Molecular Adaptation of rbcL in the Heterophyllous Aquatic Plant Potamogeton

Heterophyllous aquatic plants show marked phenotypic plasticity. They adapt to environmental changes by producing different leaf types: submerged, floating and terrestrial leaves. By contrast, homophyllous plants produce only submerged leaves and grow entirely underwater. Heterophylly and submerged homophylly evolved under selective pressure modifying the species-specific optima for photosynthesis, but little is known about the evolutionary outcome of habit. Recent evolutionary analyses suggested that rbcL, a chloroplast gene that encodes a catalytic subunit of RuBisCO, evolves under positive selection in most land plant lineages. To examine the adaptive evolutionary process linked to heterophylly or homophylly, we analyzed positive selection in the rbcL sequences of ecologically diverse aquatic plants, Japanese Potamogeton.Phylogenetic and maximum likelihood analyses of codon substitution models indicated that Potamogeton rbcL has evolved under positive Darwinian selection. The positive selection has operated specifically in heterophyllous lineages but not in homophyllous ones in the branch-site models. This suggests that the selective pressure on this chloroplast gene was higher for heterophyllous lineages than for homophyllous lineages. The replacement of 12 amino acids occurred at structurally important sites in the quaternary structure of RbcL, two of which (residue 225 and 281) were identified as potentially under positive selection.Our analysis did not show an exact relationship between the amino acid replacements and heterophylly or homophylly but revealed that lineage-specific positive selection acted on the Potamogeton rbcL. The contrasting ecological conditions between heterophyllous and homophyllous plants have imposed different selective pressures on the photosynthetic system. The increased amino acid replacement in RbcL may reflect the continuous fine-tuning of RuBisCO under varying ecological conditions

Alteration of oxalate content in rice leaves obtained from ion beam-irradiated plants

Rice straw obtained from rice leaves and stems can be used as a livestock feed. Rice leaves accumulate soluble oxalate, which easily binds to cationic ions such as calcium, iron, and magnesium ions, then becomes insoluble. It is an important agricultural topic to reduce oxalate content in crops because excess uptake of oxalate-rich plants causes mineral racking or urinary syndrome for human and livestock, although it is a useful molecule for plants to defense themselves from predators, to reduce aluminum toxicity in the acidic soil, to regulate calcium content in their body, and to provide hydrogen peroxide which acts as a signal molecule during aging or wounding. Three pathways (isocitrate, glycolate and ascorbate pathways) have been reported for oxalate synthesis in plants. However, it has been unknown which pathway contributes the oxalate accumulation in rice leaves. For reducing oxalate content in rice leaves and understanding oxalate synthesis mechanism, we first grew rice mutagenized M2 population (Koshihikari) generated from seeds irradiated by carbon ion beams and measured oxalate contents in their leaves. The oxalate measurement using CE-MS showed that there was no difference between means of oxalate contents in the population of M2 plants and those in the control plants, but wider variation of oxalate contents was observed in M2 plants compared with control plants. Metabolomic analyses of oxalate and other metabolite in the M2 plants showed that organic acids such as 2OG and succinate were decreased in the low-oxalate rice plants, although amino acids except for alanine, glutamate, and aspartate were increased. These suggested that oxalate accumulation in rice leaves would be affected by carbon flow via isocitrate pathway, and that reduction of oxalate contents might lead to increase of amino acids contents.Plant Biology 202

Impact of ion beam-irradiation on metabolisms in oxalate rich plant

Rumex plants (Polygonaceae) are widespread in the world. Some Rumex species such as sorrel (R. acetosa) are edible as baby leaf salad greens. R. obtusifolius L. (broad-leaved dock) is a perennial weed, which grows well in agricultural lands. It contains higher vitamin C (ascorbate) and amino acids and shows stress tolerance than many other Rumex species, whereas soluble oxalate is accumulated in leaves. Excess intake of Rumex leaves leads to mineral insufficiency, hypocalcaemia or kidney stones for human and livestock. Thus, the reduction of oxalate content in leaves is an important agricultural issue. In plant oxalate synthesis, three pathways (the isocitrate, glycolate, and ascorbate pathway) have been reported. However, it remains unknown which pathway or metabolite contributes the oxalate accumulation. In the present study, to clarify the mechanisms of oxalate synthesis, we focused on the metabolic alteration by ion beam-irradiation and performed metabolome analysis of the leaves of R. obtusifolius, which is one of the most oxalate rich-plant in Rumex plants, obtained from the seeds irradiated with ion beams. The results showed that oxalate contents in R. obtusifolius leaves were increased by seed irradiation of carbon ion beams. Correlation analysis of oxalate and other primary metabolite data set obtained by CE-MS (Capillary Electrophoresis-Mass Spectrometry) revealed that contents of oxalate precursors (citrate, isocitrate and ascorbate) had positive correlations with oxalate accumulation, whereas negative correlations were observed between oxalate and amino acids such as serine, glutamine, asparagine, arginine, and branched-chain amino acids. Principal component and hierarchical analyses suggested that the irradiation of ion beams affected carbon flow to the isocitrate pathway. These observations indicated that modulation of carbon flow to the isocitrate pathway is important to regulate oxalate levels in crops such as spinach and rice straw.Plant Biology 2018 (ASPB 2018

Metabolic changes associated with dark-induced leaf senescence in Arabidopsis nadk2 mutants

Arabidopsis NADK2 (NAD kinase 2) is a chloroplast-localized enzyme involved in NADP+ synthesis, which acts as the final electron acceptor in the photosynthetic electron transfer chain. The NADK2-deficient mutant (nadk2) was used to analyze the effect of NAD(P)(H) unbalance in the dark-induced leaf senescence. During senescence, WT plants and nadk2 mutants showed a similar reduction in chlorophyll content. NAD(P)(H) quantification showed that the amount of total NAD(P)(H) decreased on the day 7 in WT but on the day 3 in nadk2. The phosphorylation ratio (i.e. NADP(H)/NAD(H)) decreased on day 1 in WT. In contrast, the nadk2 showed lower phosphorylation ratio at 0 day and no change throughout the aging process. Metabolome analysis showed that the metabolic profiles of both WT plants and nadk2 mutants subjected to dark-induced senescence adopted similar patterns as the senescence progressed. However, the changes in individual metabolites in the nadk2 mutants were different from those of the WT during dark-induced senescence

Structure-guided screening strategy combining surface plasmon resonance with nuclear magnetic resonance for identification of small-molecule Argonaute 2 inhibitors.

Argonaute (AGO) proteins are the key component of the RNA interference machinery that suppresses gene expression by forming an RNA-induced silencing complex (RISC) with microRNAs (miRNAs). Each miRNA is involved in various cellular processes, such as development, differentiation, tumorigenesis, and viral infection. Thus, molecules that regulate miRNA function are expected to have therapeutic potential. In addition, the biogenesis of miRNA is a multistep process involving various proteins, although the complete pathway remains to be elucidated. Therefore, identification of molecules that can specifically modulate each step will help understand the mechanism of gene suppression. To date, several AGO2 inhibitors have been identified. However, these molecules were identified through a single screening method, and no studies have specifically evaluated a combinatorial strategy. Here, we demonstrated a combinatorial screening (SCR) approach comprising an in silico molecular docking study, surface plasmon resonance (SPR) analysis, and nuclear magnetic resonance (NMR) analysis, focusing on the strong binding between the 5'-terminal phosphate of RNA and the AGO2 middle (MID) domain. By combining SPR and NMR, we identified binding modes of amino acid residues binding to AGO2. First, using a large chemical library (over 6,000,000 compounds), 171 compounds with acidic functional groups were screened using in silico SCR. Next, we constructed an SPR inhibition system that could analyze only the 5'-terminal binding site of RNA, and nine molecules that strongly bound to the AGO2 MID domain were selected. Finally, using NMR, three molecules that bound to the desired site were identified. The RISC inhibitory ability of the "hit" compounds was analyzed in human cell lysate, and all three hit compounds strongly inhibited the binding between double-stranded RNA and AGO2

Metabolome analysis of rice leaves to obtain low-oxalate strain from ion beam-mutagenised population

Rice leaves and stems, which can be used as rice straw for livestock feed, accumulate soluble oxalate. The oxalate content often reaches 5% of the dry weight leaves. Excess uptake of oxalate-rich plants causes mineral deficiencies in vertebrates, so it is important to reduce the oxalate content in rice leaves to produce high-quality rice straw. However, the mechanism of oxalate accumulation in rice has remained unknown. In this study, we performed metabolome analysis of rice M 2 population generated by ion-beam irradiation using CE-MS to understand metabolic networks relating oxalate accumulation in rice. The result showed wide variation of oxalate contents in M 2 plants compared with those of control plants. Multivariate analyses of metabolome dataset revealed that oxalate accumulation was strongly related with anionic compounds such as 2OG and succinate. For low-oxalate plants, four patterns of metabolic alterations affected oxalate contents in the M 2 leaves were observed. In M 3 plants, we found putative low-oxalate line obtained from low-oxalate M 2 mutant. These findings would lead to produce the low-oxalate rice and to understand the oxalate synthesis in plants

Plant-Unique cis/trans Isomerism of Long-Chain Base Unsaturation is Selectively Required for Aluminum Tolerance Resulting from Glucosylceramide-Dependent Plasma Membrane Fluidity

Cis/trans isomerism of the Δ8 unsaturation of long-chain base (LCB) is found only in plant sphingolipids. This unique geometry is generated by sphingolipid LCB Δ8 desaturase SLD which produces both isomers at various ratios, resulting in diverse cis/trans ratios in plants. However, the biological significance of this isomeric diversity remains controversial. Here, we show that the plant-specific cis unsaturation of LCB selectively contributes to glucosylceramide (GlcCer)-dependent tolerance to aluminum toxicity. We established three transgenic rice lines with altered LCB unsaturation profiles. Overexpression of SLD from rice (OsSLD-OX), which preferentially exhibits cis-activity, or Arabidopsis (AtSLD-OX), showing preference for trans-activity, facilitated Δ8 unsaturation in different manners: a slight increase of cis-unsaturated glycosylinositolphosphoceramide (GIPC) in OsSLD-OX, and a drastic increase of trans-unsaturated GlcCer and GIPC in AtSLD-OX. Disruption of LCB Δ4 desaturase (des) significantly decreased the content of GlcCer. Fluorescence imaging analysis revealed that OsSLD-OX and AtSLD-OX showed increased plasma membrane fluidity, whereas des had less fluidity, demonstrating that the isomers universally contributed to increasing membrane fluidity. However, the results of a hydroponic assay showed decreased aluminum tolerance in AtSLD-OX and des compared to OsSLD-OX and the control plants, which did not correlate with membrane fluidity. These results suggest that cis-unsaturated GlcCer, not GIPC, selectively serves to maintain the membrane fluidity specifically associated with aluminum tolerance