Nondisjunction in Favor of a Chromosome: The Mechanism of Rye B Chromosome Drive during Pollen Mitosis

B chromosomes (Bs) are supernumerary components of the genome and do not confer any advantages on the organisms that harbor them. The maintenance of Bs in natural populations is possible by their transmission at higher than Mendelian frequencies. Although drive is the key for understanding B chromosomes, the mechanism is largely unknown. We provide direct insights into the cellular mechanism of B chromosome drive in the male gametophyte of rye (Secale cereale). We found that nondisjunction of Bs is accompanied by centromere activity and is likely caused by extended cohesion of the B sister chromatids. The B centromere originated from an A centromere, which accumulated B-specific repeats and rearrangements. Because of unequal spindle formation at the first pollen mitosis, nondisjoined B chromatids preferentially become located toward the generative pole. The failure to resolve pericentromeric cohesion is under the control of the B-specific nondisjunction control region. Hence, a combination of nondisjunction and unequal spindle formation at first pollen mitosis results in the accumulation of Bs in the generative nucleus and therefore ensures their transmission at a higher than expected rate to the next generation.     

Salicylic Acid Regulates Arabidopsis Microbial Pattern Receptor Kinase Levels and Signaling

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.     

Brassinosteroid Regulates Cell Elongation by Modulating Gibberellin Metabolism in Rice

Brassinosteroid (BR) and gibberellin (GA) are two predominant hormones regulating plant cell elongation. A defect in either of these leads to reduced plant growth and dwarfism. However, their relationship remains unknown in rice (Oryza sativa). Here, we demonstrated that BR regulates cell elongation by modulating GA metabolism in rice. Under physiological conditions, BR promotes GA accumulation by regulating the expression of GA metabolic genes to stimulate cell elongation. BR greatly induces the expression of D18/GA3ox-2, one of the GA biosynthetic genes, leading to increased GA₁ levels, the bioactive GA in rice seedlings. Consequently, both d18 and loss-of-function GA-signaling mutants have decreased BR sensitivity. When excessive active BR is applied, the hormone mostly induces GA inactivation through upregulation of the GA inactivation gene GA2ox-3 and also represses BR biosynthesis, resulting in decreased hormone levels and growth inhibition. As a feedback mechanism, GA extensively inhibits BR biosynthesis and the BR response. GA treatment decreases the enlarged leaf angles in plants with enhanced BR biosynthesis or signaling. Our results revealed a previously unknown mechanism underlying BR and GA crosstalk depending on tissues and hormone levels, which greatly advances our understanding of hormone actions in crop plants and appears much different from that in Arabidopsis thaliana.     

Natural Variation in Small Molecule–Induced TIR-NB-LRR Signaling Induces Root Growth Arrest via EDS1- and PAD4-Complexed R Protein VICTR in Arabidopsis

In a chemical genetics screen we identified the small-molecule [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) that triggers rapid inhibition of early abscisic acid signal transduction via PHYTOALEXIN DEFICIENT4 (PAD4)- and ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent immune signaling mechanisms. However, mechanisms upstream of EDS1 and PAD4 in DFPM-mediated signaling remain unknown. Here, we report that DFPM generates an Arabidopsis thaliana accession-specific root growth arrest in Columbia-0 (Col-0) plants. The genetic locus responsible for this natural variant, VICTR (VARIATION IN COMPOUND TRIGGERED ROOT growth response), encodes a TIR-NB-LRR (for Toll-Interleukin1 Receptor–nucleotide binding–Leucine-rich repeat) protein. Analyses of T-DNA insertion victr alleles showed that VICTR is necessary for DFPM-induced root growth arrest and inhibition of abscisic acid–induced stomatal closing. Transgenic expression of the Col-0 VICTR allele in DFPM-insensitive Arabidopsis accessions recapitulated the DFPM-induced root growth arrest. EDS1 and PAD4, both central regulators of basal resistance and effector-triggered immunity, as well as HSP90 chaperones and their cochaperones RAR1 and SGT1B, are required for the DFPM-induced root growth arrest. Salicylic acid and jasmonic acid signaling pathway components are dispensable. We further demonstrate that VICTR associates with EDS1 and PAD4 in a nuclear protein complex. These findings show a previously unexplored association between a TIR-NB-LRR protein and PAD4 and identify functions of plant immune signaling components in the regulation of root meristematic zone-targeted growth arrest.     

One Divinyl Reductase Reduces the 8-Vinyl Groups in Various Intermediates of Chlorophyll Biosynthesis in a Given Higher Plant Species,But the Isozyme Differs between Species

Divinyl reductase (DVR) converts 8-vinyl groups on various chlorophyll intermediates to ethyl groups, which is indispensable for chlorophyll biosynthesis. To date, five DVR activities have been detected, but adequate evidence of enzymatic assays using purified or recombinant DVR proteins has not been demonstrated, and it is unclear whether one or multiple enzymes catalyze these activities. In this study, we systematically carried out enzymatic assays using four recombinant DVR proteins and five divinyl substrates and then investigated the in vivo accumulation of various chlorophyll intermediates in rice (Oryza sativa), maize (Zea mays), and cucumber (Cucumis sativus). The results demonstrated that both rice and maize DVR proteins can convert all of the five divinyl substrates to corresponding monovinyl compounds, while both cucumber and Arabidopsis (Arabidopsis thaliana) DVR proteins can convert three of them. Meanwhile, the OsDVR (Os03g22780)-inactivated 824ys mutant of rice exclusively accumulated divinyl chlorophylls in its various organs during different developmental stages. Collectively, we conclude that a single DVR with broad substrate specificity is responsible for reducing the 8-vinyl groups of various chlorophyll intermediates in higher plants, but DVR proteins from different species have diverse and differing substrate preferences, although they are homologous.Chlorophyll (Chl) molecules universally exist in photosynthetic organisms. As the main component of the photosynthetic pigments, Chl molecules perform essential processes of absorbing light and transferring the light energy in the reaction center of the photosystems (Fromme et al., 2003). Based on the number of vinyl side chains, Chls are classified into two groups, 3,8-divinyl (DV)-Chl and 3-monovinyl (MV)-Chl. The DV-Chl molecule contains two vinyl groups at positions 3 and 8 of the tetrapyrrole macrocycle, whereas the MV-Chl molecule contains a vinyl group at position 3 and an ethyl group at position 8 of the macrocycle. Almost all of the oxygenic photosynthetic organisms contain MV-Chls, with the exceptions of some marine picophytoplankton species that contain only DV-Chls as their primary photosynthetic pigments (Chisholm et al., 1992; Goericke and Repeta, 1992; Porra, 1997).The classical single-branched Chl biosynthetic pathway proposed by Granick (1950) and modified by Jones (1963) assumed the rapid reduction of the 8-vinyl group of DV-protochlorophyllide (Pchlide) catalyzed by a putative 8-vinyl reductase. Ellsworth and Aronoff (1969) found evidence for both MV and DV forms of several Chl biosynthetic intermediates between magnesium-protoporphyrin IX monomethyl ester (MPE) and Pchlide in Chlorella spp. mutants. Belanger and Rebeiz (1979, 1980) reported that the Pchlide pool of etiolated higher plants contains both MV- and DV-Pchlide. Afterward, following the further detection of MV- and DV-tetrapyrrole intermediates and their biosynthetic interconversion in tissues and extracts of different plants (Belanger and Rebeiz, 1982; Duggan and Rebeiz, 1982; Tripathy and Rebeiz, 1986, 1988; Parham and Rebeiz, 1992, 1995; Kim and Rebeiz, 1996), a multibranched Chl biosynthetic heterogeneity was proposed (Rebeiz et al., 1983, 1986, 1999; Whyte and Griffiths, 1993; Kolossov and Rebeiz, 2010).Biosynthetic heterogeneity refers to the biosynthesis of a particular metabolite by an organelle, tissue, or organism via multiple biosynthetic routes. Varieties of reports lead to the assumption that Chl biosynthetic heterogeneity originates mainly in parallel DV- and MV-Chl biosynthetic routes. These routes are interconnected by 8-vinyl reductases that convert DV-tetrapyrroles to MV-tetrapyrroles by conversion of the vinyl group at position 8 of ring B to the ethyl group (Parham and Rebeiz, 1995; Rebeiz et al., 2003). DV-MPE could be converted to MV-MPE in crude homogenates from etiolated wheat (Triticum aestivum) seedlings (Ellsworth and Hsing, 1974). Exogenous DV-Pchlide could be partially converted to MV-Pchlide in barley (Hordeum vulgare) plastids (Tripathy and Rebeiz, 1988). 8-Vinyl chlorophyllide (Chlide) a reductases in etioplast membranes isolated from etiolated cucumber (Cucumis sativus) cotyledons and barley and maize (Zea mays) leaves were found to be very active in the conversion of exogenous DV-Chlide a to MV-Chlide a (Parham and Rebeiz, 1992, 1995). Kim and Rebeiz (1996) suggested that Chl biosynthetic heterogeneity in higher plants may originate at the level of DV magnesium-protoporphyrin IX (Mg-Proto) and would be mediated by the activity of a putative 8-vinyl Mg-Proto reductase in barley etiochloroplasts and plastid membranes. However, since these reports did not use purified or recombinant enzyme, it is not clear whether the reductions of the 8-vinyl groups of various Chl intermediates are catalyzed by one enzyme of broad specificity or by multiple enzymes of narrow specificity, which actually has become one of the focus issues in Chl biosynthesis.Nagata et al. (2005) and Nakanishi et al. (2005) independently identified the AT5G18660 gene of Arabidopsis (Arabidopsis thaliana) as an 8-vinyl reductase, namely, divinyl reductase (DVR). Chew and Bryant (2007) identified the DVR BciA (CT1063) gene of the green sulfur bacterium Chlorobium tepidum, which is homologous to AT5G18660. An enzymatic assay using a recombinant Arabidopsis DVR (AtDVR) on five DV substrates revealed that the major substrate of AtDVR is DV-Chlide a, while the other four DV substrates could not be converted to corresponding MV compounds (Nagata et al., 2007). Nevertheless, a recombinant BciA is able to reduce the 8-vinyl group of DV-Pchlide to generate MV-Pchlide (Chew and Bryant, 2007). Recently, we identified the rice (Oryza sativa) DVR encoded by Os03g22780 that has sequence similarity with the Arabidopsis DVR gene AT5G18660. We also confirmed that the recombinant rice DVR (OsDVR) is able to not only convert DV-Chlide a to MV-Chlide a but also to convert DV-Chl a to MV-Chl a (Wang et al., 2010). Thus, it is possible that the reductions of the 8-vinyl groups of various Chl biosynthetic intermediates are catalyzed by one enzyme of broad specificity.In this report, we extended our studies to four DVR proteins and five DV substrates. First, ZmDVR and CsDVR genes were isolated from maize and cucumber genomes, respectively, using a homology-based cloning approach. Second, enzymatic assays were systematically carried out using recombinant OsDVR, ZmDVR, CsDVR, and AtDVR as representative DVR proteins and using DV-Chl a, DV-Chlide a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto as DV substrates. Third, we examined the in vivo accumulations of various Chl intermediates in rice, maize, and cucumber. Finally, we systematically investigated the in vivo accumulations of Chl and its various intermediates in the OsDVR (Os03g22780)-inactivated 824ys mutant of rice (Wang et al., 2010). The results strongly suggested that a single DVR protein with broad substrate specificity is responsible for reducing the 8-vinyl groups of various intermediate molecules of Chl biosynthesis in higher plants, but DVR proteins from different species could have diverse and differing substrate preferences even though they are homologous.     

Acyl Editing and Headgroup Exchange Are the Major Mechanisms That Direct Polyunsaturated Fatty Acid Flux into Triacylglycerols

Triacylglycerols (TAG) in seeds of Arabidopsis (Arabidopsis thaliana) and many plant species contain large amounts of polyunsaturated fatty acids (PUFA). These PUFA are synthesized on the membrane lipid phosphatidylcholine (PC). However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the key enzymes/genes that control these fluxes known. By reverse genetics and metabolic labeling experiments, we demonstrate that two genes encoding the lysophosphatidylcholine acyltransferases LPCAT1 and LPCAT2 in Arabidopsis control the previously identified “acyl-editing” process, the main entry of fatty acids into PC. The lpcat1/lpcat2 mutant showed increased contents of very-long-chain fatty acids and decreased PUFA in TAG and the accumulation of small amounts of lysophosphatidylcholine in developing seeds revealed by [¹⁴C]acetate-labeling experiments. We also showed that mutations in LPCATs and the PC diacylglycerol cholinephosphotransferase in the reduced oleate desaturation1 (rod1)/lpcat1/lpcat2 mutant resulted in a drastic reduction of PUFA content in seed TAG, accumulating only one-third of the wild-type level. These results indicate that PC acyl editing and phosphocholine headgroup exchange between PC and diacylglycerols control the majority of acyl fluxes through PC to provide PUFA for TAG synthesis.Plant oils are an important natural resource to meet the increasing demands of food, feed, biofuel, and industrial applications (Lu et al., 2011; Snapp and Lu, 2012). The fatty acid composition in the triacylglycerols (TAG), especially the contents of polyunsaturated fatty acids (PUFA) or other specialized structures, such as hydroxy, epoxy, or conjugated groups, determines the properties and thus the uses of plant oils (Dyer and Mullen, 2008; Dyer et al., 2008; Pinzi et al., 2009; Riediger et al., 2009). To effectively modify seed oils tailored for different uses, it is necessary to understand the fundamental aspects of how plant fatty acids are synthesized and accumulated in seed oils.In developing oilseeds, fatty acids are synthesized in plastids and are exported into the cytosol mainly as oleic acid, 18:1 (carbon number:double bonds), and a small amount of palmitic acid (16:0) and stearic acid (18:0; Ohlrogge and Browse, 1995). Further modification of 18:1 occurs on the endoplasmic reticulum in two major pathways (Fig. 1): (1) the 18:1-CoA may be elongated into 20:1- to 22:1-CoA esters by a fatty acid elongase, FAE1 (Kunst et al., 1992); (2) the dominant flux of 18:1 in many oilseeds is to enter the membrane lipid phosphatidylcholine (PC; Shanklin and Cahoon, 1998; Bates and Browse, 2012), where they can be desaturated by the endoplasmic reticulum-localized fatty acid desaturases including the oleate desaturase, FAD2, and the linoleate desaturase, FAD3, to produce the polyunsaturated linoleic acid (18:2) and α-linolenic acid (18:3; Browse et al., 1993; Okuley et al., 1994). The PUFA may be removed from PC to enter the acyl-CoA pool, or PUFA-rich diacylglycerol (DAG) may be derived from PC by removal of the phosphocholine headgroup (Bates and Browse, 2012). The PUFA-rich TAG are then produced from de novo-synthesized DAG or PC-derived DAG (Bates and Browse, 2012) and PUFA-CoA by the acyl-CoA:diacylglycerol acyltransferases (DGAT; Hobbs et al., 1999; Zou et al., 1999). Alternatively, PUFA may be directly transferred from PC onto DAG to form TAG by an acyl-CoA-independent phospholipid:diacylglycerol acyltransferase (PDAT; Dahlqvist et al., 2000). Recent results demonstrated that DGAT and PDAT are responsible for the majority of TAG synthesized in Arabidopsis (Arabidopsis thaliana) seeds (Zhang et al., 2009).Open in a separate windowFigure 1.Reactions involved in the flux of fatty acids into TAG. De novo glycerolipid synthesis is shown in white arrows, acyl transfer reactions are indicated by dashed lines, and the movement of the lipid glycerol backbone through the pathway is shown in solid lines. Major reactions (in thick lines) controlling the flux of fatty acid from PC into TAG are as follows: LPC acylation reaction of acyl editing by LPCAT (A); PC deacylation reaction of acyl editing by the reverse action of LPCAT or phospholipase A (B); and the interconversion of DAG and PC by PDCT (C). Substrates are in boldface, enzymatic reactions are in italics. FAD, Fatty acid desaturase; FAS, fatty acid synthase; GPAT, acyl-CoA:G3P acyltransferase; LPA, lysophosphatidic acid; LPAT, acyl-CoA:LPA acyltransferase; PA, phosphatidic acid; PLC, phospholipase C; PLD, phospholipase D.The above TAG synthesis model highlights the importance of acyl fluxes through PC for PUFA enrichment in plant oils. However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the enzymes/genes that control these fluxes known. The traditional view is that 18:1 enters PC through de novo glycerolipid synthesis (Fig. 1; Kennedy, 1961): the sequential acylation of glycerol-3-phosphate (G3P) at the sn-1 and sn-2 positions produces phosphatidic acid; subsequent removal of the phosphate group at the sn-3 position of phosphatidic acid by phosphatidic acid phosphatases (PAPs) produces de novo DAG; finally, PC is formed from DAG by a cytidine-5′-diphosphocholine:diacylglycerol cholinephosphotransferase (CPT; Slack et al., 1983; Goode and Dewey, 1999). However, metabolic labeling experiments in many different plant tissues by us and others (Williams et al., 2000; Bates et al., 2007, 2009; Bates and Browse, 2012; Tjellström et al., 2012) have demonstrated that the majority of newly synthesized fatty acids (e.g. 18:1) enter PC by a process termed “acyl editing” rather than by proceeding through de novo PC synthesis. Acyl editing is a deacylation-reacylation cycle of PC that exchanges the fatty acids on PC with fatty acids in the acyl-CoA pool (Fig. 1, A and B). Through acyl editing, newly synthesized 18:1 can be incorporated into PC for desaturation and PUFA can be released from PC to the acyl-CoA pool to be utilized for glycerolipid synthesis.Additionally, there is accumulating evidence that many plants utilize PC-derived DAG to synthesize TAG laden with PUFA (Bates and Browse, 2012). PC-derived DAG may be synthesized through the reverse reaction of the CPT (Slack et al., 1983, 1985) or by the phospholipases C and D (followed by PAP). However, our recent discovery indicates that the main PC-to-DAG conversion is catalyzed by a phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) through the phosphocholine headgroup exchange between PC and DAG (Fig. 1C; Lu et al., 2009; Hu et al., 2012). The PDCT is encoded by the REDUCED OLEATE DESATURATION1 (ROD1) gene (At3g15820) in Arabidopsis, which is responsible for about 40% of the flux of PUFA from PC through DAG into TAG synthesis (Lu et al., 2009). Acyl editing and PC-DAG interconversion through PDCT may work together to generate PUFA-rich TAG in oilseed plants (Bates and Browse, 2012).The enzymes/genes involved in the incorporation of 18:1 into PC through acyl editing are not known. However, stereochemical localization of newly synthesized fatty acid incorporation into PC predominantly at the sn-2 position (Bates et al., 2007, 2009; Tjellström et al., 2012) strongly suggest that the acyl editing cycle proceeds through the acylation of lysophosphatidylcholine (LPC) by acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs [Enzyme Commission 2.3.1.23]; Fig. 1A). High LPCAT activity has been detected in many different oilseed plants that accumulate large amounts of PUFA in TAG (Stymne and Stobart, 1987; Bates and Browse, 2012), suggesting the potential ubiquitous involvement of LPCAT in the generation of PUFA-rich TAG. Several possible pathways for the removal of acyl groups from PC to generate the lysophosphatidylcholine within the acyl editing cycle have been proposed. The acyl groups may be released from PC to enter the acyl-CoA pool via the reverse reactions of LPCATs (Stymne and Stobart, 1984) or by reactions of phospholipase A (Chen et al., 2011) followed by the acyl-CoA synthetases (Shockey et al., 2002). The main focus of this study was to identify the genes and enzymes involved in the incorporation of fatty acids into PC through acyl editing in Arabidopsis and to quantify the contribution of acyl editing and PDCT-based PC-DAG interconversion to controlling the flux of PUFA from PC into TAG. Herein, we demonstrate that mutants of two Arabidopsis genes encoding LPCATs (At1g12640 [LPCAT1] and At1g63050 [LPCAT2]) have reduced TAG PUFA content. Analysis of the acyl-editing cycle through metabolic labeling of developing seeds with [¹⁴C]acetate indicate that the lpcat1/lpcat2 double mutant was devoid of acyl editing-based incorporation of newly synthesized fatty acids into PC, indicating that these two genes are responsible for the acylation of LPC during acyl editing. Additionally, the triple mutant rod1/lpcat1/lpcat2 indicated that PDCT-based PC-DAG interconversion and acyl editing together provide two-thirds of the flux of PUFA from PC to TAG in Arabidopsis seeds.     

Genome-Wide Characterization of Nonreference Transposons Reveals Evolutionary Propensities of Transposons in Soybean

Preferential accumulation of transposable elements (TEs), particularly long terminal repeat retrotransposons (LTR-RTs), in recombination-suppressed pericentromeric regions seems to be a general pattern of TE distribution in flowering plants. However, whether such a pattern was formed primarily by preferential TE insertions into pericentromeric regions or by selection against TE insertions into euchromatin remains obscure. We recently investigated TE insertions in 31 resequenced wild and cultivated soybean (Glycine max) genomes and detected 34,154 unique nonreference TE insertions mappable to the reference genome. Our data revealed consistent distribution patterns of the nonreference LTR-RT insertions and those present in the reference genome, whereas the distribution patterns of the nonreference DNA TE insertions and the accumulated ones were significantly different. The densities of the nonreference LTR-RT insertions were found to negatively correlate with the rates of local genetic recombination, but no significant correlation between the densities of nonreference DNA TE insertions and the rates of local genetic recombination was detected. These observations suggest that distinct insertional preferences were primary factors that resulted in different levels of effectiveness of purifying selection, perhaps as an effect of local genomic features, such as recombination rates and gene densities that reshaped the distribution patterns of LTR-RTs and DNA TEs in soybean.     

Successful Fertilization Requires the Presence of at Least One Major O-Acetylserine(thiol)lyase for Cysteine Synthesis in Pollen of Arabidopsis

The synthesis of cysteine (Cys) is a master control switch of plant primary metabolism that coordinates the flux of sulfur with carbon and nitrogen metabolism. In Arabidopsis (Arabidopsis thaliana), nine genes encode for O-acetylserine(thiol)lyase (OAS-TL)-like proteins, of which the major isoforms, OAS-TL A, OAS-TL B, and OAS-TL C, catalyze the formation of Cys by combining O-acetylserine and sulfide in the cytosol, the plastids, and the mitochondria, respectively. So far, the significance of individual OAS-TL-like enzymes is unresolved. Generation of all major OAS-TL double loss-of-function mutants in combination with radiolabeled tracer studies revealed that subcellular localization of OAS-TL proteins is more important for efficient Cys synthesis than total cellular OAS-TL activity in leaves. The absence of oastl triple embryos after targeted crosses indicated the exclusiveness of Cys synthesis by the three major OAS-TLs and ruled out alternative sulfur fixation by other OAS-TL-like proteins. Analyses of oastlABC pollen demonstrated that the presence of at least one functional OAS-TL isoform is essential for the proper function of the male gametophyte, although the synthesis of histidine, lysine, and tryptophan is dispensable in pollen. Comparisons of oastlABC pollen derived from genetically different parent plant combinations allowed us to separate distinct functions of Cys and glutathione in pollen and revealed an additional role of glutathione for pollen germination. In contrast, female gametogenesis was not affected by the absence of major OAS-TLs, indicating significant transport of Cys into the developing ovule from the mother plant.Sulfur assimilation in plants is hallmarked by two reaction sequences, namely sulfate reduction and Cys synthesis. The sulfate reduction pathway consists of three steps and produces sulfide from sulfate, which is available in the soil and transported into the roots by specific transporters (Takahashi et al., 2011). Sulfide is subsequently incorporated into the amino acid O-acetylserine (OAS) by O-acetylserine(thiol)lyase (OAS-TL; EC 2.5.1.47) to produce Cys (Hell and Wirtz, 2011). Cys then serves as the sulfur source for all organic metabolites containing reduced sulfur in plants, including proteins, cofactors, and secondary metabolites. The tripeptide glutathione (GSH) is one of the most important Cys-derived metabolites, since it has an important function in redox homeostasis and the control of development (Meyer and Rausch, 2008). Impaired GSH synthesis negatively affects growth of the shoot and root system of Arabidopsis (Arabidopsis thaliana; Vernoux et al., 2000; Xiang et al., 2001), and loss-of-function mutants for the first enzyme (GSH1, Glu-Cys ligase; EC 6.3.2.2) or the second enzyme (GSH2, glutathione synthase; EC 6.3.2.3) of the two-step pathway leading to GSH formation show an embryo- and seedling-lethal phenotype, respectively (Cairns et al., 2006; Pasternak et al., 2008).Cys synthesis by OAS-TL constitutes the direct link between carbon and nitrogen (OAS) as well as sulfur (sulfide) metabolism and, therefore, can be designated as one of the central reactions in plant primary metabolism. The genome of the model plant Arabidopsis encodes nine OAS-TL-like enzymes: OAS-TL A1 (At4g14880), OAS-TL B (At2g43750), and OAS-TL C (At3g59760) are the major isoforms and are localized in the cytosol, plastids, and mitochondria, respectively (Jost et al., 2000). OAS-TL A2 (At3g22460) encodes a truncated and nonfunctional protein (Jost et al., 2000). In the following, therefore, OAS-TL A1 is referred to as OAS-TL A. CYS D1 (At3g04940) and CYS D2 (At5g28020) show OAS-TL activity in vitro (Yamaguchi et al., 2000). Whether they contribute to net Cys synthesis in vivo is unknown (Heeg et al., 2008). CS26 (At3g03630) encodes a plastidic S-sulfocysteine synthase, which prefers thiosulfate instead of sulfide as substrate and produces S-sulfocysteine (Bermúdez et al., 2010). Whether thiosulfate is taken up from the soil or formed within the plant is unclear, but its presence in Arabidopsis was demonstrated (Tsakraklides et al., 2002). However, the synthesis of S-sulfocysteine from thiosulfate potentially constitutes an alternative sulfur fixation pathway. So far, CS26 was shown to be important for the regulation of redox homeostasis in plastids under certain stress conditions (Bermúdez et al., 2010). DES1 (At5g28030; formerly known as CS-LIKE) is a Cys desulfhydrase (EC 4.4.1.15) that releases sulfide in the cytosol (Alvarez et al., 2010). As a Cys-consuming enzyme, it contributes to Cys homeostasis, especially in late vegetative development and under certain stress conditions (Alvarez et al., 2010, 2012). CYS C1 (At3g61440), finally, encodes a mitochondrial β-cyanoalanine synthase (EC 4.4.1.9), which detoxifies cyanide by incorporation into Cys (Yamaguchi et al., 2000; Watanabe et al., 2008a; García et al., 2010). The major isoforms OAS-TL A, OAS-TL B, and OAS-TL C as well as CYS D1 and CYS D2 can interact with serine acetyltransferase (SAT; EC 2.3.1.30) in the cysteine synthase complex (CSC; Heeg et al., 2008). Although SAT acetylates Ser at the hydroxyl group to form OAS, the direct substrate of OAS-TL, formation of the CSC has no substrate-channeling function but contributes to the demand-driven regulation of Cys synthesis (Hell and Wirtz, 2011).The subcellular compartmentation of Cys precursor formation is a remarkable feature of Cys synthesis in higher plants that implies a high degree of regulation between the participating compartments: while sulfate is exclusively reduced to sulfide in plastids (Takahashi et al., 2011), the synthesis of OAS and the incorporation of sulfide take place in all three compartments where SAT and OAS-TL are present, namely in the cytosol, plastids, and mitochondria. Reverse genetics approaches proved a certain redundancy between the different SAT and OAS-TL isoforms, which demonstrates that sulfide, OAS, and Cys can be exchanged between these compartments (Haas et al., 2008; Heeg et al., 2008; Watanabe et al., 2008a, 2008b). Indeed, sulfide can easily diffuse through membranes (Mathai et al., 2009), but OAS and Cys need to be actively transported. However, the identity of these transporters is unknown. Although sulfide, OAS, and Cys can pass the mitochondrial membrane (Wirtz et al., 2012), the loss-of-function mutant for mitochondrial OAS-TL C is the only single oastl knockout mutant that displays a significant growth phenotype (Heeg et al., 2008). This result was astonishing, since OAS-TL C contributes only 5% to extractable foliar OAS-TL activity (Heeg et al., 2008). The retarded growth of the oastlC mutant, however, cannot be explained by the lack of sulfide detoxification in mitochondria by OAS-TL C, due to an alternative detoxification mechanism for sulfide in mitochondria (Birke et al., 2012). These data question the total redundancy between the different OAS-TL isoforms and suggest specific functions in the different subcellular compartments.Despite its central position in the primary metabolism of higher plants, fundamental questions about Cys synthesis are still unanswered. First, the contribution of OAS-TL-like proteins, especially CYS D1, CYS D2, and CS26, to the fixation of sulfur in planta is unknown. Second, the significance of Cys synthesis by the major OAS-TL proteins in the different subcellular compartments during sporophyte and gametophyte development is unclear. In this study, we addressed these questions using a reverse genetics approach. We were able to prove that fixation of sulfur is carried out exclusively by the major OAS-TL isoforms OAS-TL A, OAS-TL B, and OAS-TL C and elucidated specific functions for OAS-TL A in the cytosol and OAS-TL C in mitochondria of leaf cells. Furthermore, we demonstrate that Cys can be supplied by the mother plant for the development of female gametophytes lacking OAS-TL activity. In contrast, the presence of at least one functional OAS-TL isoform is essential in the male gametophyte.     

Reciprocal Control of Anaplerotic Phosphoenolpyruvate Carboxylase by in Vivo Monoubiquitination and Phosphorylation in Developing Proteoid Roots of Phosphate-Deficient Harsh Hakea

Accumulating evidence indicates important functions for phosphoenolpyruvate (PEP) carboxylase (PEPC) in inorganic phosphate (Pi)-starved plants. This includes controlling the production of organic acid anions (malate, citrate) that are excreted in copious amounts by proteoid roots of nonmycorrhizal species such as harsh hakea (Hakea prostrata). This, in turn, enhances the bioavailability of mineral-bound Pi by solubilizing Al³⁺, Fe³⁺, and Ca²⁺ phosphates in the rhizosphere. Harsh hakea thrives in the nutrient-impoverished, ancient soils of southwestern Australia. Proteoid roots from Pi-starved harsh hakea were analyzed over 20 d of development to correlate changes in malate and citrate exudation with PEPC activity, posttranslational modifications (inhibitory monoubiquitination versus activatory phosphorylation), and kinetic/allosteric properties. Immature proteoid roots contained an equivalent ratio of monoubiquitinated 110-kD and phosphorylated 107-kD PEPC polypeptides (p110 and p107, respectively). PEPC purification, immunoblotting, and mass spectrometry indicated that p110 and p107 are subunits of a 430-kD heterotetramer and that they both originate from the same plant-type PEPC gene. Incubation with a deubiquitinating enzyme converted the p110:p107 PEPC heterotetramer of immature proteoid roots into a p107 homotetramer while significantly increasing the enzyme’s activity under suboptimal but physiologically relevant assay conditions. Proteoid root maturation was paralleled by PEPC activation (e.g. reduced K_m [PEP] coupled with elevated I₅₀ [malate and Asp] values) via in vivo deubiquitination of p110 to p107, and subsequent phosphorylation of the deubiquitinated subunits. This novel mechanism of posttranslational control is hypothesized to contribute to the massive synthesis and excretion of organic acid anions that dominates the carbon metabolism of the mature proteoid roots.Phosphoenolpyruvate (PEP) carboxylase (PEPC; EC 4.1.1.31) is a ubiquitous and tightly regulated cytosolic enzyme of vascular plants that is also widely distributed in green algae and bacteria. PEPC catalyzes the irreversible β-carboxylation of PEP to form oxaloacetate (OAA) and inorganic phosphate (Pi). Vascular plant PEPCs belong to a small multigene family encoding several closely related plant-type PEPCs (PTPCs), along with a distantly related bacterial-type PEPC (BTPC; O’Leary et al., 2011a). PTPC genes encode 105- to 110-kD polypeptides that typically assemble as approximate 400-kD Class-1 PEPC homotetramers. In contrast, BTPC genes encode larger 116- to 118-kD polypeptides owing to a unique intrinsically disordered region that mediates BTPC’s tight interaction with coexpressed PTPC subunits. This association results in the formation of unusual Class-2 PEPC heterooctameric complexes that are largely desensitized to allosteric effectors and that dynamically associate with the surface of mitochondria in vivo (O’Leary et al., 2009, 2011a; Igawa et al., 2010; Park et al., 2012).The critical role of Class-1 PEPC in assimilating atmospheric CO₂ during C₄ and Crassulacean acid metabolism photosynthesis has been studied extensively. Class-1 PEPCs also fulfill a wide range of crucial nonphotosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis (O’Leary et al., 2011a). Class-1 PEPCs are subject to a complex set of posttranslational controls including allosteric effectors, covalent modification via phosphorylation or monoubiquitination, and protein-protein interactions (Uhrig et al., 2008; O’Leary et al., 2009, 2011a, 2011b). Allosteric activation by Glc-6-P and inhibition by l-malate are routinely observed, whereas phosphorylation and dephosphorylation are catalyzed by a Ca²⁺-independent PEPC protein kinase (PPCK) and a protein phosphatase type-2A (PP2A), respectively (O’Leary et al., 2011a). Phosphorylation at a conserved N-terminal seryl residue activates Class-1 PEPCs by decreasing inhibition by malate while increasing activation by Glc-6-P. By contrast, Class-1 PEPC is subject to inhibitory monoubiquitination during castor oil (Ricinus communis) seed (COS) germination, or following depodding of developing COS (Uhrig et al., 2008; O’Leary et al., 2011b). Immunoblots of germinating COS extracts revealed a 1:1 ratio of immunoreactive 110- and 107-kD PTPC polypeptides (p110 and p107, respectively). PEPC purification and mass spectrometry (MS) demonstrated that (1) p110 and p107 are subunits of a 440-kD Class-1 PEPC heterotetramer, (2) both subunits arise from the same PTPC gene (RcPpc3) that also encodes the phosphorylated 410-kD Class-1 PEPC homotetramer of intact developing COS, and (3) p110 is a monoubiquitinated form of p107 (Uhrig et al., 2008). The monoubiquitination site (Lys-628) of COS p110 is conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Incubation with a deubiquitinating enzyme converted the Class-1 PEPC p110:p107 heterotetramer into a p107 homotetramer while exerting significant effects on the enzyme’s kinetic properties (Uhrig et al., 2008). PTPC monoubiquitination rather than phosphorylation is widespread throughout the astor plant and appears to be the predominant posttranslational modification (PTM) of Class-1 PEPC that occurs in unstressed plants (O’Leary et al., 2011b). The distinctive developmental patterns of Class-1 PEPC phosphoactivation versus monoubiquitination-inhibition indicated that these PTMs might be mutually exclusive in the castor plant (O’Leary et al., 2011a, 2011b).Substantial evidence indicates that PEPC plays a pivotal role in plant acclimation to nutritional Pi deficiency (Duff et al., 1989; Vance et al., 2003; O’Leary et al., 2011a; Plaxton and Tran, 2011; Supplemental Fig. S1), a common abiotic stress that frequently limits plant growth in natural ecosystems. The marked induction of Class-1 PEPCs during Pi stress has been linked to the synthesis and excretion of large amounts of organic acid anions by roots of Pi-starved (–Pi) plants (O’Leary et al., 2011a; Uhde-Stone et al., 2003; Vance et al., 2003; Shane et al., 2004a). The excreted organic acids chelate metal cations such as Al³⁺ and Ca²⁺ that immobilize Pi in the soil, thus increasing soluble Pi concentrations by up to 1,000-fold (Vance et al., 2003). Harsh hakea (Hakea prostrata) is a perennial nonmycotroph that has evolved a host of traits that allow it to thrive in the nutrient-impoverished, ancient soils of western Australia. A crucial adaptation of harsh hakea is its proteoid roots, which excrete copious quantities of citrate and malate to mediate Pi solubilization and acquisition from the soil’s mineral-bound Pi (Supplemental Figs. S1 and S2; Shane et al., 2003, 2004a, 2004b; Shane and Lambers, 2005). Shane and coworkers (2004a) correlated proteoid root development in –Pi harsh hakea with marked increases in respiration, internal carboxylate concentrations, and rates of carboxylate exudation. Immunoblotting indicated that PEPC abundance remained relatively constant during proteoid root development, except in senescing 3-week-old roots, where it showed a marked decline. The PEPC immunoblots also revealed approximately 110- and 100-kD immunoreactive polypeptides that were of equal intensity in young proteoid roots, whereas mature proteoid roots showed a marked reduction in the p110 (Shane et al., 2004a). The possible contribution of PTMs such as phosphorylation to the in vivo activation of proteoid root PEPCs is currently unclear (e.g. see Uhde-Stone et al., 2003). However, this is feasible since the pronounced induction of PPCK genes coupled with the reversible phosphorylation-activation of a Class-1 PEPC isozyme (AtPPC1) has been conclusively demonstrated in –Pi Arabidopsis (Arabidopsis thaliana) suspension cells and seedlings (Gregory et al., 2009).The goal of the current study was to test the hypothesis that PEPC PTMs contribute to the metabolic adaptations of harsh hakea proteoid roots. We report a novel metabolic control paradigm that involves the in vivo deubiquitination and consequent kinetic activation of a phosphorylated form of a C₃ plant Class-1 PEPC.