interaction with P. nicotianae in a biphasic manner (Fig. 1D). The time course is comparable to that of cwINV activity and correlates with callose deposition and enhanced respiration.^1,11 However, repression of cwINV leads in general to a reduction of SUSY activity in source leaves of tobacco.¹ After infection the activation of SUSY was also significantly impaired (Fig. 1D). At the same time, the early defense-related callose deposition in infected mesophyll cells of SNN::cwINV plants is substantially delayed.¹ It is known that expression of SUSY isoforms is differentially controlled by sugars,¹² and there is evidence that hexoses generated by the defense-induced cwINV activity deliver sugar signals to the infected cells.¹ In this sense, the reduction of defense-related, cwINV-generated sugar signals could be responsible for the repression of SUSY activity in SNN::cwINV plants after infection with P. nicotianae.Only limited hexoses or hexose-based sugar signals could be generated by cytoplasmic Suc cleavage.¹² The reduction of soluble carbohydrates for sugar signaling and also as fuel for metabolic pathways that support defense reactions could be responsible for the impaired resistance in SNN::cwINV plants (Fig. 1A).Obviously, neither intracellular INV isoforms, nor SUSY can compensate for the reduced carbohydrate availability due to cwINV repression during plant defense. The data also suggest that the activity of SUSY is affected by cwINV and related reflux of carbohydrates. It is known that SUSY activity can be controlled, e.g., by sugar-mediated phosphorylation¹² and one may speculate that posttranslational modulation of the protein is affected by the defense-related carbohydrate status of the cell.     

Flowering time regulation by the SUMO E3 ligase SIZ1

Flowering is a developmental process, which is influenced by chemical and environmental stimuli. Recently, our research established that the Arabidopsis SUMO E3 ligase, AtSIZ1, is a negative regulator of transition to flowering through mechanisms that reduce salicylic acid (SA) accumulation and involve SUMO modification of FLOWERING LOCUS D (FLD). FLD is an autonomous pathway determinant that represses the expression of FLOWERING LOCUS C (FLC), a floral repressor. This addendum postulates mechanisms by which SIZ1-mediated SUMO conjugation regulates SA accumulation and FLD activity.Key words: SIZ1, SA, flowering, SUMO, FLD, FLCSUMO conjugation and deconjugation are post-translational processes implicated in plant defense against pathogens, abscisic acid (ABA) and phosphate (Pi) starvation signaling, development, and drought and temperature stress tolerance, albeit only a few of the modified proteins have been identified.^1–8 The Arabidopsis AtSIZ1 locus encodes a SUMO E3 ligase that regulates floral transition and leaf development.^8,9 siz1 plants accumulate substantial levels of SA, which is the primary cause for dwarfism and early short-day flowering exhibited by these plants.^1,9 How SA promotes transition to flowering is not yet known but apparently, it is through a mechanism that is independent of the known floral signaling pathways.^9,10 Exogenous SA reduces expression of AGAMOUS-like 15 (AGL15), a floral repressor that functions redundantly with AGL18.^11,12 A possible mechanism by which SA promotes transition to flowering may be by repressing expression of AGL15 and AGL18 (Fig. 1).Open in a separate windowFigure 1Model of how SUMO conjugation and deconjugation regulate plant development in Arabidopsis. SIZ1 and Avr proteins regulate biosynthesis and accumulation of SA, a plant stress hormone that is involved in plant innate immunity, leaf development and regulation of flowering time. SA promotes transition to flowering may through AGL15/AGL18 dependent and independent pathways. FLC expression is activated by FRIGIDA but repressed by the autonomous pathway gene FLD, and SIZ1-mediated sumoylation of FLD represses its activity. Lines with arrows indicate upregulation (activation), and those with bars identify downregulation (repression).siz1 mutations also cause constitutive induction of pathogenesis-related protein genes leading to enhanced resistance against biotrophic pathogens.¹ Several bacterial type III effector proteins, such as YopJ, XopD and AvrXv4, have SUMO isopeptidase activity.^13–15 PopP2, a member of YopJ/AvrRxv bacterial type III effector protein family, physically interacts with the TIR-NBS-LRR type R protein RRS1, and possibly stabilizes the RRS1 protein.¹⁶ Phytopathogen effector and plant R protein interactions lead to increased SA biosynthesis and accumulation, which in turn activates expression of pathogenesis-related proteins that facilitate plant defense.¹⁷ SIZ1 may participate in SUMO conjugation of plant R proteins to regulate Avr and R protein interactions leading to SA accumulation, which, in turn, affects phenotypes such as diseases resistance, dwarfism and flowering time (Fig. 1).Our recent work revealed also that AtSIZ1 facilitates FLC expression, negatively regulating flowering.⁹ AtSIZ1 promotes FLC expression by repressing FLD activity.⁹ Site-specific mutations that prevent SUMO1/2 conjugation to FLD result in enhanced activity of the protein to represses FLC expression, which is associated with reduced acetylation of histone 4 (H4) in FLC chromatin.⁹ FLD, an Arabidopsis ortholog of Lysine-Specific Demethylase 1 (LSD1), is a floral activator that downregulates methylation of H3K4 in FLC chromatin and represses FLC expression.^18,19 Interestingly, bacteria expressing recombinant FLD protein did not demethylate H3K4me2, inferring that the demethylase activity requires additional co-factors as are necessary for LSD1.^18,20 Together, these results suggest that SIZ1-mediated SUMO modification of FLD may affect interactions between FLD and co-factors, which is necessary for FLC chromatin modification.Despite our results that implicate SA in flowering time control, how SIZ1 regulates SA accumulation and the identity of the effectors involved remain to be discovered. In addition, it remains to be determined if SIZ1 is involved in other mechanisms that modulate FLD activity and FLC expression, or the function of other autonomous pathway determinants.     

MEK1/2 and p38-like MAP kinase successively mediate H2O2 signaling in Vicia guard cell

As a second messenger, H₂O₂ generation and signal transduction is subtly controlled and involves various signal elements, among which are the members of MAP kinase family. The increasing evidences indicate that both MEK1/2 and p38-like MAP protein kinase mediate ABA-induced H₂O₂ signaling in plant cells. Here we analyze the mechanisms of similarity and difference between MEK1/2 and p38-like MAP protein kinase in mediating ABA-induced H₂O₂ generation, inhibition of inward K⁺ currents, and stomatal closure. These data suggest that activation of MEK1/2 is prior to p38-like protein kinase in Vicia guard cells.Key words: H₂O₂ signaling, ABA, p38-like MAP kinase, MEK1/2, guard cellAn increasing number of literatures elucidate that reactive oxygen species (ROS), especially H₂O₂, is essential to plant growth and development in response to stresses,^1–4 and involves activation of various signaling events, among which are the MAP kinase cascades.^1–3,5 Typically, activation of MEK1/2 mediates NADPH oxidase-dependent ROS generation in response to stresses,^4,6–8 and the facts that MEK1/2 inhibits the expression and activation of antioxidant enzymes reveal how PD98059, the specific inhibitor of MEK1/2, abolishes abscisic acid (ABA)-induced H₂O₂ generation.^6,8,9 It has been indicated that PD98059 does not to intervene on salicylic acid (SA)-stimulated H₂O₂ signaling regardless of SA mimicking ABA in regulating stomatal closure.^2,6,8,10 Generally, activation of MEK1/2 promotes ABA-induced stomatal closure by elevating H₂O₂ generation in conjunction with inactivating anti-oxidases.Moreover, activation of plant p38-like protein kinase, the putative counterpart of yeast or mammalian p38 MAP kinase, has been reported to participate in various stress responses and ROS signaling. It has been well documented that p38 MAP kinase is involved in stress-triggered ROS signaling in yeast or mammalian cells.^11–13 Similar to those of yeast and mammals, many studies showed the activation of p38-like protein kinase in response to stresses in various plants, including Arabidopsis thaliana,^14–16 Pisum sativum,¹⁷ Medicago sativa¹⁸ and tobacco.¹⁹ The specific p38 kinase inhibitor SB203580 was found to modulate physiological processes in plant tissues or cells, such as wheat root cells,²⁰ tobacco tissue²¹ and suspension-cultured Oryza sativa cells.²² Recently, we investigate how activation of p38-like MAP kinase is involved in ABA-induced H₂O₂ signaling in guard cells. Our results show that SB203580 blocks ABA-induced stomatal closure by inhibiting ABA-induced H₂O₂ generation and decreasing K⁺ influx across the plasma membrane of Vicia guard cells, contrasting greatly with its analog SB202474, which has no effect on these events.^23,24 This suggests that ABA integrate activation of p38-like MAP kinase and H₂O₂ signaling to regulate stomatal behavior. In conjunction with SB203580 mimicking PD98059 not to mediate SA-induced H₂O₂ signaling,^23,24 these results generally reveal that the activation of p38-like MAP kinase and MEK1/2 is similar in guard cells.On the other hand, activation of p38-like MAP kinase^23,24 is not always identical to that of MEK1/2^8,25 in ABA-induced H₂O₂ signaling of Vicia guard cells. For example, H₂O₂^- and ABA-induced stomatal closure was partially reversed by SB203580. The maximum inhibition of both regent-induced stomatal closure were observed at 2 h after treatment with SB203580, under which conditions the stomatal apertures were 89% and 70% of the control values, respectively. By contrast, when PD98059 was applied together with ABA or H₂O₂, the effects of both ABA- and H₂O₂-induced stomatal closure were completely abolished (Fig. 1). These data imply that the two members of MAP kinase family are efficient in H₂O₂-stimulated stomatal closure, but p38-like MAP kinase is less susceptive than MEK1/2 to ABA stimuli.Open in a separate windowFigure 1Effects of SB203580 and PD98059 on ABA- and H₂O₂-induced stomatal closure. The experimental procedure and data analysis are according to the previous publication.^8,23,24It has been reported that ABA or NaCl activate p38 MAP kinase in the chloronema cells of the moss Funaria hygrometrica in 2∼10 min.²⁶ Similar to this, SB203580 improves H₂O₂-inhibited inward K⁺ currents after 4 min and leads it to the control level (100%) during the following 8 min (Fig. 2). However, the activation of p38-like MAP kinase in response to ABA need more time, and only recovered to 75% of the control at 8 min of treatment (Fig. 2). These results suggest that control of H₂O₂ signaling is required for the various protein kinases including p38-like MAP kinase and MEK1/2 in guard cells,^1,2,8,23,24 and the ABA and H₂O₂ pathways diverge further downstream in their actions on the K⁺ channels and, thus, on stomatal control. Other differences in action between ABA and H₂O₂ are known. For example, Köhler et al. (2001) reported that H₂O₂ inhibited the K⁺ outward rectifier in guard cells shows that H₂O₂ does not mimic ABA action on guard cell ion channels as it acts on the K⁺ outward rectifier in a manner entirely contrary to that of ABA.²⁷Open in a separate windowFigure 2Effect of SB203580 on ABA- and H₂O₂-inhibited inward K⁺ currents. The experimental procedure and data analysis are according to the previous publication.²⁴ SB203580 directs ABA- and H₂O₂-inactivated inward K⁺ currents across plasma membrane of Vicia guard cells. Here the inward K⁺ currents value is stimulated by −190 mV voltage.Based on the similarity and difference between PD98059 and SB203580 in interceding ABA and H₂O₂ signaling, we speculate the possible mechanism is that the member of MAP kinase family specially regulate signal event in ABA-triggered ROS signaling network,^1–4 and the signaling model as follows (Fig. 3).Open in a separate windowFigure 3Schematic illustration of MAP kinase-mediated H₂O₂ signaling of guard cells. The arrows indicate activation. The line indicates enhancement and the bar denotes inhibition.     

Nitric oxide and nitrite are likely mediators of pollen interactions

The ability of plants to produce nitric oxide (NO) is now well recognised. In plants, NO is involved in the control of organ development and in regulating some of their physiological functions. We have recently shown that pollen generates NO in a constitutive manner and have measured both intra- and extracellular production of this radical. Furthermore, we have shown that nitrite accumulates in the media surrounding the pollen and have suggested that the generation of these signaling molecules may be important for the normal interaction between the pollen grain and the stigma on which it alights. However, pollen grains inevitably come into contact with other tissues, including those of animals and it is likely that the NO produced will influence the behavior of the cells associated with these tissues. Such non-animal-derived, NO-mediated effects on mammalian cells may not be restricted to pollen and plant debris and fungal spore-derived NO may elicit similar effects.Key words: allergy, fungal spores, nitric oxide, nitrite, pollenNitric oxide (NO) has been recognised as a signaling molecule for 20+ years, but its roles in controlling cellular activity are far from fully understood. In plants, NO is involved in numerous biological processes¹ including seed germination,² floral development,³ the control of stomatal closure⁴ and root gravitropism⁵ and is also known to affect gene expression.⁶ Recently, we showed that pollen of Arabidopsis, Senecio and Tradescantia produces NO,⁷ and speculated that its role in this specific context is to help orchestrate early signaling events of the pollen-stigma interaction.^7,8 We subsequently showed that NO generation by pollen is more widespread among angiosperms and not just restricted to the species that were first investigated.⁹ Obviously, this intracellular generation of NO could influence the internal biochemistry of the pollen grain and pollen tube. However, for it to impact on other tissues, such as the stigma, on which the pollen grains alight during pollination, the NO generated would have to be released into the extracellular matrix.To demonstrate that pollen grains do indeed release NO to their surroundings we employed a water soluble derivative of the fluorescent NO probe, diaminofluorescein (DAF), to show that the 525 nm emission of the surrounding solution increased with time and that this fluorescence could be removed by scavenging the NO released from the pollen with compounds such as 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Thus, it is quite conceivable that, in vivo, NO produced by pollen moves into the extracellular matrix where it exerts an influence on the activity of cells in the adjacent tissues. Interestingly, in vitro rehydration of the pollen (analagous to the regulated hydration of pollen on the stigma) was needed before NO evolution could be measured. Normally, some form of specific stimulation, such as that which occurs either during pathogenesis¹⁰ or which results from the increased hormone levels observed during stomatal closure,¹¹ is required to initiate NO production by plant tissues. Thus, it is interesting here, that water appears to be the signaling cue to initiate constitutive NO release by the pollen.As a result of its free radical nature, NO is notoriously difficult to measure. As the chemistry involved in their reactivity has become better understood, doubts have been raised concerning the specificity of many of the fluorescent probes that have been used for its detection.¹² Commonly the fluorescent NO probe, DAF, is used, but similar alternative probes such as diamino-rhodamine (DAR) have recently also been described.¹³ Here, Figure 1 shows the NO-dependent fluorescence of DAR4M-AM-infused Brassica napus pollen and the associated temporal increase in the fluorescence of the extracellular medium containing a cell impermeable form of the dye. Despite the use of these different dye-based probes, it has still proved important to use other approaches to detect pollen NO production to refute the possibility that similarly reactive free radicals other than NO are responsible for the increased fluorescence observed. We have, therefore, confirmed our fluorescence measurements using electron paramagnetic resonance (EPR) techniques⁹ which have also indicated the presence of NO. Thus, the use of both fluorescent probe and EPR approaches point to the generation and release of NO from the pollen of all the plant species studied.Open in a separate windowFigure 1The diamino-rhodamine dyes, DAR4M-AM (cell permeable) and DAR4M (cell impermeable), can be used to detect intra- and extracellular pollen-derived nitric oxide (NO) respectively. Aqueous suspensions of Brassica napus sp. pollen were incubated for 15 min at room temp in 10 µM DAR4M-AM either without (A) or with (B) 200 µM of the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). In each case, after removal of the excess dye and resuspension of the pollen in 10% (v/v) glycerol, the accumulated DAR4M-AM fluorescence signals within the pollen grains were detected by spinning disc, laser scanning, confocal microscopy with excitation at 560 nm and emission detection at 575 nm. The extracellular accumulation with time of the NO-associated fluorescence signal of the dye, DAR4M, in the media was also followed spectrophotometrically (C). Using the same excitation and emission detection wavelengths, the fluorescence of aqueous suspensions of the pollen in 10 µM DAR4M either without (Ci) or with (Cii) 200 “M cPTIO was monitored over a 10 min. period at room temp. The output fluorescence signal with time is presented in relative units.An additional NO detection technique based on ozone chemiluminescence was also used to confirm the data obtained.⁹ Unlike the fluorescence and EPR approaches which measure the accumulated production of NO, this method detects the steady-state levels of NO at any given time. However, as these levels proved to be very low and not readily detectable by this approach, we altered the assay conditions so as to measure the nitrite that accumulated as a result of NO oxidation in the extracellular media. While the nitrite that accumulated in the media could have done so as a result of being directly excreted by the pollen, the results obtained were in accordance with the earlier observations that pollen evolves NO.⁹ Neither should nitrite be dismissed as a mere downstream by-product. Not only is it the substrate for the production of NO by enzymes such as nitrate reductase,¹⁴ it can also act as a cell signaling molecule in its own right¹⁵ effecting increased cGMP production, increases in different cytochrome P450 activities and the induction of specific gene expression.Having established that pollen produces NO and nitrite, the mechanisms underlying their generation and subsequent signaling require determination. In mammalian cells the production of NO by a family of nitric oxide synthase enzymes is well understood.¹⁶ However, attempts to find plant homologues have so far proved unsuccessful, with the sole proposed candidate¹⁷ having now been shown to be a G protein.^18–20 Nitrate reductase is clearly one source of NO in plants,^11,14 but whether other enzymes exist which are similarly involved remains a matter for debate and discovery. Obviously, as plant NO synthesising enzymes are identified their function in the generation of NO and nitrite in pollen will need to be established.Originally,⁷ we suggested that pollen-derived NO is integral to the pollen-stigma interaction and this now needs to be determined. Nevertheless, the NO and nitrite released externally by pollen may also affect the cells of any moist tissues on which pollen grains land. Such cells may include, for example, those lining mammalian nasal passages. It is well established that NO helps orchestrate the activity of cells involved in human immune responses¹⁶ and this begs the question as to whether or not pollen-produced NO alters these responses during, for example, the onset of the symptoms of hayfever? Many plant cells produce NO, particular during stress and after wounding²¹ and damaged plant tissues that come into contact with human cells in environments that create such debris also have the potential to elicit similar responses. The reaction of mammalian cells to fungi, which are known to possess NOS enzymes²² and whose spores are a main contributor to asthma,²³ may also be similarly mediated.To conclude, pollen grains appear to generate both NO and nitrite constitutively. Determining the functional significance and ramifications of this production in terms of both endogenous and exogenous cell signaling is an important focus for future research.     

Reactive oxygen species as transducers of sphinganine-mediated cell death pathway

Long chain bases or sphingoid bases are building blocks of complex sphingolipids that display a signaling role in programmed cell death in plants. So far, the type of programmed cell death in which these signaling lipids have been demonstrated to participate is the cell death that occurs in plant immunity, known as the hypersensitive response. The few links that have been described in this pathway are: MPK6 activation, increased calcium concentrations and reactive oxygen species (ROS) generation. The latter constitute one of the more elusive loops because of the chemical nature of ROS, the multiple possible cell sites where they can be formed and the ways in which they influence cell structure and function.Key words: hydrogen peroxide, long chain bases, programmed cell death, reactive oxygen species, sphinganine, sphingoid bases, superoxideA new transduction pathway that leads to programmed cell death (PCD) in plants has started to be unveiled.^1,2 Sphingoid bases or long chain bases (LCBs) are the distinctive elements in this PCD route that naturally operates in the entrance site of a pathogen as a way to contend its spread in the plant tissues.^2,3 This defense strategy has been known as the hypersensitive response (HR).^4,5As a lately discovered PCD signaling circuit, three connected transducers have been clearly identified in Arabidopsis: the LCB sphinganine (also named dihydrosphingosine or d18:0); MPK6, a mitogen activated kinase and superoxide and hydrogen peroxide as reactive oxygen species (ROS).^1,2 In addition, calcium transients have been recently allocated downstream of exogenously added sphinganine in tobacco cells.⁶Contrary to the signaling lipids derived from complex glycerolipid degradation, sphinganine, a metabolic precursor of complex sphingolipids, is raised by de novo synthesis in the endoplasmic reticulum to mediate PCD.^1,2 Our recent work demonstrated that only MPK6 and not MPK3 (commonly functionally redundant kinases) acts in this pathway and is positioned downstream of sphinganine elevation.² Although ROS have been identified downstream of LCBs in the route towards PCD,¹ the molecular system responsible for this ROS generation, their cellular site of formation and their precise role in the pathway have not been unequivocally identified. ROS are produced in practically all cell compartments as a result of energy transfer reactions, leaks from the electron transport chains, and oxidase and peroxidase catalysis.⁷Similar to what is observed in pathogen defense,³ increases in endogenous LCBs may be elicited by addition of fumonisin B1 (FB1) as well; FB1 is a mycotoxin that inhibits ceramide synthase. This inhibition results in an accumulation of its substrate, sphinganine and its modified forms, leading to the activation of PCD.^1,2,8 The application of FB1 is a commonly used approach for the study of PCD elicitation in Arabidopsis.^1,2,9–11An early production of ROS has been linked to an increase of LCBs. For example, an H₂O₂ burst is found in tobacco cells after 2–20 min of sphinganine supplementation,¹² and superoxide radical augmented in the medium 60 min after FB1 or sphinganine addition to Arabidopsis protoplasts (Fig. 1A). In consonance with this timing, both superoxide and H₂O₂ were detected in Arabidopsis leaves after 3–6 h exposure to FB1 or LCBs.¹ However, the source of ROS generation associated with sphinganine elevation seems to not be the same in both species: in tobacco cells, ROS formation is apparently dependent on a NADPH oxidase activity, a ROS source consistently implicated in the HR,^13,14 while in Arabidopsis, superoxide formation was unaffected by diphenyliodonium (DPI), a NADPH oxidase inhibitor (Fig. 1A). It is possible that the latter oxidative burst is due to an apoplastic peroxidase,¹⁵ or to intracellular ROS that diffuse outwards.^16,17 These results also suggest that both tobacco and Arabidopsis cells could produce ROS from different sources.Open in a separate windowFigure 1ROS are produced at early and long times in the FB1-induced PCD in Arabidopsis thaliana (Col-0). (A) Superoxide formation by Arabidopsis protoplasts is NADPH oxidase-independent and occurs 60 min after FB1 or sphinganine (d18:0) exposure. Protoplasts were obtained from a cell culture treated with cell wall lytic enzymes. Protoplasts were incubated with 10 µM FB1 or 10 µM sphinganine for 1 h. Then, cells were vacuum-filtered and the filtrate was used to determine XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt] reduction as described in references ²⁸ and ²⁹. DPI was used at 50 µM. (B) H₂O₂ formation in Arabidopsis wt and lcb2a-1 mutant in the presence and absence of FB1. Arabidopsis seedlings were exposed to 10 µM FB1 and after 48 h seedlings were treated with DA B (3,3-diaminobencidine) to detect H₂O₂ according to Thordal-Christensen et al.³⁰It has been suggested that the H₂O₂ burst associated with the sphinganine signaling pathway leads to the expression of defense-related genes but not to the PCD itself in tobacco cells.¹² It is possible that ROS are involved in the same way in Arabidopsis, since defense gene expression is also induced by FB1 in Arabidopsis.⁹ In this case, it will be important to define how the early ROS that are DPI-insensitive could contribute to the PCD manifestation mediated by sphinganine.The generation of ROS (4–60 min) found in Arabidopsis was associated to three conditions: the addition of sphinganine (Fig. 1A), FB1 (Fig. 1A) or pathogen elicitors.¹⁵ This is consistent with the MPK6 activation time, which is downstream of sphinganine elevation and occurs as early as 15 min of FB1 or sphinganine exposure.² All of them are events that appear as initial steps in the relay pathway that produces PCD.In order to explore a possible participation of ROS at more advanced times of PCD progression, we detected in situ H₂O₂ formation in Arabidopsis seedlings previously exposed to FB1 for 48 h. As shown in Figure 1B, formation of the brown-reddish precipitate corresponding to the reaction of H₂O₂ with 3,3′-diaminobenzidine (DAB) was only visible in the FB1-exposed wild type plants, as compared to the non-treated plants. However, when lcb2a-1 mutant seedlings were used, FB1 exposure had a subtle effect in ROS formation. This mutant has a T-DNA insertion in the gene encoding subunit LCB2a from serine palmitoyltransferase (SPT), which catalyzes the first step in sphingolipid synthesis¹⁸ and the mutant has a FB1-resistant phenotype.² These results indicate that mutations in the LCB1¹ and LCB2a² genes (coding for the subunits of the heterodimeric SPT) that lead to a non-PCD phenotype upon the FB1 treatment, are unable to produce H₂O₂. In addition, they suggest that high levels of hydrogen peroxide are produced at advanced times in the PCD mediated by LCBs in Arabidopsis.Exposure of Arabidopsis to an avirulent strain of Pseudomonas syringae produces an endogenous elevation of LCBs as a way to implement defense responses that include HR-PCD.³ In this condition, we clearly detected H₂O₂ formation inside chloroplasts (Fig. 2A). When ultrastructure of the seedlings tissues exposed to FB1 for 72 h was analyzed, integrity of the chloroplast membrane system was severely affected in Arabidopsis wild-type seedlings exposed to FB1.² Therefore, we suggest that ROS generation-LCB induced in the chloroplast could be responsible of the observed membrane alteration, as noted by Liu et al. who found impairment in chloroplast function as a result of H₂O₂ formation in this organelle from tobacco plants. Interestingly, these plants overexpressed a MAP kinase kinase that activated the kinase SIPK, which is the ortholog of the MPK6 from Arabidopsis, a transducer in the PCD instrumented by LCBs.²Open in a separate windowFigure 2Conditions of LCBs elevation produce H₂O₂ formation in the chloroplast and perturbation in the membrane morphology of mitochondria. (A) Exposure of Arabidopsis leaves to the avirulent strain Pseudomonas syringae pv. tomato DC3000 (avrRPM1) (or Pst avrRPM1) induces H₂O₂ formation in the chloroplast. Arabidopsis leaves were infiltrated with 1 × 10⁸ UFC/ml Pst avrRPM1 and after 18 h, samples were treated to visualize H₂O₂ formation with the DAB reaction. Controls were infiltrated with 10 mM MgCl₂ and then processed for DAB staining. Then, samples were analyzed in an optical photomicroscope Olympus Provis Model AX70. (B) Effect of FB1 on mitochondria ultrastructure. Wild type Arabidopsis seedlings were treated with FB1 for 72 h and tissues were processed and analyzed according to Saucedo et al.² Ch, chloroplast; M, mitochondria; PM, plasma membrane. Arrows show mitochondrial cisternae. Bars show the correspondent magnification.In addition, we have detected alterations in mitochondria ultrastructure as a result of 72 h of FB1 exposure (Fig. 2B). These alterations mainly consist in the reduced number of cristae, the membrane site of residence of the electron transport complexes. In this sense, it has been shown that factors that induce PCD such as the victorin toxin, methyl jasmonate and H₂O₂ produce alterations in mitochondrial morphology.^20–22 In fact, some of these studies propose that ROS are formed in the mitochondria and then diffuse to the chloroplasts.^22–24It is reasonable to envisage that damage of the membrane integrity of these two organelles reflects the effects of vast amounts of ROS produced by the electron transport chains.^25,26 Recent evidence supports the destruction of the photosynthetic apparatus associated to the generation of ROS in the HR.²⁶ At this time of PCD progression, ROS could be contributing to shut down the energy machinery in the cell, which ultimately would become the point of no-return of PCD²⁷ as part of the execution program of the cell death mediated by LCBs.In conclusion, we propose that ROS can display two different functional roles in the PCD process driven by LCBs. These roles depend on the time of ROS expression, the cellular site where they are generated, the enzymes that produce them, and the magnitude in which they are formed.     

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