Galactonolactone Dehydrogenase Requires a Redox-Sensitive Thiol for Optimal Production of Vitamin C

The mitochondrial flavoenzyme l-galactono-γ-lactone dehydrogenase (GALDH) catalyzes the ultimate step of vitamin C biosynthesis in plants. We found that recombinant GALDH from Arabidopsis (Arabidopsis thaliana) is inactivated by hydrogen peroxide due to selective oxidation of cysteine (Cys)-340, located in the cap domain. Electrospray ionization mass spectrometry revealed that the partial reversible oxidative modification of Cys-340 involves the sequential formation of sulfenic, sulfinic, and sulfonic acid states. S-Glutathionylation of the sulfenic acid switches off GALDH activity and protects the enzyme against oxidative damage in vitro. C340A and C340S GALDH variants are insensitive toward thiol oxidation, but exhibit a poor affinity for l-galactono-1,4-lactone. Cys-340 is buried beneath the protein surface and its estimated pK_a of 6.5 suggests the involvement of the thiolate anion in substrate recognition. The indispensability of a redox-sensitive thiol provides a rationale why GALDH was designed as a dehydrogenase and not, like related aldonolactone oxidoreductases, as an oxidase.l-Ascorbate (vitamin C) is the most consumed vitamin on earth. It is a multifunctional antioxidant that is particularly abundant in plants where it can reach millimolar concentrations, representing over 10% of the soluble carbohydrate content. l-Ascorbate is a cofactor for a number of enzymes and it is a major constituent of the intracellular redox buffer. Its main function in plants is to scavenge reducing equivalents originating from respiration and photosynthetic activity, protecting proteins, unsaturated fatty acids, and DNA from irreversible oxidative damage (Smirnoff and Wheeler, 2000).The terminal step of l-ascorbate biosynthesis in plants is catalyzed by the mitochondrial flavoenzyme l-galactono-γ-lactone dehydrogenase (GALDH; l-galactono-1,4-lactone:ferricytochrome c oxidoreductase; EC 1.3.2.3). GALDH mediates the two-electron oxidation of l-galactono-1,4-lactone into l-ascorbic acid with the concomitant reduction of cytochrome c (Scheme 1):Open in a separate windowScheme 1.Besides from producing l-ascorbate, the exploitation of the electron transport chain by GALDH is important for the proper functioning of plant mitochondria (Alhagdow et al., 2007). Furthermore, it has been reported that GALDH is required for the correct assembly of respiratory complex I (Pineau et al., 2008).GALDH and other aldonolactone oxidoreductases are two-domain proteins with a conserved vanillyl-alcohol oxidase (VAO)-type FAD domain (Fraaije et al., 1998; Leferink et al., 2008a). Most aldonolactone oxidoreductases are hydrogen peroxide-producing oxidases containing covalently bound FAD, while GALDH reacts poorly with molecular oxygen and contains noncovalently bound FAD (Leferink et al., 2008b). Aldonolactone oxidoreductases have been isolated from various sources, but they are not well characterized. No crystal structure is available, and little is known about the nature of the active site and the catalytic mechanism. Several aldonolactone oxidoreductases, including GALDH from plants (Mapson and Breslow, 1958; Ôba et al., 1995; Østergaard et al., 1997; Imai et al., 1998; Yabuta et al., 2000), l-gulono-1,4-lactone oxidase from animals (Nishikimi, 1979), d-arabinono-1,4-lactone oxidase from yeast (Huh et al., 1994), and trypanosomal aldonolactone oxidoreductases (Logan et al., 2007), are sensitive toward inactivation by thiol-modifying agents. In line with this, we previously found that recombinant GALDH from Arabidopsis (Arabidopsis thaliana) is slowly inactivated during storage and that the activity can be completely restored by treatment with the reducing agent dithiothreitol (DTT; Leferink et al., 2008b).So far nothing is known about the nature of the thiol inactivation, and to our knowledge the effect of oxidants on the activity of aldonolactone oxidoreductases has not been studied before. Here we investigated the susceptibility of GALDH to oxidative stress and identified a critical Cys in the substrate binding site that makes the enzyme vulnerable toward irreversible inactivation.     

A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin β-glycoside of a xylogluco-oligosaccharide (XXXG-β-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK_a (5.8) and large extinction coefficient (ε 62,000 m⁻¹cm⁻¹), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-β-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-β-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.The development and application of molecular probes for the localization of biomolecules in planta continues to have a profound impact on the field of plant physiology. A number of elegant techniques have been devised for the detection of nucleic acids, polypeptides, and polysaccharides in situ, including DNA/RNA hybridization (Jin and Lloyd, 1997), reporter protein fusions (Jefferson et al., 1987; Ehrhardt, 2003; Chapman et al., 2005; Stewart, 2005; Berg and Beachy, 2008; Nelson et al., 2008), immunohistochemical methods (Walker et al., 2001; Chapman et al., 2005), applications of natural carbohydrate-binding proteins (Knox, 2008), and direct spectroscopy (Vicente et al., 2007). While there now exists a considerable toolbox to identify the location to which biomolecules are directed in the cell, elucidation of specific biochemical function at the site of localization often remains challenging.Presently, there is a growing interest in the roles of glycoside hydrolases (GHs) and transglycosylases in plant cell wall biogenesis, remodeling, and degradation (Minic and Jouanin, 2006; Vicente et al., 2007; Gilbert et al., 2008; Lopez-Casado et al., 2008). A technical limitation of many studies, however, is that enzyme activities can only be measured for crude whole-tissue extracts, or purified or recombinant enzymes, and thus cannot be directly correlated with the high-resolution in situ localization of other biomacromolecules. As such, the in situ analysis of GH activities responsible for the degradation of plant cell wall polysaccharides has received comparatively little attention, primarily due to a paucity of convenient assay methods (Vicente et al., 2007). Some notable exceptions include the use of commercially available X (5-bromo-4-chloro-3-indolyl glycoside) substrates for the detection of exoglycosidase activity (Monroe et al., 1999; Chantarangsee et al., 2007; Macquet et al., 2007; Wen et al., 2008). Likewise, transglycosylase activity has been visualized in higher plant and yeast cell walls using sulforhodamine-oligosaccharide acceptor substrates (Vissenberg et al., 2000; Bourquin et al., 2002; Nishikubo et al., 2007; Cabib et al., 2008). Both types are examples of end point, or stopped, assays, in which precipitated indigoid dyes or incorporated fluorescent oligosaccharide conjugate, respectively, are observed after a terminal incubation time.In this study, we have developed the use of resorufin glycosides as substrates for the real-time, continuous observation of GH activity in situ (Fig. 1). Enzymatic hydrolysis of such substrates releases the resorufin aglycone, which is distinguished by a low pK_a value (5.8) and a large extinction coefficient (ε 62,000 m⁻¹cm⁻¹), long-wavelength fluorescence (excitation/emission maxima, 571 nm/585 nm), and high quantum yield (0.74) of the resorufinyl anion (Bueno et al., 2002). The pK_a value and spectral properties make resorufin glycosides particularly suitable for high sensitivity in muro enzyme activity assays due to significant ionization of resorufin at typical apoplastic pH values (Felle, 2005). To highlight the potential of this class of substrates in cell wall morphological studies, we have chemically synthesized a xylogluco-oligosaccharide (XGO) resorufin β-glycoside (XXXG-β-Res; Fig. 1 [1]; XGO nomenclature according to Fry et al., 1993) and demonstrated its use for the real-time imaging of xyloglucan endohydrolase (XEH) activity in plant tissues from nasturtium (Tropaeolum majus) and Arabidopsis (Arabidopsis thaliana) by confocal fluorescence microscopy.Open in a separate windowFigure 1.Use of resorufin glycosides as fluorogenic substrates for glycosidases. R = saccharide or hydrogen; [1] and [2], substrates for determination of (xylo)glucanase activity. Oligosaccharide nomenclature is according to Fry et al. (1993). [See online article for color version of this figure.]     

Genetics of Sexual Development: An Evolutionary Playground for Fish

Teleost fishes are the most species-rich clade of vertebrates and feature an overwhelming diversity of sex-determining mechanisms, classically grouped into environmental and genetic systems. Here, we review the recent findings in the field of sex determination in fish. In the past few years, several new master regulators of sex determination and other factors involved in sexual development have been discovered in teleosts. These data point toward a greater genetic plasticity in generating the male and female sex than previously appreciated and implicate novel gene pathways in the initial regulation of the sexual fate. Overall, it seems that sex determination in fish does not resort to a single genetic cascade but is rather regulated along a continuum of environmental and heritable factors.IN contrast to mammals and birds, cold-blooded vertebrates, and among them teleost fishes in particular, show a variety of strategies for sexual reproduction (Figure 1), ranging from unisexuality (all-female species) to hermaphroditism (sequential, serial, and simultaneous, including outcrossing and selfing species) to gonochorism (two separate sexes at all life stages). The underlying phenotypes are regulated by a variety of sex determination (SD) mechanisms that have classically been divided into two main categories: genetic sex determination (GSD) and environmental sex determination (ESD) (Figure 2).Open in a separate windowFigure 1Reproductive strategies in fish. Fish can be grouped according to their reproductive strategy into unisexuals, hermaphrodites, and gonochorists. Further subdivisions of these three categories are shown with pictures of species exemplifying the strategies. Fish images: Amphiprion clarkii courtesy of Sara Mae Stieb; Hypoplectrus nigricans courtesy of Oscar Puebla; Scarus ferrugineus courtesy of Moritz Muschick; Astatotilapia burtoni courtesy of Anya Theis; Poecilia formosa and Kryptolebias marmoratus courtesy of Manfred Schartl; Trimma sp. courtesy of Rick Winterbottom [serial hermaphroditism has been described in several species of the genus Trimma (Kuwamura and Nakashima 1998; Sakurai et al. 2009; and references therein)].Open in a separate windowFigure 2Sex-determining mechanisms in fish. Sex-determining systems in fish have been broadly classified into environmental and genetic sex determination. For both classes, the currently described subsystems are shown.Environmental factors impacting sex determination in fish are water pH, oxygen concentration, growth rate, density, social state, and, most commonly, temperature (for a detailed review on ESD see, e.g., Baroiller et al. 2009b and Stelkens and Wedekind 2010). As indicated in Figure 2, GSD systems in fish compose a variety of different mechanisms and have been reviewed in detail elsewhere (e.g., Devlin and Nagahama 2002; Volff et al. 2007).The GSD systems that have received the most scientific attention so far are those involving sex chromosomes, which either may be distinguishable cytologically (heteromorphic) or appear identical (homomorphic). In both cases, one sex is heterogametic (possessing two different sex chromosomes and hence producing two types of gametes) and the other one homogametic (a genotype with two copies of the same sex chromosome, producing only one type of gamete). A male-heterogametic system is called an XX-XY system, and female-heterogametic systems are denoted as ZZ-ZW. Both types of heterogamety exist in teleosts and are even found side by side in closely related species [e.g., tilapias (Cnaani et al. 2008), ricefishes (Takehana et al. 2008), or sticklebacks (Ross et al. 2009)]; for more details on the phylogenetic distribution of GSD mechanisms in teleost fish, see Mank et al. (2006). Note that sex chromosomes in fish are mostly homomorphic and not differentiated (Ohno 1974), which is in contrast to the degenerated Y and W chromosomes in mammals (Graves 2006) and birds (Takagi and Sasaki 1974), respectively. This is one possible explanation for the viable combination of different sex chromosomal systems within a single species or population of fish (Parnell and Streelman 2013) and could be a mechanistic reason why sex chromosome turnovers occur easily and frequently in this group (Mank and Avise 2009). Additionally, fish can have more complex sex chromosomal systems involving more than one chromosome pair (see Figure 2). Even within a single fish species, more than two sex chromosomes may occur at the same time, or more than two types of sex chromosomes may co-exist in the same species (Schultheis et al. 2006; Cioffi et al. 2013), which can sometimes be due to chromosome fusions (Kitano and Peichel 2012).Detailed insights on the gene level for GSD/sex chromosomal systems are currently available for only a limited number of fish species, and all but one of these cases involve a rather simple genetic system with male heterogamety and one major sex determiner (see below). The only exception is the widely used model species zebrafish (Danio rerio), which has a polyfactorial SD system implicating four different chromosomes (chromosomes 3, 4, 5, and 16) (Bradley et al. 2011; Anderson et al. 2012) and also environmental cues (Shang et al. 2006).In this review, we focus on newly described genetic sex-determining systems and possible mechanisms allowing their emergence in fishes, which are the most successful group of vertebrates with ∼30,000 species.     

Genetic Evidence for an Essential Oscillation of Transmembrane-Spanning Segment 5 in the Escherichia coli Ammonium Channel AmtB

Ammonium channels, called Amt or Mep, concentrate against a gradient. Each monomer of the trimer has a pore through which substrate passes and a C-terminal cytoplasmic extension. The importance of the C-terminal extension to AmtB activity remains unclear. We have described lesions in conserved C-terminal residues that inactivate AmtB and here characterize 38 intragenic suppressors upstream of the C terminus (∼1/3 of total suppressors). Three that occurred repeatedly, including the previously characterized W148L at the pore entry, restored growth at low NH₃ to nearly wild-type levels and hence restored high activity. V116L completely restored function to two of the mutant proteins and, when separated from other lesions, did not damage wild-type AmtB. A179E notably altered folding of AmtB, compensated for all inactivating C-terminal lesions, and damaged wild-type AmtB. V116L and A179E lie at the cytoplasmic end of transmembrane-spanning segments (TM) 3 and 5, respectively, and the proximal part of the C-terminal tail makes intimate contacts with the loops following them before crossing to the adjacent monomer. Collectively, the properties of intragenic suppressor strains lead us to postulate that the C-terminal tail facilitates an oscillation of TM 5 that is required for coordinated pore function and high AmtB activity. Movement of TM 5 appears to control the opening of both the periplasmic entry and the cytoplasmic exit to the pore.Amt proteins are trimeric inner membrane channels for the hydrated gas (Andrade and Einsle 2007; Fong et al. 2007; Ludewig et al. 2007). They concentrate their substrate against a gradient (Kleiner and Fitzke 1981; Boussiba et al. 1984) and, to our knowledge, are the only active channels described. Each monomer of the Amt trimer contains a pore through which the substrate passes. Although the substrate for Amt channels appears to be , structural studies and molecular dynamics simulations indicate that neutral NH₃ passes through the pore (Khademi et al. 2004; Zheng et al. 2004; Andrade et al. 2005; Lin et al. 2006; Nygaard et al. 2006; Javelle et al. 2007). Hence the pathway for the proton accompanying NH₃ is not clear, but the two appear to separate during their passage through the channel (Mayer et al. 2006). Despite general similarities in charge and size to , K⁺ is neither a substrate for Amt proteins nor an inhibitor of their function (Fong et al. 2007; Javelle et al. 2008). It is for this reason that we refer to as a hydrated gas.The partly stacked phenyl rings of F107 and F215 block entry of NH₃ into the Amt pore, and F215 appears to play a critical role in deprotonation of (Javelle et al. 2008). It has a high structural temperature (B) factor, indicating that it is mobile. The constriction at the periplasmic opening to the pore has been referred to as the “phe gate,” but we prefer the term “phe flap” because GlnK serves as a gate in the classical sense (Andrade et al. 2005; Javelle and Merrick 2005; Durand and Merrick 2006; Conroy et al. 2007; Gruswitz et al. 2007; and see below) and the two should not be confused. We will use the mechanical analogy that the “phe flap” is “open” as a shorthand way of designating that can somehow enter the channel. Although pores were sterically open when F215 (see discussion and Figure 7) was replaced with A, the channel was inactive (Javelle et al. 2008) and hence the mechanical analogy is not sufficient. Above the phe flap is a collar of residues that appears to bind [at the site designated S1 (Khademi et al. 2004)]. This collar, which includes the aromatic residues W148 and F107, has been proposed to play an essential role in recruitment of by π-cation interactions. However, we have shown that W148 restricts entry of into the channel and have proposed instead that the role of the collar is to restrict movement of through the channel (Fong et al. 2007). As the external concentration declines, increased flexibility of the collar/phe flap may allow more rapid entry of into the channel. At this time, it is not clear how much the functions of the collar and the phe flap overlap or can be distinguished.Open in a separate windowFigure 7.—Three-dimensional locations of intragenic suppressors of AmtB^L394A (A) and AmtB^fs (B). Stick models of AmtB were created using PyMOL as in Figure 1. Sections along the pore are perpendicular to the membrane. The periplasmic entry to the pore, which is marked by W148 in lime green, is at the left, and the cytoplasmic exit, which is marked by R47 of GlnK in red stick, is at the right. The threefold axis of the trimer is at the top and the lipid interface is at the bottom. The twin histidine residues at the center of the pore are in green stick, and there is a dot between them. F107 and F215 at the pore entrance are in green and are marked with asterisks. TM 3 is in cyan and TM 5 is in bright pink. TMs 1, 2, and 4 are in light shades of gray, TMs 6–10 are in dark shades of gray, and TM 11 is in brown. Interior loops are in shades of cyan and exterior loops are in shades of light pink. The C terminus is not in the plane of the section, but the C termini of adjacent monomers are in blue and gold. Positions at which suppressor lesions were obtained are space-filled and numbered in order. (A) Numbers correspond to the following: 1, Y32; 2, G113; 3, V116; 4, A118; 5, L119, 6, W148; 7, G175; and 8, A179. F125 is not visible. (B) Numbers correspond to the following: 1, L35; 2, G113; 3, G117; 4, A120; 5, W148; 6, V166; 7, A179; 8, R185; 9, P199; 10, G211; 11, R307; 12, C312; 13, V314; and 14, I359. Suppressors 2, 3, 9, 10, 12, and 13 are in yellow to make the numbers more visible.In addition to the pore, each monomer of the Amt trimer carries a cytoplasmic C-terminal extension of somewhat mysterious function. The cytoplasmic C-terminal extension of the Escherichia coli AmtB protein is ∼25 residues long and can fold precisely against cytoplasmic loops of the same monomer (loops 3, 5, and 1) and the adjacent monomer (loops 7 and 5) (Andrade et al. 2005; Conroy et al. 2007; Gruswitz et al. 2007) (Figure 1). After crossing between monomers, the distal end of the C-terminal extension completes the cytoplasmic vestibule of the adjacent monomer. Although it is known that C-terminal extensions are required for binding of the regulatory protein GlnK, which gates the channel when sufficient internal glutamine is available, it is not known precisely how the extension contributes to AmtB activity. A protein lacking the entire C-terminal tail (AmtB^ΔC-term) fails to bind GlnK but retains intermediate levels of activity, despite the fact that its three pores must be acting independently of one another (Coutts et al. 2002; Severi et al. 2007; Inwood et al. 2009).Open in a separate windowFigure 1.—View of the cytoplasmic face of AmtB and locations of polar connections at the cytoplasmic pore exit. (A) Space-filling model of the cytoplasmic face of E. coli AmtB. The model was created using PyMOL (Delano 2002) from Protein Data Bank entry 2NUU deposited by Conroy et al. (2007) and is similar to that of Neuhäuser et al. (2007). The cytoplasmic C-terminal tails are brown and the three monomers are in different shades of gray. Loop 5 of a single monomer is in pink, loop 1 is in yellow, and the cytoplasmic pore exit is indicated by a large black dot. Note the contacts between the tail and both the proximal and distal ends of loop 5 within a monomer. The tails cross from one monomer to another (counterclockwise) and make an additional contact with the distal end of loop 5 in the adjacent monomer (Conroy et al. 2007; Gruswitz et al. 2007). (B) Polar connections between cytoplasmic loop residues and R185 at the proximal end of loop 5. The stick representation, which is indicated by a box in A, was created using PyMOL. The C-terminal tail is in aqua, with the exception of L394, which is in gold. Residues making polar connections are numbered, and hydrogen bonds are indicated with dotted yellow lines. The cytoplasmic pore exit is indicated by R47 of GlnK, which is in red. Y404, which contacts the distal end of loop 5 in the monomer to which it is covalently attached, is indicated for all three monomers and marks the threefold symmetry axis for the trimer.Surprisingly, mutations that change residues in a kink about half way through the C-terminal tail inactivate AmtB. The kink is the point at which the tail crosses from one monomer to the next, and altering it even conservatively (e.g., the L394A substitution) inactivates AmtB. We have shown that the damaging effects of mutant C-terminal tails are relieved if the chaperone HflB cannot “tack” them (Inwood et al. 2009). Preventing “tacking,” or abnormal folding, can be effected either by extragenic lesions in the ATPase domain of HflB or by intragenic lesions that shorten the AmtB tail so that HflB cannot bind it. Both types of lesions mimic complete deletion of the tail and yield the intermediate level of activity of AmtB^ΔC-term.In this work, we examine intragenic suppressors of inactivating C-terminal lesions in amtB that affect regions upstream of the C terminus. Those that occur most frequently differ from C-terminal truncations and hflB suppressors in restoring activity toward wild type, i.e., to a much higher level than deletion of the C terminus. They indicate that the proximal portion of the C-terminal tail, which can bind precisely to cytoplasmic loops 3, 5, and 1 of the monomer to which it is covalently attached (Figure 1), plays a central role in channel activity. Analysis of upstream intragenic suppressors leads us to postulate that the proximal region of the tail may facilitate a movement of TM 5 (Andrade et al. 2005) that allows opening of the “phe flap” at the periplasmic entry to the pore. For this communication between the bottom and the top of the pore, the distal portion of the tail need not be normal; its sequence can apparently be randomized as long as it can be “tacked” by HflB to the adjacent monomer (Inwood et al. 2009). Inactivating C-terminal lesions in amtB appear to restrict the oscillation of TM 5, which is essential for channel function.     

AUXIN RESPONSE FACTOR17 Is Essential for Pollen Wall Pattern Formation in Arabidopsis

In angiosperms, pollen wall pattern formation is determined by primexine deposition on the microspores. Here, we show that AUXIN RESPONSE FACTOR17 (ARF17) is essential for primexine formation and pollen development in Arabidopsis (Arabidopsis thaliana). The arf17 mutant exhibited a male-sterile phenotype with normal vegetative growth. ARF17 was expressed in microsporocytes and microgametophytes from meiosis to the bicellular microspore stage. Transmission electron microscopy analysis showed that primexine was absent in the arf17 mutant, which leads to pollen wall-patterning defects and pollen degradation. Callose deposition was also significantly reduced in the arf17 mutant, and the expression of CALLOSE SYNTHASE5 (CalS5), the major gene for callose biosynthesis, was approximately 10% that of the wild type. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that ARF17 can directly bind to the CalS5 promoter. As indicated by the expression of DR5-driven green fluorescent protein, which is an synthetic auxin response reporter, auxin signaling appeared to be specifically impaired in arf17 anthers. Taken together, our results suggest that ARF17 is essential for pollen wall patterning in Arabidopsis by modulating primexine formation at least partially through direct regulation of CalS5 gene expression.In angiosperms, the pollen wall is the most complex plant cell wall. It consists of the inner wall, the intine, and the outer wall, the exine. The exine is further divided into sexine and nexine layers. The sculptured sexine includes three major parts: baculum, tectum, and tryphine (Heslop-Harrison, 1971; Piffanelli et al., 1998; Ariizumi and Toriyama, 2011; Fig. 1A). Production of a functional pollen wall requires the precise spatial and temporal cooperation of gametophytic and sporophytic tissues and metabolic events (Blackmore et al., 2007). The intine layer is controlled gametophytically, while the exine is regulated sporophytically. The sporophytic tapetum cells provide material for pollen wall formation, while primexine determines pollen wall patterning (Heslop-Harrison, 1968).Open in a separate windowFigure 1.Schematic representation of the pollen wall and primexine development. A, The innermost layer adjacent to the plasma membrane is the intine. The bacula (Ba), tectum (Te), and tryphine (T) make up the sexine layer. The nexine is located between the intine and the sexine layers. The exine includes the nexine and sexine layers. B, Primexine (Pr) appears between callose (Cl) and plasma membrane (Pm) at the early tetrad stage (left panel). Subsequently, the plasma membrane becomes undulated (middle panel) and sporopollenin deposits on the peak of the undulated plasma membrane to form bacula and tectum (right panel).After meiosis, four microspores were encased in callose to form a tetrad. Subsequently, the primexine develops between the callose layer and the microspore membrane (Fig. 1B), and the microspore plasma membrane becomes undulated (Fig. 1B; Fitzgerald and Knox, 1995; Southworth and Jernstedt, 1995). Sporopollenin precursors then accumulate on the peak of the undulated microspore membrane to form the bacula and tectum (Fig. 1B; Fitzgerald and Knox, 1995). After callose degradation, individual microspores are released from the tetrad, and the bacula and tectum continue to grow into exine with further sporopollenin deposition (Fitzgerald and Knox, 1995; Blackmore et al., 2007).The callose has been reported to affect primexine deposition and pollen wall pattern formation. The peripheral callose layer, secreted by the microsporocyte, acts as the mold for primexine (Waterkeyn and Bienfait, 1970; Heslop-Harrison, 1971). CALLOSE SYNTHASE5 (CalS5) is the major enzyme responsible for the biosynthesis of the callose peripheral of the tetrad (Dong et al., 2005; Nishikawa et al., 2005). Mutation of Cals5 and abnormal CalS5 pre-mRNA splicing resulted in defective peripheral callose deposition and primexine formation (Dong et al., 2005; Nishikawa et al., 2005; Huang et al., 2013). Besides CalS5, four membrane-associated proteins have also been reported to be involved in primexine formation: DEFECTIVE EXINE FORMATION1 (DEX1; Paxson-Sowders et al., 1997, 2001), NO EXINE FORMATION1 (NEF1; Ariizumi et al., 2004), RUPTURED POLLEN GRAIN1 (RPG1; Guan et al., 2008; Sun et al., 2013), and NO PRIMEXINE AND PLASMA MEMBRANE UNDULATION (NPU; Chang et al., 2012). Mutation of DEX1 results in delayed primexine formation (Paxson-Sowders et al., 2001). The primexine in nef1 is coarse compared with the wild type (Ariizumi et al., 2004). The loss-of-function rpg1 shows reduced primexine deposition (Guan et al., 2008; Sun et al., 2013), while the npu mutant does not deposit any primexine (Chang et al., 2012). Recently, it was reported that Arabidopsis (Arabidopsis thaliana) CYCLIN-DEPENDENT KINASE G1 (CDKG1) associates with the spliceosome to regulate the CalS5 pre-mRNA splicing for pollen wall formation (Huang et al., 2013). Clearly, disrupted primexine deposition leads to aberrant pollen wall patterning and ruptured pollen grains in these mutants.The plant hormone auxin has multiple roles in plant reproductive development (Aloni et al., 2006; Sundberg and Østergaard, 2009). Knocking out the two auxin biosynthesis genes, YUC2 and YUC6, caused an essentially sterile phenotype in Arabidopsis (Cheng et al., 2006). Auxin transport is essential for anther development; defects in auxin flow in anther filaments resulted in abnormal pollen mitosis and pollen development (Feng et al., 2006). Ding et al. (2012) showed that the endoplasmic reticulum-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis. Evidence for the localization, biosynthesis, and transport of auxin indicates that auxin regulates anther dehiscence, pollen maturation, and filament elongation during late anther development (Cecchetti et al., 2004, 2008). The role of auxin in pollen wall development has not been reported.The auxin signaling pathway requires the auxin response factor (ARF) family proteins (Quint and Gray, 2006; Guilfoyle and Hagen, 2007; Mockaitis and Estelle, 2008; Vanneste and Friml, 2009). ARF proteins can either activate or repress the expression of target genes by directly binding to auxin response elements (AuxRE; TGTCTC/GAGACA) in the promoters (Ulmasov et al., 1999; Tiwari et al., 2003). The Arabidopsis ARF family contains 23 members. A subgroup in the ARF family, ARF10, ARF16, and ARF17, are targets of miRNA160 (Okushima et al., 2005b; Wang et al., 2005). Plants expressing miR160-resistant ARF17 exhibited pleiotropic developmental defects, including abnormal stamen structure and reduced fertility (Mallory et al., 2005). This indicates a potential role for ARF17 in plant fertility, although the detailed function remains unknown. In addition, ARF17 was also proposed to negatively regulate adventitious root formation (Sorin et al., 2005; Gutierrez et al., 2009), although an ARF17 knockout mutant was not reported and its phenotype is unknown.In this work, we isolated and characterized a loss-of-function mutant of ARF17. Results from cytological observations suggest that ARF17 controls callose biosynthesis and primexine deposition. Consistent with this, the ARF17 protein is highly abundant in microsporocytes and tetrads. Furthermore, we demonstrate that the ARF17 protein is able to bind the promoter region of CalS5. Our results suggest that ARF17 regulates pollen wall pattern formation in Arabidopsis.     

Endogenous Diterpenes Derived from ent-Kaurene,a Common Gibberellin Precursor,Regulate Protonema Differentiation of the Moss Physcomitrella patens

Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurene via ent-kaurenoic acid. GAs are ubiquitously present in seed plants. The GA signal is perceived and transduced by the GID1 GA receptor/DELLA repressor pathway. The lycopod Selaginella moellendorffii biosynthesizes GA and has functional GID1-DELLA signaling components. In contrast, no GAs or functionally orthologous GID1-DELLA components have been found in the moss Physcomitrella patens. However, P. patens produces ent-kaurene, a common precursor for GAs, and possesses a functional ent-kaurene synthase, PpCPS/KS. To assess the biological role of ent-kaurene in P. patens, we generated a PpCPS/KS disruption mutant that does not accumulate ent-kaurene. Phenotypic analysis demonstrates that the mutant has a defect in the protonemal differentiation of the chloronemata to caulonemata. Gas chromatography-mass spectrometry analysis shows that P. patens produces ent-kaurenoic acid, an ent-kaurene metabolite in the GA biosynthesis pathway. The phenotypic defect of the disruptant was recovered by the application of ent-kaurene or ent-kaurenoic acid, suggesting that ent-kaurenoic acid, or a downstream metabolite, is involved in protonemal differentiation. Treatment with uniconazole, an inhibitor of ent-kaurene oxidase in GA biosynthesis, mimics the protonemal phenotypes of the PpCPS/KS mutant, which were also restored by ent-kaurenoic acid treatment. Interestingly, the GA₉ methyl ester, a fern antheridiogen, rescued the protonemal defect of the disruption mutant, while GA₃ and GA₄, both of which are active GAs in angiosperms, did not. Our results suggest that the moss P. patens utilizes a diterpene metabolite from ent-kaurene as an endogenous developmental regulator and provide insights into the evolution of GA functions in land plants.GAs are a large family of tetracyclic diterpenoids, and bioactive GAs play crucial roles in aspects of plant growth and development, including seed germination, stem elongation, leaf expansion, trichome development, and flower and fruit development (Olszewski et al., 2002). GAs are biosynthesized from ent-kaurene, the key intermediate of the GA biosynthetic pathway (Olszewski et al., 2002; Yamaguchi, 2008; Fig. 1). ent-Kaurene is synthesized via sequential cyclization steps of geranylgeranyl diphosphate (GGDP) by ent-copalyl diphosphate synthase (CPS; Sun and Kamiya, 1994) and ent-kaurene synthase (KS; Yamaguchi et al., 1996, 1998). The bioactive GAs (GA₁ and GA₄) are synthesized through a series of oxidation reactions of ent-kaurene by two types of oxidases. Both ent-kaurene oxidase and ent-kaurenoic acid oxidase are cytochrome P450 monooxygenases that successively convert ent-kaurene to GA₁₂. GA₁₂ is further converted to bioactive GAs by two 2-oxoglutarate-dependent dioxygenases, GA 20-oxidase and GA 3-oxidase (Phillips et al., 1995; Olszewski et al., 2002; Yamaguchi, 2008; Fig. 1). GA 2-oxidase is another member of the 2-oxoglutarate-dependent dioxygenase family and is responsible for GA inactivation (Fig. 1). The active GAs can bind to the soluble GA receptor, GID1, and promote the interaction of GID1 with DELLA repressors, which are negative regulators of GA signaling (Ueguchi-Tanaka et al., 2005; Nakajima et al., 2006). This GA-promoted GID1-DELLA interaction triggers the degradation of DELLA repressors via the SCF^GID2/SLY1 proteasome pathway and consequently activates GA signaling (Ueguchi-Tanaka et al., 2007).Open in a separate windowFigure 1.The biosynthetic pathway of GA. The enzyme names are shown in boldface below or to the right of each arrow. AMO-1618 is an angiosperm inhibitor of CPS. Uniconazole, a GA biosynthesis inhibitor, blocks ent-kaurene oxidase activity. GA₁ and GA₄ are the bioactive GAs, and GA₈ and GA₃₄ are their inactive catabolites, respectively. KAO, ent-Kaurenoic acid oxidase.In nonseed land vascular plants, auxin, cytokinin, and abscisic acid function as regulators of plant growth and development (Chopra and Kumra, 1988; Raghavan, 1989). Various physiological responses to these phytohormones are investigated in nonseed land plants, especially in the model moss Physcomitrella patens (Cove et al., 2006). Auxin and cytokinin function in developmental phase changes of chloronemata, caulonemata, and gametophores as well as in cellular growth regulation in P. patens (Imaizumi et al., 2002; Sakakibara et al., 2003; Decker et al., 2006). Abscisic acid mediates the establishment of tolerance to dehydration, cold temperature, and osmotic stresses in P. patens as in angiosperms (Decker et al., 2006; Cho et al., 2009; Khandelwal et al., 2010). In contrast to these hormones, there are only a few studies on the physiological activity of GA in mosses (Von Maltzahn and Macquarrie, 1958; Chopra and Mehta, 1987; Vandenbussche et al., 2007), and the GA function and signaling pathways are still unclear.Recent progress in plant molecular biology and chemical analysis of GA revealed the biosynthesis, perception, and signaling of GA in P. patens and the lycopod Selaginella moellendorffii (Hirano et al., 2007; Vandenbussche et al., 2007; Yasumura et al., 2007). Genome sequence for these organisms has enabled the identification of genes orthologous to flowering plant genes encoding GA biosynthetic enzymes and GA signaling components involved in the GID1-DELLA pathway (Hirano et al., 2007; Vandenbussche et al., 2007). Recently, two reports demonstrated that GID1-DELLA-mediated signaling is functionally conserved in the fern Selaginella and in angiosperms (Hirano et al., 2007; Yasumura et al., 2007). GA-dependent protein-protein interactions were observed between SmGID1 and SmDELLA proteins, the S. moellendorffii proteins orthologous to the rice (Oryza sativa) GID1 and DELLA proteins, respectively. The introduction of either the SmGID1a or SmGID1b gene rescued the rice Osgid1-3 mutant, and the overproduction of SmDELLA1 suppressed GA action in the wild-type background. These reports indicate that the GID1 and DELLA proteins function similarly in S. moellendorffii and in angiosperms. Additionally, S. moellendorffii has functional GA biosynthetic enzymes similar to the angiosperm GA 20- and GA 3-oxidases and endogenous active GA₄ detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. However, endogenous GAs were not detected in P. patens by LC-MS/MS analysis, and the putative P. patens GA oxidases did not show any enzymatic activity on the known substrate for the orthologous angiosperm GA oxidases (Hirano et al., 2007). Furthermore, the PpGID1-like and PpDELLA-like proteins did not interact in the presence of active GA in yeast cells, and the PpDELLA-like protein did not complement the rice DELLA function. These findings suggest that GID1-DELLA-mediated GA signaling evolved in the vascular plant lineage after bryophyte divergence (Hirano et al., 2008).GA₁ and GA₄ are recognized as major biologically active GAs in angiosperms. S. moellendorffii biosynthesizes GA₄ as an active GA. Additionally, the Schizaeaceae family of ferns utilize GA methyl esters (methyl esters of GA₉, GA₂₀, and GA₇₃) as regulators of antheridium development, whereas these GA methyl esters are inactive in angiosperms (Yamauchi et al., 1996, 1997; Kurumatani et al., 2001). The biologically active GAs present in angiosperms were not detected in P. patens (Hirano et al., 2007). Although diverse GA metabolites have been found in plants and fungi, all the GA metabolites are thought to be derived from ent-kaurene, a common intermediate in early GA biosynthetic steps in both land plants and fungi (Kawaide, 2006). In angiosperms, two separate enzymes (CPS and KS) are involved in ent-kaurene synthesis from GGDP via ent-copalyl diphosphate as a reaction intermediate (Fig. 1). We have reported that PpCPS/KS, catalyzing the direct cyclization of GGDP to ent-kaurene, was a bifunctional diterpene cyclase with both CPS and KS activities in a single polypeptide (Hayashi et al., 2006). This type of bifunctional ent-kaurene synthase was also found in GA-producing fungi but was not identified in angiosperms (Kawaide et al., 1997; Toyomasu et al., 2000). The P. patens genome contains a single CPS/KS homolog, and no diterpene cyclase gene was found on the basis of sequence similarity in this organism. Anterola et al. (2009) reported that AMO-1618, an inhibitor of CPS, suppressed spore germination in P. patens; the suppression was recovered by exogenous ent-kaurene application. These results led the authors to hypothesize a role for ent-kaurene in regulating spore germination (Anterola et al., 2009). However, the hypothesis should be examined because the AMO-1618 inhibitory effect was not fully recovered by ent-kaurene application, probably because of the unspecific inhibitory effect of AMO-1618 on spore germination (Anterola et al., 2009).To assess the biological role of ent-kaurene and its metabolites in P. patens, we performed an insertional knockout of the ent-kaurene synthase gene, CPS/KS, in P. patens; the loss of ent-kaurene production was confirmed by gas chromatography-mass spectrometry (GC-MS) analysis. We also determined the abundance of all possible GAs and their precursors in P. patens by LC-MS/MS analysis. The PpCPS/KS disruption mutant (Ppcps/ks KO) lines have a defect in protonemal development. The differentiation of chloronemata to caulonemata was suppressed in the Ppcps/ks KO mutants, and the defect was recovered by the exogenous application of ent-kaurene or ent-kaurenoic acid. Furthermore, the GA₉ methyl ester, an antheridiogen of schizaeaceous ferns, rescued the protonemal defect of the mutants, but GA₃ and GA₄, the representative active GAs for angiosperm, did not. Our results demonstrate that P. patens utilizes GA-type diterpenes synthesized from ent-kaurene as an endogenous growth regulator in protonemal development.