Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration

Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)–mediated double-strand DNA break (DSB) repair. Here, we report a negative role of X-RAY CROSS COMPLEMENTATION GROUP4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate.     

CENTRAL REGION COMPONENT1, a Novel Synaptonemal Complex Component,Is Essential for Meiotic Recombination Initiation in Rice

In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated CENTRAL REGION COMPONENT1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID RECEPTOR-INTERACTING PROTEIN13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13.     

The Axial Element Protein DESYNAPTIC2 Mediates Meiotic Double-Strand Break Formation and Synaptonemal Complex Assembly in Maize

During meiosis, homologous chromosomes pair and recombine via repair of programmed DNA double-strand breaks (DSBs). DSBs are formed in the context of chromatin loops, which are anchored to the proteinaceous axial element (AE). The AE later serves as a framework to assemble the synaptonemal complex (SC) that provides a transient but tight connection between homologous chromosomes. Here, we showed that DESYNAPTIC2 (DSY2), a coiled-coil protein, mediates DSB formation and is directly involved in SC assembly in maize (Zea mays). The dsy2 mutant exhibits homologous pairing defects, leading to sterility. Analyses revealed that DSB formation and the number of RADIATION SENSITIVE51 (RAD51) foci are largely reduced, and synapsis is completely abolished in dsy2 meiocytes. Super-resolution structured illumination microscopy showed that DSY2 is located on the AE and forms a distinct alternating pattern with the HORMA-domain protein ASYNAPTIC1 (ASY1). In the dsy2 mutant, localization of ASY1 is affected, and loading of the central element ZIPPER1 (ZYP1) is disrupted. Yeast two-hybrid and bimolecular fluorescence complementation experiments further demonstrated that ZYP1 interacts with DSY2 but does not interact with ASY1. Therefore, DSY2, an AE protein, not only mediates DSB formation but also bridges the AE and central element of SC during meiosis.     

Blocking Single-Stranded Transferred DNA Conversion to Double-Stranded Intermediates by Overexpression of Yeast DNA REPLICATION FACTOR A

Agrobacterium tumefaciens delivers its single-stranded transferred DNA (T-strand) into the host cell nucleus, where it can be converted into double-stranded molecules. Various studies have revealed that double-stranded transfer DNA (T-DNA) intermediates can serve as substrates by as yet uncharacterized integration machinery. Nevertheless, the possibility that T-strands are themselves substrates for integration cannot be ruled out. We attempted to block the conversion of T-strands into double-stranded intermediates prior to integration in order to further investigate the route taken by T-DNA molecules on their way to integration. Transgenic tobacco (Nicotiana benthamiana) plants that overexpress three yeast (Saccharomyces cerevisiae) protein subunits of DNA REPLICATION FACTOR A (RFA) were produced. In yeast, these subunits (RFA1–RFA3) function as a complex that can bind single-stranded DNA molecules, promoting the repair of genomic double strand breaks. Overexpression of the RFA complex in tobacco resulted in decreased T-DNA expression, as determined by infection with A. tumefaciens cells carrying the β-glucuronidase intron reporter gene. Gene expression was not blocked when the reporter gene was delivered by microbombardment. Enhanced green fluorescent protein-assisted localization studies indicated that the three-protein complex was predominantly nuclear, thus indicating its function within the plant cell nucleus, possibly by binding naked T-strands and blocking their conversion into double-stranded intermediates. This notion was further supported by the inhibitory effect of RFA expression on the cell-to-cell movement of Bean dwarf mosaic virus, a single-stranded DNA virus. The observation that RFA complex plants dramatically inhibited the transient expression level of T-DNA and only reduced T-DNA integration by 50% suggests that double-stranded T-DNA intermediates, as well as single-stranded T-DNA, play significant roles in the integration process.Genetic transformation of plant cells by Agrobacterium tumefaciens represents a unique case of transkingdom DNA transfer (Stachel and Zambryski, 1989; Tzfira and Citovsky, 2002; Lacroix and Citovsky, 2013). This bacterium creates crown galls by transforming plant cells with a part of its Ti plasmid, the transfer DNA (T-DNA), at the infection site. The T-DNA, which integrates into the plant genome, contains genes encoding for enzymes involved in the production, modification, and alteration of plant growth regulators‎ and in the production of tumor-specific metabolites called opines. Expression of the integrated T-DNA, therefore, results in uncontrolled cell division and the formation of tumors (Gaudin et al., 1994; Das, 1998).The route that takes the T-DNA from the bacterium to the plant cell nucleus occurs in three major steps. It begins inside A. tumefaciens, where the virulence proteins VirD1 and VirD2 are responsible for processing the single-stranded T-DNA. The T-strand with one VirD2 molecule covalently attached to its 5′ end (Herrera-Estrella et al., 1988; Young and Nester, 1988) is then exported, together with several other virulence proteins, through the bacterial type IV secretion system, where it is most likely coated with many VirE2 molecules, becoming the transported form of the T-DNA, the transport complex (Dumas et al., 2001). VirE2 is probably translocated to the plant cell independently of the VirD2-conjugated T-strand along with other A. tumefaciens effector proteins (Otten et al., 1984; Sundberg et al., 1996). Moreover, VirE2 binds to the T-strand in the plant and thus provides protection from nuclease attack inside the plant cells (Yusibov et al., 1994). Abu-Arish et al. (2004) and Citovsky et al. (1997) used the bacteriophage M13 single-stranded DNA (ssDNA) mixed with purified VirE2 molecules to gain direct insight into the transport complex structure. Electron microscopy and single-particle image-processing methods yielded a better understanding of the transport complex structure. During the last step on the way to the nucleus, VirD2 and VirE2 are thought to assist the import of the transport complex into the host cell nucleus (Howard et al., 1992; Rossi et al., 1993; Citovsky, 1994; Ziemienowicz, 2001), whereas phosphorylated VirE2 INTERACTED PROTEIN1 (VIP1; Djamei et al., 2007; Dafny-Yelin et al., 2008) functions as a molecular linker between VirE2 and KARYOPHERIN-α (KAPα; Tzfira and Citovsky, 2002), allowing VirE2 to use the host cell nuclear import machinery (Tzfira et al., 2001; Citovsky et al., 2004; Anand et al., 2007).Once inside the nucleus, the transport complex is the substrate destined for integration. Two different models have been suggested for the T-DNA integration, double-stranded break (DSB) repair and single-stranded gap repair (SSGR; Gheysen et al., 1991; Mayerhofer et al., 1991). The DSB model predicts that unwound ends of a double-stranded T-DNA molecule anneal with single-stranded overhangs of DSBs in the plant DNA, the residual 5′ and 3′ overhangs are removed, and the inserted T-DNA is ligated. Mutations in the Arabidopsis (Arabidopsis thaliana) RADIATION-SENSITIVE52 (RAD52) gene that plays an essential role in homology-dependent DSB repair and recombination (Symington, 2002) decrease the levels of intrachromosomal recombination compared with wild-type plants (Samach et al., 2011). Recent studies further supported the notion that the T-strand molecule becomes double stranded prior to integration. For example, a direct indication of the involvement of double-stranded integration intermediates came from the detection of T-DNA inserts within the sites of DSB repair in the host DNA, which suggested ligation of double-stranded T-DNA molecules to both sides of the break made using a rear-cutting restriction enzyme (Salomon and Puchta, 1998; Chilton and Que, 2003; Tzfira et al., 2003). Recent evidence shows that T-DNA molecules can be converted into double-stranded T-DNA molecules using T-strand priming (Liang and Tzfira, 2013), and while double-stranded T-DNA molecules may serve as substrates for integration (Chilton and Que, 2003; Tzfira et al., 2003), a recent study showed that double-stranded T-DNA molecules can circularize and may end up as a dead ends for the integration process (Singer ‎et al., 2012). The SSGR model was established as the dominant model for T-DNA integration in plant cells based on two major observations. First, VirD2 is able not only to digest but also to rejoin single-stranded substrates in vitro (Pansegrau et al., 1993). This led to the suggestion that VirD2 may function as the T-DNA ligase in plant cells and, by implication, that T-DNAs integrate as single-stranded molecules. Second, specific mutations at the VirD2 putative integrase motif resulted in small deletions at the 5′ end of the T-DNA molecules (Tinland et al., 1995). Therefore, Tinland et al. (1995) revisited the SSGR integration model to the microhomology-dependent integration model, suggesting that only short sequences of the T-DNA molecule actually anneal to preintegration genomic sites (Tzfira et al., 2004a; Windels et al., 2008; Ziemienowicz et al., 2008).The DSB model suggests that the transport complex is first stripped of its attached protein in the nucleus and then arrives at the integration site as double-stranded DNA (dsDNA). However, in the SSGR model, the transport complex itself reaches the site of integration and then probably degrades during the integration process. Evidence for a physical connection between the transport complex and the chromatin was presented by Loyter et al. (2005), who found that VIP1 acts as a molecular linker between the transport complex and the histone constituents of the host chromatin. When the transport complex reaches the chromatin, VIP1 becomes polyubiquitinated by VirF or Skp1/Cullin/F-box protein VIP1-binding F-box [SCF(VBF); Zaltsman et al., 2010, 2013] containing a proteolysis complex, and after the protein complex is degraded, the single-stranded T-DNA remains naked, with only VirD2 attached to its right border (Magori and Citovsky, 2011).We overexpressed the ssDNA-binding protein REPLICATION FACTOR A (RFA) from yeast (Saccharomyces cerevisiae) in order to further characterize the route that takes the transport complex to the integration site through double- or single-stranded forms. Previous work shows that overexpression of an ssDNA-binding protein from Escherichia coli (i.e. the recombination protein A) can function in plant cells and can interact with or supplement the endogenous plant recombination machinery (Reiss et al., 1996).RFA from yeast is a tightly associated complex composed of three subunits of 69, 36, and 13 kD, termed RFA1, RFA2, and RFA3, respectively (Longhese et al., 1994). Hays et al. (1998) suggested a role for RFA in recruiting DNA polymerase to the site of a DSB via simultaneous interactions with RAD52 and a repair polymerase, in order to protect the exposed single-stranded ends from cellular nucleases (Smith and Rothstein, 1995; Park et al., 1996; Hays et al., 1998). RFA heterologous proteins have been found in plants, but their function in T-DNA integration and their involvement in the DNA-repair mechanism are still unknown. In this study, we investigated the effect of the RFA complex from yeast on the T-DNA integration mechanism in the plant cell nucleus and report that overexpression of the RFA complex (Rex) in tobacco (Nicotiana benthamiana) plants almost completely blocks the T-DNA’s transient and reduced stable transformation, probably by binding to single-stranded T-DNA, and prevents its expression and integration through the DSB pathway. Moreover, we show that the movement of Bean dwarf mosaic virus (BDMV; Begomovirus: Geminiviridae), a single-stranded virus, is inhibited in the Rex-transformed plant.     

The Compromised Recognition of Turnip Crinkle Virus1 Subfamily of Microrchidia ATPases Regulates Disease Resistance in Barley to Biotrophic and Necrotrophic Pathogens

MORC1 and MORC2, two of the seven members of the Arabidopsis (Arabidopsis thaliana) Compromised Recognition of Turnip Crinkle Virus1 subfamily of microrchidia Gyrase, Heat Shock Protein90, Histidine Kinase, MutL (GHKL) ATPases, were previously shown to be required in multiple layers of plant immunity. Here, we show that the barley (Hordeum vulgare) MORCs also are involved in disease resistance. Genome-wide analyses identified five MORCs that are 37% to 48% identical on the protein level to AtMORC1. Unexpectedly, and in clear contrast to Arabidopsis, RNA interference-mediated knockdown of MORC in barley resulted in enhanced basal resistance and effector-triggered, powdery mildew resistance locus A12-mediated resistance against the biotrophic powdery mildew fungus (Blumeria graminis f. sp. hordei), while MORC overexpression decreased resistance. Moreover, barley knockdown mutants also showed higher resistance to Fusarium graminearum. Barley MORCs, like their Arabidopsis homologs, contain the highly conserved GHKL ATPase and S5 domains, which identify them as members of the MORC superfamily. Like AtMORC1, barley MORC1 (HvMORC1) binds DNA and has Mn²⁺-dependent endonuclease activities, suggesting that the contrasting function of MORC1 homologs in barley versus Arabidopsis is not due to differences in their enzyme activities. In contrast to AtMORCs, which are involved in silencing of transposons that are largely restricted to pericentromeric regions, barley MORC mutants did not show a loss-of-transposon silencing regardless of their genomic location. Reciprocal overexpression of MORC1 homologs in barley and Arabidopsis showed that AtMORC1 and HvMORC1 could not restore each other’s function. Together, these results suggest that MORC proteins function as modulators of immunity, which can act negatively (barley) or positively (Arabidopsis) dependent on the species.The evolution of a complex defense system has been the consequence of plants being constantly exposed to pathogenic microbes and pests. One of the first lines of active defense is based on a perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors located in the plant cell membrane. The defense response to PAMP recognition is called PAMP-triggered immunity (PTI). While PTI is sufficient to stop colonization by many microbes, some microorganisms overcome this immune response by releasing effectors (formerly called virulence factors). In a coevolutionary process, some plants have evolved resistance (R) proteins for direct or indirect recognition of microbial effectors (avirulence [Avr] factors) leading to effector-triggered immunity (ETI). ETI is frequently characterized by a rapid and locally restricted programmed cell death response (also known as hypersensitive reaction [HR]), which helps to limit pathogen proliferation and disease symptoms. On the contrary, the absence of an Avr-R protein interaction results in virulence of the pathogen. In addition, ETI is counteracted by some microbes by the release of additional virulence factors that block or overcome effector recognition and ensure pathogenicity. The mutual evolution of host and microbe leading to elicitation or suppression of ETI is summarized by the “zigzag” model proposed by Jones and Dangl (2006). PTI and ETI are associated with activation of various defense responses both at infection sites and in distal tissue, including production and accumulation of reactive oxygen species, salicylic acid, and pathogenesis-related proteins. Systemic activation of such responses, triggered in the uninfected tissue, leads to long-lasting, broad-based resistance to subsequent pathogen infections, termed systemic acquired resistance.A genetic screen in Arabidopsis (Arabidopsis thaliana) searching for mutants with compromised resistance mediated by the R protein HR to Turnip Crinkle Virus (HRT) against Turnip Crinkle Virus (TCV) led to the discovery of the Compromised Recognition of TCV1 (CRT1) subfamily of the microrchidia (MORC) subclade of the GHKL (for Gyrase, Heat Shock Protein90, Histidine Kinase, MutL) ATPase superfamily (Watson et al., 1998; Iyer et al., 2008; Kang et al., 2008). Genome analysis of Arabidopsis revealed that MORC1 (formerly named CRT1 in Kang et al., 2008, 2010, 2012) has two close (>70% sequence similarity on amino acid [aa] level) and four distant (<50% aa similarity) homologs. A double knockout mutant, morc1-2 morc2-1, lacking MORC1 and its closest homolog MORC2 also displayed compromised ETI to avirulent Pseudomonas syringae, suppressed basal resistance, systemic acquired resistance, and/or PTI to TCV and virulent P. syringae and compromised nonhost resistance to Phytophthora infestans (Kang et al., 2012). Arabidopsis MORC1 physically interacts with at least eleven R proteins belonging to three different structural classes (Martin et al., 2003), including HRT, the R protein involved in recognition of TCV. This interaction is a dynamic process, as MORC1 bound inactive R proteins, while little or no interaction was observed when the R proteins were activated (Kang et al., 2010). Taken together, these results argued that MORC1 protein family members in Arabidopsis are key components in multiple layers of resistance against a variety of pathogens. Recently, it was shown that a small fraction of AtMORC1 translocates to the plant nucleus after ETI and PTI activation (Kang et al., 2012). Because Arabidopsis MORC1 possesses DNA/RNA-binding capacity and endonuclease activity in vitro, these findings suggest a potential role in DNA recombination and repair (Kang et al., 2012). In addition, three recent independent studies identified Arabidopsis MORC1 and its homolog MORC6 (also named Defective in Meristem Silencing11) as novel factors involved in gene silencing and/or chromatin superstructure remodeling in response to epigenetic signals (Lorković et al., 2012; Moissiard et al., 2012; Brabbs et al., 2013).Given that the CRT1 subfamily of MORC ATPases is involved in multiple layers of disease resistance against various pathogens, these genes may have relevance for agronomic applications. To assess whether MORCs are involved in crop plant resistance and thus could be exploited in breeding strategies, MORC1 homologous genes were identified in the model cereal crop barley (Hordeum vulgare). We show here that all five barley MORCs, discovered in the not yet fully annotated barley genome, are involved in resistance to agronomically important diseases. Unexpectedly, however, and in clear contrast to Arabidopsis, barley plants silenced for MORC genes were more resistant, while overexpression compromised resistance to infections by both biotrophic and necrotrophic fungal pathogens. Moreover, reciprocal overexpression in Arabidopsis and barley showed that AtMORC1 and HvMORC1 homologs are not functionally interchangeable.     

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.     

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.     

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.     

MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice

F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression.