Ancestral and more recently acquired syntenic relationships of MADS-box genes uncovered by the <Emphasis Type="Italic">Physcomitrella patens</Emphasis> pseudochromosomal genome assembly

Key message

The Physcomitrella pseudochromosomal genome assembly revealed previously invisible synteny enabling realisation of the full potential of shared synteny as a tool for probing evolution of this plant’s MADS-box gene family.

Abstract

Assembly of the sequenced genome of Physcomitrella patens into 27 mega-scaffolds (pseudochromosomes) has confirmed the major predictions of our earlier model of expansion of the MADS-box gene family in the Physcomitrella lineage. Additionally, microsynteny has been conserved in the immediate vicinity of some recent duplicates of MADS-box genes. However, comparison of non-syntenic MIKC MADS-box genes and neighbouring genes indicates that chromosomal rearrangements and/or sequence degeneration have destroyed shared synteny over longer distances (macrosynteny) around MADS-box genes despite subsets comprising two or three MIKC genes having remained syntenic. In contrast, half of the type I MADS-box genes have been transposed creating new syntenic relations with MIKC genes. This implies that conservation of ancient ancestral synteny of MIKC genes and of more recently acquired synteny of type I and MIKC genes may be selectively advantageous. Our revised model predicts the birth rate of MIKC genes in Physcomitrella is higher than that of type I genes. However, this difference is attributable to an early tandem duplication and an early segmental duplication of MIKC genes prior to the two polyploidisations that account for most of the expansion of the MADS-box gene family in Physcomitrella. Furthermore, this early segmental duplication spawned two chromosomal lineages: one with a MIKC ^C gene, belonging to the PPM2 clade, in close proximity to one or a pair of MIKC* genes and another with a MIKC ^C gene, belonging to the PpMADS-S clade, characterised by greater separation from syntenic MIKC* genes. Our model has evolutionary implications for the Physcomitrella karyotype.

     

<Emphasis Type="Italic">SOC1</Emphasis> and <Emphasis Type="Italic">AGL24</Emphasis> interact with AGL18-1, not the other family members AGL18-2 and AGL18-3 in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Many MCM1-AGAMOUS-DEFICIENS-SRF (MADS) genes have been proved to play an important role in the flowering time regulation of plants. The flowering-inhibiting factor AGAMOUS-LIKE 18 (AGL18) integrates into the two flowering-activating factors SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and AGAMOUS-LIKE 24 (AGL24), which play an important role during the plant developmental stages of the flowering pathway. However, it remains unknown whether and how the AGL18 protein directly interacts with SOC1 and/or AGL24 genes to regulate flowering time in Brassica juncea. In this study, three members (AGL18-1 in florescence, AGL18-2 and AGL18-3 in young seedlings) of the AGL18 family, and SOC1 and AGL24 in florescence were cloned in Brassica juncea. Yeast One-Hybrid assays and Dual-Glo^® Luciferase assays showed that the SOC1 and AGL24 promoters interacted only with AGL18-1 protein, not AGL18-2 and AGL18-3. The typical conserved structure of the M-domain of AGL18-1 was the key region that mediated the interaction between the AGL18-1 protein and SOC1 promoter, and the I-domain, K-domain and C-domain did not regulate the interaction of AGL18-1/SOC1. In contrast, the K-domain and M-domain in AGL18-1 could mediate the interaction between the AGL18-1 protein and AGL24 promoter. This indicated that the AGL18-1 protein must have its unique functions that differed from AGL18-2 and AGL18-3. This work provides valuable information for in-depth studies into the molecular mechanisms of the AGL18 protein with SOC1 and AGL24 for flowering time control of Brassica juncea.     

Synthesis, characterization of [{(N-methyl-N-benzyl)amino}methyl]ferrocenes and their cyclopalladated complexes: Crystal structure of σ-Pd[(η-C5H5)Fe(η-C5H3CH2N(CH3)CH2C6H4NO2-4)]Cl(PPh3)

Condensation of aminomethylferrocene (1) and substituted benzaldehydes resulted in aldimines 2a-c which followed by reduction with sodium borohydride to give 3a-c. N-methylation of 3a-c with HCHO/NaCNBH₃/HOAc led to 4a-c. Treatment of 4a-c with sodium palladium tetrachloride in the presence of sodium acetate afforded cleanly cyclopalladated 5a-c in which configurations consisted of the R_NR_C, S_NS_C. The preferable activation of C_Ferrocenyl-H bond over C_Phenyl-H bond was also observed. All compounds 2-5 were characterized by elemental analysis, IR and ¹H NMR. In addition, the molecular structure of 5c was confirmed by single crystal X-ray diffraction. The possible mechanism for the formation of 5 was also discussed.     

The Vaccinium corymbosum FLOWERING LOCUS T-like gene (VcFT): a flowering activator reverses photoperiodic and chilling requirements in blueberry

Key message

The blueberry FLOWERING LOCUS T ( FT )-like gene ( VcFT ) cloned from the cDNA of a tetraploid, northern highbush blueberry ( Vaccinium corymbosum L.) is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.

Abstract

Blueberry is a woody perennial bush with a longer juvenile period than annual crops, requiring vernalization to flower normally. Few studies have been reported on the molecular mechanism of flowering in blueberry or other woody plants. Because FLOWERING LOCUS T (FT) from Arabidopsis thaliana plays a multifaceted role in generating mobile molecular signals to regulate plant flowering time, isolation and functional analysis of the blueberry (Vaccinium corymbosum L.) FT-like gene (VcFT) will facilitate the elucidation of molecular mechanisms of flowering in woody plants. Based on EST sequences, a 525-bpVcFT was identified and cloned from the cDNA of a tetraploid, northern highbush blueberry cultivar, Bluecrop. Ectopic expression of 35S:VcFT in tobacco induced flowering an average of 28 days earlier than wild-type plants. Expression of the 35S:VcFT in the blueberry cultivar Aurora resulted in an extremely early flowering phenotype, which flowered not only during in vitro culture, a growth stage when nontransgenic shoots had not yet flowered, but also in 6–10-week old, soil-grown transgenic plants, in contrast to the fact that at least 1 year and 800 chilling hours are required for the appearance of the first flower of both nontransgenic ‘Aurora’ and transgenic controls with the gusA. These results demonstrate that the VcFT is a functional floral activator and overexpression of the VcFT is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.     

Synthesis of Pyrrolo[2′,3′:4,5]Furo[3,2-c]Pyridine-2-Carboxylic Acids

1H-Pyrrolo[2′,3′:4,5]furo[3,2-c]pyridine-2-carboxylic acid (6a) and its 1-methyl (6b) and 1-benzyl (6c) derivatives were synthesized. 3-(5-Methoxycarbonyl-4H-furo[3,2-b]-pyrrole-2-yl)propenoic acid (1) was converted to the corresponding azide 2, which in turn was cyclized to give 3 by heating in diphenylether. The pyridone 3 obtained was aromatized with phosphorus oxychloride, then reduced with zinc in acetic acid to give methyl 1H-pyrrolo[2′,3′:4,5]furo[3,2-c]pyridine-2-carboxylate (5), which by hydrolysis gave the corresponding carboxylic acid 6a.     

Increased gibberellin contents contribute to accelerated growth and development of transgenic tobacco overexpressing a wheat ubiquitin gene

Key message

Overexpressing TaUb2 promoted stem growth and resulted in early flowering in transgenic tobacco plants. Ubiquitin are involved in the production, metabolism and proper function of gibberellin.

Abstract

The ubiquitin–26S proteasome system (UPS), in which ubiquitin (Ub) functions as a marker, is a post-translational regulatory system that plays a prominent role in various biological processes. To investigate the impact of different Ub levels on plant growth and development, transgenic tobacco (Nicotiana tabacum L.) plants were engineered to express an Ub gene (TaUb2) from wheat (Triticum aestivum L.) under the control of cauliflower mosaic virus 35S promoter. Transgenic tobacco plants overexpressing TaUb2 demonstrated an accelerated growth rate at early stage and an early flowering phenotype in development. The preceding expression of MADS-box genes also corresponded to the accelerated developmental phenotypes of the transgenic tobacco plants compared to that of wild-type (WT). Total gibberellin (GA) and active GA contents in transgenic tobacco plants were higher than those in WT at the corresponding developmental stages, and some GA metabolism genes were upregulated. Treatment with GA₃ conferred a similarly accelerated grown rate in WT plants to that of transgenic tobacco plants, while growth was inhibited when transgenic tobacco plants were treated with a GA biosynthesis inhibitor. Thus, the results suggest that Ub are involved in the production, metabolism and proper function of GA, which is important in the regulation of plant growth and development.     

Density functional studies on photophysical properties and chemical reactivities of the triarylboranes: effect of the constraint of planarity

The geometric and electronic structures, absorption spectra, transporting properties, chemical reactivity indices and electrostatic potentials of the planar three-coordinate organoboron compounds 1-2 and twisted reference compound Mes ₃ B, have been investigated by employing density functional theory (DFT) and conceptual DFT methods to shed light on the planarity effects on the photophysical properties and the chemical reactivity. The results show that the planar compounds 1-2 exhibit significantly lower HOMO level than Mes ₃ B, owing to the stronger electronic induction effect of boron centers. This feature conspicuously induces a blue shifted absorption for 1, although 1 seemingly possesses more extended conjugation framework than Mes ₃ B. Importantly, the reactivity strength of the boron atoms in 1-2 is much lower than that in Mes ₃ B, despite the fact that the tri-coordinate boron centers of 1-2 are completely naked. The interesting and abnormal phenomenon is caused by the strong p-π electronic interactions, that is, the empty p-orbital of boron center is partly filled by π-electron of the neighbor carbon atoms in 1-2, which are confirmed by the analysis of Laplacian of the electron density and natural bond orbitals. Furthermore, the negative electrostatic potentials of the boron centers in 1-2 also interpret that they are not the most preferred sites for incoming nucleophiles. Moreover, it is also found that the planar compounds 1-2 can act as promising electron transporting materials since the internal reorganization energies for electron are really small.

2-(1-(Dimethylamino)ethyl)phenylpalladium(II) complexes 5-functionalized with fluorous silyl tails

New fluorous-organometallics based on the chiral ligand α-methyl-N,N-dimethylbenzylamine (TMBA) were prepared by treatment of fluorous silyl bromide reagents with in situ 4-lithiated TMBA to give fluorous N,N-dimethyl(α-methyl-4-trialkylsilylbenzyl)amine ligands 1a-1c that vary in the number of fluorous tails attached to the Si atom. Ligands 1a-1c were successfully cyclo-palladated by treatment with Pd(OAc)₂/LiCl in methanol to furnish the corresponding chloride-bridged dimeric arylpalladium(II) complexes 2a-2c in good yields. The latter derivatives could be converted into monomeric Lewis-base adducts by complexation with pyridine (3a-3c), or triphenylphosphine (4a-4c). The crystal structure of triphenylphosphine complex 4a has been elucidated. To probe their fluorophilicity, the partition coefficient of each of the derivatives in the fluorous biphasic solvent (FBS) system perfluoromethylcyclohexane/n-octane has been determined.     

MADS about MOSS

Classic MIKC-type MADS-box genes (MIKC^c) play diverse and crucial roles in angiosperm development, the most studied and best understood of which is the specification of floral organ identities. To shed light on how the flower evolved, phylogenetic and functional analyses of genes involved in its ontogeny, such as the MIKC^c genes, must be undertaken in as broad a selection as possible of plants with disparate ancestries. Since little is known about the functions of these genes in non-seed plants, we investigated the developmental roles of a subset of the MIKC^c genes present in the moss, Physcomitrella patens, which is positioned informatively near the base of the land plant evolutionary tree. We observed that transgenic lines possessing an antisense copy of a MIKC^c gene characteristically displayed knocked-down expression of the corresponding native MIKC^c gene as well as multiple diverse phenotypic alterations to the haploid gametophytic and diploid sporophytic generations of the life cycle.¹ In this addendum, we re-examine our findings in the light of recent pertinent literature and provide additional data concerning the effects of simultaneously knocking out multiple MIKC^c genes in this moss.Key words: Physcomitrella, moss, MADS-box gene functions, gene knock-down, gene knockout, gene expression, evo-devoThe moss, Physcomitrella patens, is the only non-seed plant that is amenable to an investigation of MADS-box gene function comparable to that achieved in angiosperms. P. patens possesses six MIKC^c genes which cluster into two distinct phylogenetic clades.² We recently reported a functional genetic analysis of the three genes (PPM1, PPM2 and PpMADS1) within the PPM2-like clade as an initial contribution towards gaining an understanding of the role(s) of MIKC^c genes in this moss.¹By fusing the respective MIKC^c promoters to a GUS reporter gene, we found that both PPM1 and PpMADS1 exhibited fairly ubiquitous expression patterns in both gametophytic and sporophytic tissues. The levels of PPM1 expression were generally higher than those of PpMADS1, and PpMADS1 was not expressed in antheridia, suggesting subtle differences in the functions of these genes. The observed patterns of widespread expression resemble those characterising the majority of vascular, non-seed plant MIKC^c genes^3–7 and accord with RT-PCR results of Quodt and coworkers.² Our in situ GUS expression and RT-PCR results¹ showed that PPM2 was not expressed or was expressed at levels too low to be detected by these methods. Conversely, the original isolation of PPM2 cDNA⁸ and data from more recent expression studies² indicated that PPM2 is expressed (albeit inconsistently and weakly) ubiquitously with elevated levels of expression sometimes observed in gametangia, sporophytic feet and basal portions of sporophytic setae.² The contradictory expression data for PPM2 may derive from differences between the PPM2-reporter gene constructs used by the respective research groups^1,2 or perhaps from variations in moss culture conditions.We also employed an antisense approach designed to knock down expression of PPM1, and perhaps closely related MIKC^c genes, in order to discern MADS-box gene function in P. patens. Knocked-down strains displayed a complex mutant phenotype comprising delayed gametangia formation and sporophyte production, diminished sporophyte yields, and morphological abnormalities in both leaves and sporophytes, findings that are generally consistent with the ubiquitous expression pattern of PPM1¹ and PPM2''s expression as described by Quodt et al.²The phenotypes of strains with single gene knockouts of PPM1, PPM2 or PpMADS1 appeared to be perfectly normal, not displaying any of the phenotypic alterations observed in PPM1 gene knock-down mutants. While it is possible that subtle, transient or conditional phenotypic changes went unnoticed, it seems more probable that genetic redundancy is responsible for these results since the PPM2-like genes exhibit a very high level of sequence similarity. In an effort to circumvent the problem of functional redundancy, we generated all double knockout combinations for PPM1, PPM2 and PpMADS1. However, the double mutants were also phenotypically unchanged. Finally we attempted to produce triple mutants by co-transforming single PPM2 knockout lines with PPM1 and PpMADS1 linear knockout constructs. Of the 31 stable transformants from two transformation experiments, 55% were shown to be double mutants in which the original PPM2 knockout was accompanied by a second gene knockout in either PPM1 or PpMADS1. However, no triple knockouts were obtained. Given the knockout frequencies generally observed in batch transformation experiments in our laboratory and those of others,⁹ between two and five of the transformants had been expected to be triple mutants. These preliminary data, albeit involving a relatively small sample of transformants, suggest that PPM1, PPM2 and PpMADS1 triple knockouts may be lethal.We have related compelling evidence that functionally redundant PPM2-like MIKC^c genes are involved in several aspects of the moss developmental program. It has been argued that broad expression patterns like theirs represent the ancestral state of MADS-box genes in land plants, and that the sporophytic- and organ-specific expression patterns that characterise many MIKC^c genes in extant spermatophytes, including those that specify floral organ identity, correspond to a derived condition that evolved in the spermatophyte lineage following its separation from lineages that led to bryophytes and ferns and fern allies.¹⁰ Nevertheless, it is the apparent participation of PPM2-like genes in the formation of gametangia (the differentiation of reproductive organs from non-reproductive tissues at the gametophore apex) that is particularly interesting and assumes a special significance because of its analogy to the proposed role for ancestors of seed plant C-function MADS-box genes (identifying those regions of the vegetative SAM that will become reproductive organs).11 Furthermore, expression studies of MIKC^c genes in two charophycean algae, the presumed progenitors of all terrestrial plants,^12–14 suggest that they too are involved in haploid reproductive cell differentiation.¹⁵ While these functional similarities do not infer orthology and may be coincidental, we should not discount yet the admittedly controversial hypothesis that some MIKC^c genes in non-seed plants, for example PPM2-like genes of Physcomitrella, are homologous to spermatophyte class C genes and that the ancient role proposed for ancestral class C genes¹¹ has been conserved, in some form, in all major terrestrial plant taxa.     

Nicotinamidase from the thermophilic archaeon Acidilobus saccharovorans: Structural and functional characteristics

Nicotinamidase is involved in the maintenance of NAD⁺ homeostasis and in the NAD⁺ salvage pathway of most prokaryotes, and it is considered as a possible drug target. The gene (ASAC_0847) encoding a hypothetical nicotinamidase has been found in the genome of the thermophilic archaeon Acidilobus saccharovorans. The product of this gene, NA_As0847, has been expressed in Escherichia coli, isolated, and characterized as a Fe²⁺-containing nicotinamidase (k _cat/K _m = 427 mM^?1·sec^?1)/pyrazinamidase (k _cat/K _m = 331 mM^?1·sec^?1). NA_As0847 is a homodimer with molecular mass 46.4 kDa. The enzyme has high thermostability (T_1/2 (60°C) = 180 min, T_1/2 (80°C) = 35 min) and thermophilicity (T_opt = 90°C, E_a = 30.2 ± 1.0 kJ/mol) and broad pH interval of activity, with the optimum at pH 7.5. Special features of NA_As084 are the presence of Fe²⁺ instead of Zn²⁺ in the active site of the enzyme and inhibition of the enzyme activity by Zn²⁺ at micromolar concentrations. Analysis of the amino acid sequence revealed a new motif of the metal-binding site (DXHXXXDXXEXXXWXXH) for homological archaeal nicotinamidases.     

QTL-seq identifies an early flowering QTL located near Flowering Locus T in cucumber

Key message

Next-generation sequencing enabled a fast discovery of a major QTL controlling early flowering in cucumber, corresponding to the FT gene conditioning flowering time in Arabidopsis.

Abstract

Next-generation sequencing technologies are making it faster and more efficient to establish the association of agronomic traits with molecular markers or candidate genes, which is the requirement for marker-assisted selection in molecular breeding. Early flowering is an important agronomic trait in cucumber (Cucumis sativus L.), but the underlying genetic mechanism is unknown. In this study, we identified a candidate gene for early flowering QTL, Ef1.1 through QTL-seq. Segregation analysis in F₂ and BC₁ populations derived from a cross between two inbred lines “Muromskij” (early flowering) and “9930” (late flowering) suggested quantitative nature of flowering time in cucumber. Genome-wide comparison of SNP profiles between the early and late-flowering bulks constructed from F₂ plants identified a major QTL, designated Ef1.1 on cucumber chromosome 1 for early flowering in Muromskij, which was confirmed by microsatellite marker-based classical QTL mapping in the F₂ population. Joint QTL-seq and traditional QTL analysis delimited Ef1.1 to an 890 kb genomic region. A cucumber gene, Csa1G651710, was identified in this region, which is a homolog of the FLOWERING LOCUS T (FT), the main flowering switch gene in Arabidopsis. Quantitative RT-PCR study of the expression level of Csa1G651710 revealed significantly higher expression in early flowering genotypes. Data presented here provide support for Csa1G651710 as a possible candidate gene for early flowering in the cucumber line Muromskij.     

Protocol for the BAG-RECALL clinical trial: a prospective, multi-center, randomized, controlled trial to determine whether a bispectral index-guided protocol is superior to an anesthesia gas-guided protocol in reducing intraoperative awareness with explicit recall in high risk surgical patients

Background

Sevoflurane has been demonstrated to vasodilate the foeto-placental vasculature. We aimed to determine the contribution of modulation of potassium and calcium channel function to the vasodilatory effect of sevoflurane in isolated human chorionic plate arterial rings.

Methods

Quadruplicate ex vivo human chorionic plate arterial rings were used in all studies. Series 1 and 2 examined the role of the K⁺ channel in sevoflurane-mediated vasodilation. Separate experiments examined whether tetraethylammonium, which blocks large conductance calcium activated K⁺ (K_Ca++) channels (Series 1A+B) or glibenclamide, which blocks the ATP sensitive K⁺ (K_ATP) channel (Series 2), modulated sevoflurane-mediated vasodilation. Series 3 – 5 examined the role of the Ca⁺⁺ channel in sevoflurane induced vasodilation. Separate experiments examined whether verapamil, which blocks the sarcolemmal voltage-operated Ca⁺⁺ channel (Series 3), SK&F 96365 an inhibitor of sarcolemmal voltage-independent Ca⁺⁺ channels (Series 4A+B), or ryanodine an inhibitor of the sarcoplasmic reticulum Ca⁺⁺ channel (Series 5A+B), modulated sevoflurane-mediated vasodilation.

Results

Sevoflurane produced dose dependent vasodilatation of chorionic plate arterial rings in all studies. Prior blockade of the K_Ca++ and K_ATP channels augmented the vasodilator effects of sevoflurane. Furthermore, exposure of rings to sevoflurane in advance of TEA occluded the effects of TEA. Taken together, these findings suggest that sevoflurane blocks K⁺ channels. Blockade of the voltage-operated Ca⁺⁺channels inhibited the vasodilator effects of sevoflurane. In contrast, blockade of the voltage-independent and sarcoplasmic reticulum Ca⁺⁺channels did not alter sevoflurane vasodilation.

Conclusion

Sevoflurane appears to block chorionic arterial K_Ca++ and K_ATP channels. Sevoflurane also blocks voltage-operated calcium channels, and exerts a net vasodilatory effect in the in vitro foeto-placental circulation.     

Introduction of chromosome segment carrying the seed storage protein genes from chromosome 1V of Dasypyrum villosum showed positive effect on bread-making quality of common wheat

Key message

Development of wheat- D. villosum 1V#4 translocation lines; physically mapping the Glu - V1 and Gli - V1 / Glu - V3 loci; and assess the effects of the introduced Glu - V1 and Gli - V1 / Glu - V3 on wheat bread-making quality.

Abstract

Glu-V1 and Gli-V1/Glu-V3 loci, located in the chromosome 1V of Dasypyrum villosum, were proved to have positive effects on grain quality. However, there are very few reports about the transfer of the D. villosum-derived seed storage protein genes into wheat background by chromosome manipulation. In the present study, a total of six CS-1V#4 introgression lines with different alien-fragment sizes were developed through ionizing radiation of the mature female gametes of CS––D. villosum 1V#4 disomic addition line and confirmed by cytogenetic analysis. Genomic in situ hybridization (GISH), chromosome C-banding, twelve 1V#4-specific EST–STS markers and seed storage protein analysis enabled the cytological physical mapping of Glu-V1 and Gli-V1/Glu-V3 loci to the region of FL 0.50–1.00 of 1V#4S of D. villosum. The Glu-V1 allele of D. villosum was Glu-V1a and its coded protein was V71 subunit. Quality analysis indicated that Glu-V1a together with Gli-V1/Glu-V3 loci showed a positive effect on protein content, Zeleny sedimentation value and the rheological characteristics of wheat flour dough. In addition, the positive effect could be maintained when specific Glu-V1 and Gli-V1/Glu-V3 loci were transferred to the wheat genetic background as in the case of T1V#4S-6BS·6BL, T1V#4S·1BL and T1V#4S·1DS translocation lines. These results showed that the chromosome segment carrying the Glu-V1 and Gli-V1/Glu-V3 loci in 1V#4S of D. villosum had positive effect on bread-making quality, and the T1V#4S-6BS·6BL and T1V#4S·1BL translocation lines could be useful germplasms for bread wheat improvement. The developed 1V#4S-specific molecular markers could be used to rapidly identify and trace the alien chromatin of 1V#4S in wheat background.     

Prodrugs of fumarate esters for the treatment of psoriasis and multiple sclerosis—a computational approach

Density functional theory (DFT) calculations at B3LYP/6-31 G (d,p) and B3LYP/6-311?+?G(d,p) levels for the substituted pyridine-catalyzed isomerization of monomethyl maleate revealed that isomerization proceeds via four steps, with the rate-limiting step being proton transfer from the substituted pyridinium ion to the C=C double bond in INT1. In addition, it was found that the isomerization rate (maleate to fumarate) is solvent dependent. Polar solvents, such as water, tend to accelerate the isomerization rate, whereas apolar solvents, such as chloroform, act to slow down the reaction. A linear correlation was obtained between the isomerization activation energy and the dielectric constant of the solvent. Furthermore, linearity was achieved when the activation energy was plotted against the pK _a value of the catalyst. Substituted-pyridine derivatives with high pK _a values were able to catalyze isomerization more efficiently than those with low pK _a values. The calculated relative rates for prodrugs 1–6 were: 1 (406.7), 2 (7.6?×?10⁶), 3 (1.0), 4 (20.7), 5 (13.5) and 6 (2.2?×?10³). This result indicates that isomerizations of prodrugs 1 and 3–5 are expected to be slow and that of prodrugs 2 and 6 are expected to be relatively fast. Hence, prodrugs 2 and 3–5 have the potential to be utilized as prodrugs for the slow release of monomethylfumarate in the treatment of psoriasis and multiple sclerosis.

Albino midrib 1, encoding a putative potassium efflux antiporter,affects chloroplast development and drought tolerance in rice

Key message

Mutation of the AM1 gene causes an albino midrib phenotype and enhances tolerance to drought in rice

Abstract

K⁺ efflux antiporter (KEA) genes encode putative potassium efflux antiporters that are mainly located in plastid-containing organisms, ranging from lower green algae to higher flowering plants. However, little genetic evidence has been provided on the functions of KEA in chloroplast development. In this study, we isolated a rice mutant, albino midrib 1 (am1), with green- and white-variegation in the first few leaves, and albino midrib phenotype in older tissues. We found that AM1 encoded a putative KEA in chloroplast. AM1 was highly expressed in leaves, while lowly in roots. Chloroplast gene expression and proteins accumulation were affected during chlorophyll biosynthesis and photosynthesis in am1 mutants. Interestingly, AM1 was induced by salt and PEG, and am1 showed enhanced sensitivity to salinity in seed germination and increased tolerance to drought. Taken together, we concluded that KEAs were involved in chloroplast development and played important roles in drought tolerance.     

Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 1: Identification of its inhibitory subunit complexes and their roles

Cyclic GMP phosphodiesterase (PDE) in bovine rod photoreceptor outer segments (OS) comprises a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγ) and is regulated by the α subunit of transducin (Tα). Here, we show an overall mechanism for PDE regulation by identifying Pγ complexes in OS homogenates prepared with an isotonic buffer. Before Tα activation, three Pγ complexes exist in the soluble fraction. Complex a, a minor complex, contains Pαβ, Tα, and a protein named Pδ. Complex b, Pαβγγ ^b , has a PDE activity similar to that of membranous Pαβγγ, Pαβγγ ^M , and its level, although its large portion is Pδ-free, is estimated to be 20–30% of the total Pαβγγ. Complex c, (Pγ·GDP-Tα) ₂ ^c , appears to be a dimer of Pγ·GDP-Tα. Upon Tα activation, (1) complex a stays unchanged, (2) Pαβγγ ^b binds to membranes, (3) the level of (Pγ·GDP-Tα) ₂ ^c is reduced as its GTP-form is produced, (4) complex d, Pγ·GTP-Tα ^d , is formed on membranes and its substantial amount is released to the soluble fraction, and (5) membranous Pαβγγ, Pαβγγ ^M and/or Pαβγγ ^b , becomes Pγ-depleted. These observations indicate that Pγ as a complex with GTP-Tα dissociates from Pαβγγ on membranes and is released to the soluble fraction and that Pγ-depleted PDE is the GTP-Tα-activated PDE. After GTP hydrolysis, both (Pγ·GDP-Tα) ₂ ^c and Pγ·GDP-Tα ^d , without liberating Pγ, deactivate Pγ-depleted PDE. The preferential order to be used for the deactivation is membranous Pγ·GDP-Tα ^d , solubilized Pγ·GDP-Tα ^d and (Pγ·GDP-Tα) ₂ ^c . Release of Pγ·GTP-Tα complexes to the soluble fraction is relevant to light adaptation.     

The Arabidopsis kinesin gene AtKin-1 plays a role in the nuclear division process during megagametogenesis

Key message

Atkin - 1 , the only Kinesin-1 member of Arabidopsis thaliana , plays a role during female gametogenesis through regulation of nuclear division cycles.

Abstract

Kinesins are microtubule-dependent motor proteins found in eukaryotic organisms. They constitute a superfamily that can be further classified into at least 14 families. In the Kinesin-1 family, members from animal and fungi play roles in long-distance transport of organelles and vesicles. Although Kinesin-1-like sequences have been identified in higher plants, little is known about their function in plant cells, other than in a recently identified Kinesin-1-like protein in a rice pollen semi-sterile mutant. In this study, the gene encoding the only Kinesin-1 member in Arabidopsis, AtKin-1 was found to be specifically expressed in ovules and anthers. AtKin-1 loss-of-function mutants showed substantially aborted ovules in siliques, and this finding was supported by complementation testing. Reciprocal crossing between mutant and wild-type plants indicated that a defect in AtKin-1 results in partially aborted megagametophytes, with no observable effects on pollen fertility. Further observation of ovule development in the mutant pistils indicated that the enlargement of the megaspore was blocked and nuclear division arrested at the one-nucleate stage during embryo sac formation. Our data suggest that AtKin-1 plays a role in the nuclear division cycles during megagametogenesis.