Distribution of Fitness Effects Caused by Single-Nucleotide Substitutions in Bacteriophage f1

Empirical knowledge of the fitness effects of mutations is important for understanding many evolutionary processes, yet this knowledge is often hampered by several sources of measurement error and bias. Most of these problems can be solved using site-directed mutagenesis to engineer single mutations, an approach particularly suited for viruses due to their small genomes. Here, we used this technique to measure the fitness effect of 100 single-nucleotide substitutions in the bacteriophage f1, a filamentous single-strand DNA virus. We found that approximately one-fifth of all mutations are lethal. Viable ones reduced fitness by 11% on average and were accurately described by a log-normal distribution. More than 90% of synonymous substitutions were selectively neutral, while those affecting intergenic regions reduced fitness by 14% on average. Mutations leading to amino acid substitutions had an overall mean deleterious effect of 37%, which increased to 45% for those changing the amino acid polarity. Interestingly, mutations affecting early steps of the infection cycle tended to be more deleterious than those affecting late steps. Finally, we observed at least two beneficial mutations. Our results confirm that high mutational sensitivity is a general property of viruses with small genomes, including RNA and single-strand DNA viruses infecting animals, plants, and bacteria.MUTATIONAL fitness effects are relevant to many evolutionary processes. For instance, they determine the fraction of mutations that evolves neutrally (Ohta 1992), the amount of genetic variation at the mutation–selection balance (Haldane 1937), processes of fitness decay, such as Muller''s ratchet (Butcher 1995), mutational meltdown (Lynch et al. 1993), or lethal mutagenesis (Bull et al. 2007), the ability of organisms to fix beneficial mutations and evolve novel functions (Wagner 2005), or the origin of sex and recombination (Peck et al. 1997; de Visser et al. 2003). Considerable progress has been made in characterizing mutational fitness effects using model organisms or studying genetic variation in natural populations (Eyre-Walker and Keightley 2007). For instance, mutation–accumulation experiments suggest that the average effect of spontaneous deleterious mutations is 1% or lower (Kibota and Lynch 1996) in Escherichia coli, while roughly 90% of engineered gene knockouts are viable (Baba et al. 2006) and transposon insertions reduce fitness by 3% or less on average (Elena et al. 1998). In yeast, mutation–accumulation and chemical mutagenesis experiments have shown that mutations reduce fitness by 1–4% on average in diploid strains (Zeyl and de Visser 2001; Szafraniec et al. 2003; Joseph and Hall 2004). In nematodes most mutations have fitness effects lower than 1% (Keightley and Caballero 1997; Davies et al. 1999), in Drosophila the average effect of mutations ranges from 0.5 to 3.5% (Mukai et al. 1972; Ohnishi 1977; Fernández and López-Fanjul 1996; Fry et al. 1999), and, in humans, most segregating amino acid substitutions have fitness effects lower than 10% (Eyre-Walker and Keightley 2007).Although mutation–accumulation studies provide valuable information about the average effects of deleterious mutations, their power to infer the entire distribution of mutational effects, including neutral and lethal mutations, is more limited. Also, excluding bias due to selection can be problematic, and the precise location and nature of each mutation is often unknown. On the other hand, studies based on engineering mutations have been generally restricted to large deletions or insertions, which are probably infrequent in nature compared to point mutations. A direct and powerful approach that helps us to solve these difficulties consists of introducing single-nucleotide substitutions by site-directed mutagenesis. Due to their small genome sizes, viruses are excellent systems for achieving this goal. In previous work, this technique has been used for studying mutational fitness effects in several RNA viruses (Sanjuán et al. 2004; Carrasco et al. 2007; Domingo-Calap et al. 2009). However, less is known for DNA viruses—but see Domingo-Calap et al. (2009). Here, we use this approach to characterize the distribution of mutational fitness effects in the bacteriophage f1, an inovirus of the bacteriophage m13 clade, making two important improvements over previous work: first, the number of mutations tested is higher (100) and second, the contribution of experimental error to the observed distribution is explicitly accounted for. We show that one-fifth of single-nucleotide substitutions are lethal, while viable ones reduce fitness by 11% on average and can be described by a heavy-tail two-parameter distribution such as the log-normal. Interestingly, the fraction of beneficial mutations is unexpectedly high. We also compare the average effects of different mutation types and of mutations affecting different genes.     

A High Frequency of Beneficial Mutations Across Multiple Fitness Components in Saccharomyces cerevisiae

Mutation-accumulation experiments are widely used to estimate parameters of spontaneous mutations affecting fitness. In many experiments only one component of fitness is measured. In a previous study involving the diploid yeast Saccharomyces cerevisiae, we measured the growth rate of 151 mutation-accumulation lines to estimate parameters of mutation. We found that an unexpectedly high frequency of fitness-altering mutations was beneficial. Here, we build upon our previous work by examining sporulation efficiency, spore viability, and haploid growth rate and find that these components of fitness also show a high frequency of beneficial mutations. We also examine whether mutation-acycumulation (MA) lines show any evidence of pleiotropy among accumulated mutations and find that, for most, there is none. However, MA lines that have zero fitness (i.e., lethality) for any one fitness component do show evidence for pleiotropy among accumulated mutations. We also report estimates of other parameters of mutation based on each component of fitness.ADAPTATION can occur from standing genetic variation or from newly arising mutations. The relative importance of these two sources of adaptive mutations is affected by a variety of factors, including those that alter standing levels of genetic variation (see Barrett and Schluter 2008) and those that generate new mutations. Predicting how quickly a population will adapt and the type of beneficial mutations that will fuel that adaptation requires estimates of the additive genetic variance in fitness and of the beneficial mutation rate and the distribution of beneficial effects. While additive genetic variance for fitness has been estimated in a variety of organisms (Mousseau and Roff 1987), the beneficial mutation rate and the distribution of beneficial effects have only been estimated in a few studies (Shaw et al. 2002; Joseph and Hall 2004; Perfeito et al. 2007; Dickinson 2008; Hall et al. 2008). Surprisingly, these studies estimate that between 6 (Joseph and Hall 2004) and 50% (Shaw et al. 2002) of fitness-altering mutations are beneficial. In contrast, most mutation-accumulation (MA) experiments identify few, if any, beneficial mutations. Such wildly different estimates have even been generated from studies of the same species in similar environments (Zeyl and Devisser 2001; Joseph and Hall 2004; Dickinson 2008; Hall et al. 2008). If these estimates are correct, then they would suggest that the genotypes used in these experiments have vastly different evolutionary potential with respect to their capacity to exhibit rapid adaptation from new mutations.A more likely scenario is that much of the variation in estimates of the beneficial mutation rate is due to methodological differences between studies. One possibility is the fitness component being analyzed. The beneficial mutation rate may be under- or overestimated if the fitness component is under stabilizing selection or subject to antagonistic pleiotropy. Analyses of mutation-accumulation data typically assume that selection is directional. As a result, analyses of phenotypes under stabilizing selection may falsely conclude that mutations that increase a phenotype are beneficial and mutations that lower values are deleterious (see Keightley and Lynch''s 2003 criticism of Shaw et al. 2002). Alternatively, the beneficial mutation rate may be over- (or under) estimated if mutations increase fitness in regard to one component, but lower fitness in regard to lifetime fitness or another fitness component (i.e., antagonistic pleiotropy). Here, we explore these possibilities by investigating whether the high beneficial mutation rates estimated from our previous experiments are specific to the fitness component that we examined.In two previous studies we accumulated mutations in 152 yeast, MA lines and used measures of their effects on diploid growth rate to estimate parameters of beneficial and deleterious mutations. In the first study we estimated that 6% of mutations accumulated during the first 1012 generations of accumulation improved diploid growth (Joseph and Hall 2004). To determine whether this high beneficial mutation rate was due to sampling error, we passaged the lines for an additional 1050 generations and found that 13% of mutations improved diploid growth (Hall et al. 2008). Similarly, another yeast MA experiment (Dickinson 2008) estimated an uncorrected frequency of beneficial mutations of 25%, although correction for within-colony selection reduces this estimate by approximately half. Together, these studies indicate that a substantial proportion of mutations accumulated in these yeast MA lines are beneficial for a single fitness component and that this observation cannot be explained by the chance sampling of a few beneficial mutations.In this study we return to our yeast MA lines (Joseph and Hall 2004) and examine whether the high beneficial mutation rate that we estimated after 1012 generations is an artifact of the fitness component that we examined. To test this hypothesis we examined whether our MA lines carry mutations that are beneficial across multiple fitness components: diploid growth, sporulation efficiency, spore viability, and haploid growth rate. If our previous results are due to us analyzing a fitness component that is either subject to stabilizing selection or antagonistic pleiotropy, then mutations accumulated in our lines will be conditionally beneficial and analyses of additional fitness components would yield different estimates of the beneficial mutation rate. We found that three of the four fitness components yield high estimates of the beneficial mutation rate. This suggests that multiple MA lines have accumulated beneficial mutations and that the high beneficial mutation rate that we previously estimated is not an artifact of the fitness component that we examined.Measuring multiple components of fitness also allowed us to examine the pleiotropic effects of beneficial and deleterious mutations. In general, we found that mutations altering one component of fitness have little effect on other components. However, lethal mutations were typically pleiotropic.

Conclusions:

We find that for three of four fitness components examined, a high frequency of spontaneous, fitness-altering mutations in diploid yeast is beneficial. Further, we do not detect pleiotropy of small-effect mutations, while lethal mutations show high levels of pleiotropy. In most cases, pleiotropy is positive. Two lines show evidence of antagonistic pleiotropy, indicating trade-offs, although heterozygote advantage cannot be ruled out.     

Ribosomal Protein RPL27a Promotes Female Gametophyte Development in a Dose-Dependent Manner

Ribosomal protein mutations in Arabidopsis (Arabidopsis thaliana) result in a range of specific developmental phenotypes. Why ribosomal protein mutants have specific phenotypes is not fully known, but such defects potentially result from ribosome insufficiency, ribosome heterogeneity, or extraribosomal functions of ribosomal proteins. Here, we report that ovule development is sensitive to the level of Ribosomal Protein L27a (RPL27a) and is disrupted by mutations in the two paralogs RPL27aC and RPL27aB. Mutations in RPL27aC result in high levels of female sterility, whereas mutations in RPL27aB have a significant but lesser effect on fertility. Progressive reduction in RPL27a function results in increasing sterility, indicating a dose-dependent relationship between RPL27a and female fertility. RPL27a levels in both the sporophyte and gametophyte affect female gametogenesis, with different developmental outcomes determined by the dose of RPL27a. These results demonstrate that RPL27aC and RPL27aB act redundantly and reveal a function for RPL27a in coordinating complex interactions between sporophyte and gametophyte during ovule development.Eukaryotic cytoplasmic ribosomes are comprised of two subunits, a large 60S and a small 40S subunit. The 60S subunit includes 25S or 28S, 5.8S, and 5S ribosomal RNA (rRNA) and approximately 47 ribosomal proteins, whereas the 40S subunit includes an 18S rRNA and approximately 33 ribosomal proteins. In plants and animals, reduced ribosomal protein function results in specific developmental phenotypes (Byrne, 2009; Warner and McIntosh, 2009; McCann and Baserga, 2013; Terzian and Box, 2013; Tsukaya et al., 2013). Currently, it is not known how ribosomal proteins modulate development. Potentially specific developmental phenotypes in ribosomal protein mutants are an outcome of ribosome haploinsufficiency and reduced global protein synthesis or reduced translation of specific proteins. Alternatively, ribosomal proteins, in addition to their role in translation, may have extraribosomal function required for specific developmental processes.In Arabidopsis (Arabidopsis thaliana), cytoplasmic ribosomal proteins are encoded by two to five genes (Barakat et al., 2001; Giavalisco et al., 2005; Carroll et al., 2008). Mutations in single ribosomal protein genes are sometimes gametophyte or embryo lethal (Weijers et al., 2001; Tzafrir et al., 2004). However, many ribosomal protein mutants are viable. These mutants typically display a subtle change in leaf shape and may also have distinct developmental defects affecting embryo morphogenesis, inflorescence development, the transition to flowering, and plant stature (Van Lijsebettens et al., 1994; Ito et al., 2000; Pinon et al., 2008; Yao et al., 2008; Byrne, 2009; Fujikura et al., 2009; Falcone Ferreyra et al., 2010; Rosado et al., 2010; Horiguchi et al., 2011; Szakonyi and Byrne, 2011a, 2011b; Stirnberg et al., 2012). Female fertility is also reduced in several ribosomal protein mutants. Mutations in the ribosomal protein genes SHORT VALVE1 (STV1)/RPL24B, SUPPRESSOR OF ACAULIS52 (SAC52)/RPL10A, ARABIDOPSIS MINUTE-LIKE1 (AML1)/RPS5B, and the Ribosomal Protein L27a gene RPL27aC reduce female fertility (Weijers et al., 2001; Nishimura et al., 2005; Imai et al., 2008; Szakonyi and Byrne, 2011b). aml1 and sac52-t1 are partially and fully gametophyte lethal, respectively. Although lower fertility in stv1 and rpl27ac is associated with defective ovules, the nature of the fertility defect in these mutants has not been fully explored.Female gametophyte development is also disrupted by mutations in a number of genes predicted to be involved in ribosome biogenesis. SLOW WALKER1 (SWA1), SWA3/Arabidopsis thaliana RNA HELICASE36 (AtRH36), and NUCLEOLAR FACTOR1 (NOF1) encode nucleolar-localized proteins required for processing 18S pre-rRNA (Shi et al., 2005; Harscoët et al., 2010; Huang et al., 2010; Liu et al., 2010). Mutations in other genes encoding proteins predicted to be involved in pre-rRNA processing and ribosome maturation or in export of preribosomes from the nucleus to the cytoplasm also reduce female fertility (Li et al., 2009, 2010; Chantha et al., 2010; Wang et al., 2012; Missbach et al., 2013). These mutants share similar phenotypes, where female gametophyte development is delayed and there is a failure in progression through gametophyte mitotic cell divisions. Transmission of these ribosome biogenesis mutants through the female is often reduced. This ostensibly reflects a requirement for active ribosome synthesis and sufficient ribosome levels to support morphogenesis of the gametophyte.Here, we show that mutations in a number of different ribosomal protein genes lead to reduced seed set and an increase in the number of defective ovules in siliques. This is particularly apparent in mutants affecting ribosomal protein RPL27a. We show the two RPL27a genes, RPL27aC and RPL27aB, act redundantly and that ovule development is sensitive to the dose of RPL27a. rpl27ac and rpl27ab mutations are together female and male gametophyte lethal. Single rpl27ac mutants also result in some female gametophyte lethality. In the homozygous rpl27ac-2 mutant, the mature embryo sac is frequently expelled from the ovule, suggesting RPL27a is necessary for maintaining a viable gametophyte. However, in the heterozygous rpl27ac-2/+, gametogenesis frequently fails early in development. This occurs independent of the genotype of the gametophyte, indicating somatic sporophyte cells in the mutant affect gametophyte development. Together, our data demonstrate that appropriate levels of RPL27a in the sporophyte and gametophyte are required for female gametophyte development and plant fertility.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.     

Class XI Myosins Move Specific Organelles in Pollen Tubes and Are Required for Normal Fertility and Pollen Tube Growth in Arabidopsis

Pollen tube growth is an essential aspect of plant reproduction because it is the mechanism through which nonmotile sperm cells are delivered to ovules, thus allowing fertilization to occur. A pollen tube is a single cell that only grows at the tip, and this tip growth has been shown to depend on actin filaments. It is generally assumed that myosin-driven movements along these actin filaments are required to sustain the high growth rates of pollen tubes. We tested this conjecture by examining seed set, pollen fitness, and pollen tube growth for knockout mutants of five of the six myosin XI genes expressed in pollen of Arabidopsis (Arabidopsis thaliana). Single mutants had little or no reduction in overall fertility, whereas double mutants of highly similar pollen myosins had greater defects in pollen tube growth. In particular, myo11c1 myo11c2 pollen tubes grew more slowly than wild-type pollen tubes, which resulted in reduced fitness compared with the wild type and a drastic reduction in seed set. Golgi stack and peroxisome movements were also significantly reduced, and actin filaments were less organized in myo11c1 myo11c2 pollen tubes. Interestingly, the movement of yellow fluorescent protein-RabA4d-labeled vesicles and their accumulation at pollen tube tips were not affected in the myo11c1 myo11c2 double mutant, demonstrating functional specialization among myosin isoforms. We conclude that class XI myosins are required for organelle motility, actin organization, and optimal growth of pollen tubes.Pollen tubes play a crucial role in flowering plant reproduction. A pollen tube is the vegetative cell of the male gametophyte. It undergoes rapid polarized growth in order to transport the two nonmotile sperm cells to an ovule. This rapid growth is supported by the constant delivery of secretory vesicles to the pollen tube tip, where they fuse with the plasma membrane to enlarge the cell (Bove et al., 2008; Bou Daher and Geitmann, 2011; Chebli et al., 2013). This vesicle delivery is assumed to be driven by the rapid movement of organelles and cytosol throughout the cell, a process that is commonly referred to as cytoplasmic streaming (Shimmen, 2007). Cytoplasmic streaming in angiosperm pollen tubes forms a reverse fountain: organelles moving toward the tip travel along the cell membrane, while organelles moving away from the tip travel through the center of the tube (Heslop-Harrison and Heslop-Harrison, 1990; Derksen et al., 2002). Drug treatments revealed that pollen tube cytoplasmic streaming and tip growth depend on actin filaments (Franke et al., 1972; Mascarenhas and Lafountain, 1972; Heslop-Harrison and Heslop-Harrison, 1989; Parton et al., 2001; Vidali et al., 2001). Curiously, very low concentrations of actin polymerization inhibitors can prevent growth without completely stopping cytoplasmic streaming, indicating that cytoplasmic streaming is not sufficient for pollen tube growth (Vidali et al., 2001). At the same time, however, drug treatments have not been able to specifically inhibit cytoplasmic streaming; thus, it is unknown whether cytoplasmic streaming is necessary for pollen tube growth.Myosins are actin-based motor proteins that actively transport organelles throughout the cell and are responsible for cytoplasmic streaming in plants (Shimmen, 2007; Sparkes, 2011; Madison and Nebenführ, 2013). Myosins can be grouped into at least 30 different classes based on amino acid sequence similarity of the motor domain, of which only class VIII and class XI myosins are found in plants (Odronitz and Kollmar, 2007; Sebé-Pedrós et al., 2014). Class VIII and class XI myosins have similar domain architecture. The N-terminal motor domain binds actin and hydrolyzes ATP (Tominaga et al., 2003) and is often preceded by an SH3-like (for sarcoma homology3) domain of unknown function. The neck domain, containing IQ (Ile-Gln) motifs, acts as a lever arm and is bound by calmodulin-like proteins that mediate calcium regulation of motor activity (Kinkema and Schiefelbein, 1994; Yokota et al., 1999; Tominaga et al., 2012). The coiled-coil domain facilitates dimerization (Li and Nebenführ, 2008), and the globular tail functions as the cargo-binding domain (Li and Nebenführ, 2007). Class VIII myosins also contain an N-terminal extension, MyTH8 (for myosin tail homology8; Mühlhausen and Kollmar, 2013), and class XI myosins contain a dilute domain in the C-terminal globular tail (Kinkema and Schiefelbein, 1994; Odronitz and Kollmar, 2007; Sebé-Pedrós et al., 2014). Recently, Mühlhausen and Kollmar (2013) proposed a new nomenclature for plant myosins based on a comprehensive phylogenetic analysis of all known plant myosins that clearly identifies paralogs and makes interspecies comparisons easier (Madison and Nebenführ, 2013).The localization of class VIII myosins, as determined by immunolocalization and the expression of fluorescently labeled full-length or tail constructs, has implicated these myosins in cell-to-cell communication, cell division, and endocytosis in angiosperms and moss (Reichelt et al., 1999; Van Damme et al., 2004; Avisar et al., 2008; Golomb et al., 2008; Sattarzadeh et al., 2008; Yuan et al., 2011; Haraguchi et al., 2014; Wu and Bezanilla, 2014). On the other hand, class XI myosin mutants have been studied extensively in Arabidopsis (Arabidopsis thaliana), which revealed roles for class XI myosins in cell expansion and organelle motility (Ojangu et al., 2007, 2012; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008; Park and Nebenführ, 2013). Very few studies have examined the reproductive tissues of class XI myosin mutants. In rice (Oryza sativa), one myosin XI was shown to be required for normal pollen development under short-day conditions (Jiang et al., 2007). In Arabidopsis, class XI myosins are required for stigmatic papillae elongation, which is necessary for normal fertility (Ojangu et al., 2012). Even though pollen tubes of myosin XI mutants have not been examined, the tip growth of another tip-growing plant cell has been thoroughly examined in myosin mutants. Root hairs are tubular outgrowths of root epidermal cells that function to increase the surface area of the root for water and nutrient uptake. Two myosin XI mutants have shorter root hairs, of which the myo11e1 (xik; myosin XI K) mutation has been shown to be associated with a slower root hair growth rate and reduced actin dynamics compared with the wild type (Ojangu et al., 2007; Peremyslov et al., 2008; Park and Nebenführ, 2013). Higher order mutants have a further reduction in root hair growth and have altered actin organization (Prokhnevsky et al., 2008; Peremyslov et al., 2010). Disruption of actin organization was also observed in myosin XI mutants of the moss Physcomitrella patens (Vidali et al., 2010), where these motors appear to coordinate the formation of actin filaments in the apical dome of the tip-growing protonemal cells (Furt et al., 2013). Interestingly, organelle movements in P. patens are much slower than in angiosperms and do not seem to depend on myosin motors (Furt et al., 2012).The function of myosins in pollen tubes is currently not known, although it is generally assumed that they are responsible for the prominent cytoplasmic streaming observed in these cells by associating with organelle surfaces (Kohno and Shimmen, 1988; Shimmen, 2007). Myosin from lily (Lilium longiflorum) pollen tubes was isolated biochemically and shown to move actin filaments with a speed of about 8 µm s⁻¹ (Yokota and Shimmen, 1994) in a calcium-dependent manner (Yokota et al., 1999). Antibodies against this myosin labeled small structures in both the tip region and along the shank (Yokota et al., 1995), consistent with the proposed role of this motor in moving secretory vesicles to the apex.In Arabidopsis, six of 13 myosin XI genes are highly expressed in pollen: Myo11A1 (XIA), Myo11A2 (XID), Myo11B1 (XIB), Myo11C1 (XIC), Myo11C2 (XIE), and Myo11D (XIJ; Peremyslov et al., 2011; Sparkes, 2011). The original gene names (Reddy and Day, 2001) are given in parentheses. Myo11D is the only short-tailed myosin XI in Arabidopsis (Mühlhausen and Kollmar, 2013) and lacks the typical myosin XI globular tail involved in cargo binding (Li and Nebenführ, 2007). The remaining genes have the same domain architecture as the conventional class XI myosins that have been shown to be involved in the elongation of trichomes, stigmatic papillae, and root hairs (Ojangu et al., 2007, 2012; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008; Park and Nebenführ, 2013). Therefore, we predicted that these five pollen-expressed, conventional class XI myosins are required for the rapid elongation of pollen tubes. In this study, we examined transfer DNA (T-DNA) insertion mutants of Myo11A1, Myo11A2, Myo11B1, Myo11C1, and Myo11C2 for defects in fertility and pollen tube growth. Organelle motility and actin organization were also examined in myo11c1 myo11c2 pollen tubes.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Patterns of Metabolite Changes Identified from Large-Scale Gene Perturbations in Arabidopsis Using a Genome-Scale Metabolic Network

Metabolomics enables quantitative evaluation of metabolic changes caused by genetic or environmental perturbations. However, little is known about how perturbing a single gene changes the metabolic system as a whole and which network and functional properties are involved in this response. To answer this question, we investigated the metabolite profiles from 136 mutants with single gene perturbations of functionally diverse Arabidopsis (Arabidopsis thaliana) genes. Fewer than 10 metabolites were changed significantly relative to the wild type in most of the mutants, indicating that the metabolic network was robust to perturbations of single metabolic genes. These changed metabolites were closer to each other in a genome-scale metabolic network than expected by chance, supporting the notion that the genetic perturbations changed the network more locally than globally. Surprisingly, the changed metabolites were close to the perturbed reactions in only 30% of the mutants of the well-characterized genes. To determine the factors that contributed to the distance between the observed metabolic changes and the perturbation site in the network, we examined nine network and functional properties of the perturbed genes. Only the isozyme number affected the distance between the perturbed reactions and changed metabolites. This study revealed patterns of metabolic changes from large-scale gene perturbations and relationships between characteristics of the perturbed genes and metabolic changes.Rational and quantitative assessment of metabolic changes in response to genetic modification (GM) is an open question and in need of innovative solutions. Nontargeted metabolite profiling can detect thousands of compounds, but it is not easy to understand the significance of the changed metabolites in the biochemical and biological context of the organism. To better assess the changes in metabolites from nontargeted metabolomics studies, it is important to examine the changed metabolites in the context of the genome-scale metabolic network of the organism.Metabolomics is a technique that aims to quantify all the metabolites in a biological system (Nikolau and Wurtele, 2007; Nicholson and Lindon, 2008; Roessner and Bowne, 2009). It has been used widely in studies ranging from disease diagnosis (Holmes et al., 2008; DeBerardinis and Thompson, 2012) and drug discovery (Cascante et al., 2002; Kell, 2006) to metabolic reconstruction (Feist et al., 2009; Kim et al., 2012) and metabolic engineering (Keasling, 2010; Lee et al., 2011). Metabolomic studies have demonstrated the possibility of identifying gene functions from changes in the relative concentrations of metabolites (metabotypes or metabolic signatures; Ebbels et al., 2004) in various species including yeast (Saccharomyces cerevisiae; Raamsdonk et al., 2001; Allen et al., 2003), Arabidopsis (Arabidopsis thaliana; Brotman et al., 2011), tomato (Solanum lycopersicum; Schauer et al., 2006), and maize (Zea mays; Riedelsheimer et al., 2012). Metabolomics has also been used to better understand how plants interact with their environments (Field and Lake, 2011), including their responses to biotic and abiotic stresses (Dixon et al., 2006; Arbona et al., 2013), and to predict important agronomic traits (Riedelsheimer et al., 2012). Metabolite profiling has been performed on many plant species, including angiosperms such as Arabidopsis, poplar (Populus trichocarpa), and Catharanthus roseus (Sumner et al., 2003; Rischer et al., 2006), basal land plants such as Selaginella moellendorffii and Physcomitrella patens (Erxleben et al., 2012; Yobi et al., 2012), and Chlamydomonas reinhardtii (Fernie et al., 2012; Davis et al., 2013). With the availability of whole genome sequences of various species, metabolomics has the potential to become a useful tool for elucidating the functions of genes using large-scale systematic analyses (Fiehn et al., 2000; Saito and Matsuda, 2010; Hur et al., 2013).Although metabolomics data have the potential for identifying the roles of genes that are associated with metabolic phenotypes, the biochemical mechanisms that link functions of genes with metabolic phenotypes are still poorly characterized. For example, we do not yet know the principles behind how perturbing the expression of a single gene changes the metabolic system as a whole. Large-scale metabolomics data have provided useful resources for linking phenotypes to genotypes (Fiehn et al., 2000; Roessner et al., 2001; Tikunov et al., 2005; Schauer et al., 2006; Lu et al., 2011; Fukushima et al., 2014). For example, Lu et al. (2011) compared morphological and metabolic phenotypes from more than 5,000 Arabidopsis chloroplast mutants using gas chromatography (GC)- and liquid chromatography (LC)-mass spectrometry (MS). Fukushima et al. (2014) generated metabolite profiles from various characterized and uncharacterized mutant plants and clustered the mutants with similar metabolic phenotypes by conducting multidimensional scaling with quantified metabolic phenotypes. Nonetheless, representation and analysis of such a large amount of data remains a challenge for scientific discovery (Lu et al., 2011). In addition, these studies do not examine the topological and functional characteristics of metabolic changes in the context of a genome-scale metabolic network. To understand the relationship between genotype and metabolic phenotype, we need to investigate the metabolic changes caused by perturbing the expression of a gene in a genome-scale metabolic network perspective, because metabolic pathways are not independent biochemical factories but are components of a complex network (Berg et al., 2002; Merico et al., 2009).Much progress has been made in the last 2 decades to represent metabolism at a genome scale (Terzer et al., 2009). The advances in genome sequencing and emerging fields such as biocuration and bioinformatics enabled the representation of genome-scale metabolic network reconstructions for model organisms (Bassel et al., 2012). Genome-scale metabolic models have been built and applied broadly from microbes to plants. The first step toward modeling a genome-scale metabolism in a plant species started with developing a genome-scale metabolic pathway database for Arabidopsis (AraCyc; Mueller et al., 2003) from reference pathway databases (Kanehisa and Goto, 2000; Karp et al., 2002; Zhang et al., 2010). Genome-scale metabolic pathway databases have been built for several plant species (Mueller et al., 2005; Zhang et al., 2005, 2010; Urbanczyk-Wochniak and Sumner, 2007; May et al., 2009; Dharmawardhana et al., 2013; Monaco et al., 2013, 2014; Van Moerkercke et al., 2013; Chae et al., 2014; Jung et al., 2014). Efforts have been made to develop predictive genome-scale metabolic models using enzyme kinetics and stoichiometric flux-balance approaches (Sweetlove et al., 2008). de Oliveira Dal’Molin et al. (2010) developed a genome-scale metabolic model for Arabidopsis and successfully validated the model by predicting the classical photorespiratory cycle as well as known key differences between redox metabolism in photosynthetic and nonphotosynthetic plant cells. Other genome-scale models have been developed for Arabidopsis (Poolman et al., 2009; Radrich et al., 2010; Mintz-Oron et al., 2012), C. reinhardtii (Chang et al., 2011; Dal’Molin et al., 2011), maize (Dal’Molin et al., 2010; Saha et al., 2011), sorghum (Sorghum bicolor; Dal’Molin et al., 2010), and sugarcane (Saccharum officinarum; Dal’Molin et al., 2010). These predictive models have the potential to be applied broadly in fields such as metabolic engineering, drug target discovery, identification of gene function, study of evolutionary processes, risk assessment of genetically modified crops, and interpretations of mutant phenotypes (Feist and Palsson, 2008; Ricroch et al., 2011).Here, we interrogate the metabotypes caused by 136 single gene perturbations of Arabidopsis by analyzing the relative concentration changes of 1,348 chemically identified metabolites using a reconstructed genome-scale metabolic network. We examine the characteristics of the changed metabolites (the metabolites whose relative concentrations were significantly different in mutants relative to the wild type) in the metabolic network to uncover biological and topological consequences of the perturbed genes.     

New Evidence for Differential Roles of L10 Ribosomal Proteins from Arabidopsis

The RIBOSOMAL PROTEIN L10 (RPL10) is an integral component of the eukaryotic ribosome large subunit. Besides being a constituent of ribosomes and participating in protein translation, additional extraribosomal functions in the nucleus have been described for RPL10 in different organisms. Previously, we demonstrated that Arabidopsis (Arabidopsis thaliana) RPL10 genes are involved in development and translation under ultraviolet B (UV-B) stress. In this work, transgenic plants expressing Pro_RPL10:β-glucuronidase fusions show that, while AtRPL10A and AtRPL10B are expressed both in the female and male reproductive organs, AtRPL10C expression is restricted to pollen grains. Moreover, the characterization of double rpl10 mutants indicates that the three AtRPL10s differentially contribute to the total RPL10 activity in the male gametophyte. All three AtRPL10 proteins mainly accumulate in the cytosol but also in the nucleus, suggesting extraribosomal functions. After UV-B treatment, only AtRPL10B localization increases in the nuclei. We also here demonstrate that the three AtRPL10 genes can complement a yeast RPL10 mutant. Finally, the involvement of RPL10B and RPL10C in UV-B responses was analyzed by two-dimensional gels followed by mass spectrometry. Overall, our data provide new evidence about the nonredundant roles of RPL10 proteins in Arabidopsis.In eukaryotes, the cytosolic ribosomes consist of large 60S and small 40S subunits. In Arabidopsis (Arabidopsis thaliana), ribosomal protein genes exist as families composed of two to seven members that could be differentially incorporated into the cytosolic ribosome under specific situations (Schmid et al., 2005; Byrne, 2009). In this way, ribosomal heterogeneity would allow selective translation of specific mRNAs under particular cell conditions (Barakat et al., 2001; Szick-Miranda and Bailey-Serres, 2001; Giavalisco et al., 2005; Carroll et al., 2008; Carroll, 2013). Arabidopsis mutants in ribosomal proteins exhibit a large range of developmental phenotypes with extreme abnormalities, including embryonic lethality, suggesting that ribosomes also have specific functions regulating the expression of developmental genes (Van Lijsebettens et al., 1994; Degenhardt and Bonham-Smith, 2008; Byrne, 2009; Horiguchi et al., 2011, 2012; Szakonyi and Byrne, 2011). Furthermore, it has been recently demonstrated that ribosomal proteins control auxin-mediated developmental programs by translational regulation of auxin response factors (Rosado et al., 2012). In addition, the characterization of single, double, and, in certain cases, triple mutants as well as complementation by paralog genes have demonstrated full, partial, and no redundancy between members of ribosomal protein families (Briggs et al., 2006; Guo and Chen, 2008; Guo et al., 2011; Horiguchi et al., 2011; Stirnberg et al., 2012).RIBOSOMAL PROTEIN L10 (RPL10) was initially identified in humans as a putative suppressor of Wilms’ tumor (Dowdy et al., 1991). Since then, RPL10 has been studied in different organisms from archaea and bacteria to eukaryotes such as mammals, insects, yeast, and plants (Marty et al., 1993; Mills et al., 1999; Hwang et al., 2000; Zhang et al., 2004; Wen et al., 2005; Singh et al., 2009). A remarkable property of this protein is its high degree of amino acid conservation, suggesting fundamental and critical conserved functions of RPL10 in different organisms (Farmer et al., 1994; Eisinger et al., 1997; Hofer et al., 2007; Nishimura et al., 2008). Likewise, the crystallographic structural similarity observed among RPL10 orthologs in eukaryotes, bacteria, and archaea (called L16) established the conservation of this universal ribosomal protein family and provided evidence of the inalterability of the ribosome during evolution (Spahn et al., 2001; Nishimura et al., 2008). Nevertheless, besides being a constituent of ribosomes and participating in protein translation, additional extraribosomal functions have been described for RPL10 (Mills et al., 1999; Hwang et al., 2000; Chávez-Rios et al., 2003; Zhang et al., 2004; Singh et al., 2009). In yeast, RPL10 is essential for viability, organizes the union site of the aminoacyl-tRNA, and its incorporation into the 60S subunit is a prerequisite for subunit joining and the initiation of translation (West et al., 2005; Hofer et al., 2007). Extensive analysis of the in vivo assembly of ribosomes revealed that RPL10 is loaded to the ribosome in the cytosol with the assistance of its chaperone suppressor of QSR1 truncations (Hedges et al., 2005; West et al., 2005).Arabidopsis has three genes encoding RPL10 proteins, AtRPL10A, AtRPL10B, and, AtRPL10C. Recently, we demonstrated that Arabidopsis RPL10 genes are differentially regulated by UV-B radiation: RPL10B is down-regulated, RPL10C is up-regulated, while RPL10A is not UV-B regulated. Arabidopsis single mutants showed that RPL10 genes are not functionally equivalent. Heterozygous rpl10a mutant plants are translation deficient under UV-B conditions, knockout rpl10A mutants are not viable, and knockdown homozygous rpl10B mutants show abnormal growth. Conversely, knockout homozygous rpl10C mutants do not exhibit any visible phenotype. Overall, RPL10 genes are involved in development and translation under UV-B stress (Falcone Ferreyra et al., 2010b). Furthermore, coimmunoprecipitation studies showed an association of RPL10 with nuclear proteins, suggesting that at least one of the RPL10 isoforms could have an extraribosomal function in the nucleus (Falcone Ferreyra et al., 2010a).The aim of this work was to further investigate the contribution of each Arabidopsis RPL10 to plant development and UV-B responses. We examined the spatiotemporal expression of each AtRPL10 using transgenic plants expressing Pro_RPL10:GUS fusions. By AtRPL10-GFP fusions, we analyzed the subcellular localization of each RPL10, demonstrating that the three isoforms are mainly localized in the cytosol but also in the nucleus. In order to investigate the functional redundancy between AtRPL10 genes in more detail, we generated and characterized double rpl10 mutants. We also here demonstrate that the three AtRPL10 genes can complement a yeast RPL10 mutant. Finally, the involvement of RPL10B and RPL10C in UV-B responses was analyzed by two-dimensional (2D) gels followed by mass spectrometry. Overall, our data provide new insights into the role of each RPL10 in Arabidopsis.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.