Statistical Verification of Anomaly in Chiral Angle Distribution of Air-Suspended Carbon Nanotubes.

Single-walled carbon nanotubes (SWCNT) have long attracted attention due to their distinct physical properties, depending on their chiral structures (chiralities). Clarifying their growth mechanism is important toward perfect chirality-controlled bulk synthesis. Although a correlation between the chirality distribution and the carbon atom configuration at an open tube edge has been predicted theoretically, lack of sufficient statistical data on metallic and semiconducting SWCNTs prohibited its verification. Here, we report statistical verification of the chirality distribution of 413 as-grown individual air-suspended SWCNTs with a length of over 20 µm using broadband Rayleigh spectroscopy. After excluding the impact of the difference in the number of possible SWCNT structures per chiral angle interval, the abundance profile with chiral angle exhibits an increasing trend with a distinct anomaly at a chiral angle of approximately 20°. These results are well explained considering the growth rate depending on armchair-shaped site configurations at the catalyst-nanotube interface.

Retraction Note: Living annulative π-extension polymerization for graphene nanoribbon synthesis.

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1038/s41586-020-2950-0 .

Living annulative π-extension polymerization for graphene nanoribbon synthesis.

The properties of graphene nanoribbons (GNRs)1-5-such as conductivity or semiconductivity, charge mobility and on/off ratio-depend greatly on their width, length and edge structure. Existing bottom-up methods used to synthesize GNRs cannot achieve control over all three of these parameters simultaneously, and length control is particularly challenging because of the nature of step-growth polymerization6-18. Here we describe a living annulative π-extension (APEX)19 polymerization technique that enables rapid and modular synthesis of GNRs, as well as control over their width, edge structure and length. In the presence of palladium/silver salts, o-chloranil and an initiator (phenanthrene or diphenylacetylene), the benzonaphthosilole monomer polymerizes in an annulative manner to furnish fjord-type GNRs. The length of these GNRs can be controlled by simply changing the initiator-to-monomer ratio, achieving the synthesis of GNR block copolymers. This method represents a type of direct C-H arylation polymerization20 and ladder polymerization21, activating two C-H bonds of polycyclic aromatic hydrocarbons and constructing one fused aromatic ring per chain propagation step.

Formation of Clusters in Whiskies During the Maturation Process.

Maturation provides whisky with a mild and smooth texture by removing the irritating alcoholic flavor. However, the precise mechanism by which the whisky flavor is improved through the maturation process remains unknown. In this study, we performed mesoscopic structural measurements-dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS)-to elucidate the relationship between the liquid structure and flavor maturation of whiskies. Both techniques detected two scattering components corresponding to the clusters formed by the extractives from oak casks during maturation, which are not present in the new make (freshly distilled whisky). Analyzing the scattering profiles revealed that only the small clusters increase in concentration during maturation. It is concluded the small cluster component is crucial for obtaining flavorful whiskies, while the large cluster component, whose concentration is independent of the maturation time, is related to the alcoholic irritation of the whiskies, as demonstrated by the sonication test.

Identification and Characterization of Novel Nemophila menziesii Flavone Glucosyltransferases that Catalyze Biosynthesis of Flavone 7,4'-O-Diglucoside, a Key Component of Blue Metalloanthocyanins.

The brilliant blue color of the Nemophila menziesii flower is derived from metalloanthocyanin, which consists of anthocyanin {petunidin 3-O-[6-O-(trans-p-coumaroyl)-ß-glucoside]-5-O-[6-O-(malonyl)-ß-glucoside]}, flavone [apigenin 7-O-ß-glucoside-4'-O-(6-O-malonyl)-O-ß-glucoside] and metal ions (Mg2+, Fe3+). Although the two glucosyl moieties at the apigenin 7-O and 4'-O positions are essential for metalloanthocyanin formation, the mechanism of glucosylation has not yet been clarified. In this study, we used crude protein extract prepared from N. menziesii petals to determine that apigenin is sequentially glucosylated by the catalysis of UDP-glucose:flavone 4'-O-glucosyltrasferase (F4'GT) and UDP-glucose:flavone 4'-O-glucoside 7-O-glucosyltransferase (F4'G7GT). We identified 150 contigs exhibiting homology with a UDP-glucose-dependent GT in the N. menziesii petal transcriptome and isolated 24 putative full-length GT cDNAs which were then subjected to functional analysis. Two GT cDNAs, NmF4'GT and NmF4'G7GT, which are highly expressed during the early stages of petal development and rarely in leaves, were shown to encode F4'GT and F4'G7GT activities, respectively. Biochemical characterization of the recombinant enzymes revealed that NmF4'GT specifically catalyzed 4'-glucosylation of flavonoids and that NmF4'G7GT specifically catalyzed 7-glucosylation of flavone 4'-O-glucosides and flavones. Apigenin 7,4'-O-diglucoside was efficiently synthesized from apigenin in the presence of recombinant NmF4'GT and NmF4'G7GT. Transgenic tobacco BY-2 cells expressing NmF4'GT and NmF4'G7GT converted apigenin into apigenin 7,4'-O-diglucoside, confirming their activities in vivo. Based on these results, we conclude that these two GTs act co-ordinately to catalyze apigenin 7,4'-O-diglucoside biosynthesis in N. menziesii.

Molecular cloning of flavonoid biosynthetic genes and biochemical characterization of anthocyanin O-methyltransferase of Nemophila menziesii Hook. and Arn.

Blue flower color of Nemophila menziesii Hook. and Arn. is derived from a metalloanthocyanin, nemophilin, which comprises petunidin-3-O-[6-O-(trans-p-coumaroyl)-ß-glucoside]-5-O-[6-O-(malonyl)-ß-glucoside], apigenin-7-O-ß-glucoside-4'-O-(6-O-malonyl)-ß-glucoside, and Mg2+ and Fe3+ ions. The flavonoid biosynthetic pathway of nemophilin has not yet been characterized. RNA-Seq analysis of the petals yielded 61,491 contigs. These were searched using BLAST against petunia or torenia flavonoid biosynthetic proteins, which identified 11 putative full-length protein sequences belonging to the flavonoid biosynthetic pathway. RT-PCR using primers designed on the basis of these sequences yielded 14 sequences. Spatio-temporal transcriptome analysis indicated that genes involved in the early part of the pathway are strongly expressed during early-petal development and that those in the late part at late-flower opening stages, but they are rarely expressed in leaves. Flavanone 3-hydroxylase and flavonoid 3',5'-hydroxylase cDNAs were successfully expressed in yeast to confirm their activities. Recombinant anthocyanin O-methyltransferase cDNA (NmAMT6) produced using Escherichia coli was subjected to biochemical characterization. Km of NmAMT6 toward delphinidin 3-O-glucoside was 22 µM, which is comparable with Km values of anthocyanin O-methyltransferases from other plants. With delphinidin 3-O-glucoside as substrate, NmAMT6 almost exclusively yielded petunidin 3-O-glucoside rather than malvidin 3-O-glucoside. This specificity is consistent with the anthocyanin composition of Nemophila petals.

Enhancement of phosphate absorption by garden plants by genetic engineering: a new tool for phytoremediation.

Although phosphorus is an essential factor for proper plant growth in natural environments, an excess of phosphate in water sources causes serious pollution. In this paper we describe transgenic plants which hyperaccumulate inorganic phosphate (Pi) and which may be used to reduce environmental water pollution by phytoremediation. AtPHR1, a transcription factor for a key regulator of the Pi starvation response in Arabidopsis thaliana, was overexpressed in the ornamental garden plants Torenia, Petunia, and Verbena. The transgenic plants showed hyperaccumulation of Pi in leaves and accelerated Pi absorption rates from hydroponic solutions. Large-scale hydroponic experiments indicated that the enhanced ability to absorb Pi in transgenic torenia (AtPHR1) was comparable to water hyacinth a plant that though is used for phytoremediation causes overgrowth problems.

Arabidopsis RPT2a encoding the 26S proteasome subunit is required for various aspects of root meristem maintenance, and regulates gametogenesis redundantly with its homolog, RPT2b.

The 26S proteasome plays fundamental roles in the degradation of short-lived regulatory proteins, thereby controlling diverse cellular processes. In Arabidopsis, the essential RPT2 subunit is encoded by two highly homologous genes: RPT2a and RPT2b. Currently, only RPT2a has been reported to regulate various developmental processes, including the maintenance of the root apical meristem (RAM), although the roles of RPT2a in the RAM are still obscure. Here, we analyzed the cell type-specific requirement for RPT2a. When RPT2a was expressed locally in the rpt2a mutant, pleiotropic defects in the RAM, such as cell death and distorted cellular organization, were rescued differently, suggesting that RPT2a regulates various specific activities, which converge to maintain the RAM. On the other hand, the homologous RPT2b was also expressed in meristems, and the expression of RPT2b protein under the control of the RPT2a promoter complemented the rpt2a RAM defects, although the rpt2b mutant showed no obvious defect in all developmental aspects we examined. These results show that RPT2b might work in the RAM, but is dispensable for RAM maintenance in the presence of RPT2a. In contrast, the rpt2a rpt2b double mutant was lethal in male and female gametophytes, suggesting that RPT2a and RPT2b are redundantly required for gametogenesis. Furthermore, we showed that similar meristematic and gametophytic defects were caused by mutations in other subunit genes, RPT5a and RPT5b, suggesting that proper activity of the proteasome, not an RPT2-specific function, is required. Taken together, our results suggest that RPT2a and RPT2b contribute differently to the proteasome activity required for each developmental context.

Production of arachidonic and eicosapentaenoic acids in plants using bryophyte fatty acid Delta6-desaturase, Delta6-elongase, and Delta5-desaturase genes.

The liverwort Marchantia polymorpha L. synthesizes arachidonic (ARA) and eicosapentaenoic acids (EPA) from linoleic and alpha-linolenic acids respectively by a series of reactions catalyzed by Delta6-desaturase, Delta6-elongase, and Delta5-desaturase. Overexpression of the M. polymorpha genes encoding these enzymes in transgenic M. polymorpha plants resulted in 3- and 2-fold accumulation of ARA and EPA respectively, as compared to those in the wild type. When these three genes were introduced and co-expressed in tobacco plants, in which long-chain polyunsaturated fatty acids (LCPUFAs) are not native cellular components, ARA and EPA represented up to 15.5% and 4.9% respectively of the total fatty acid in the leaves. Similarly in soybean, C20-LCPUFAs represented up to 19.5% of the total fatty acids in the seeds. These results suggest that M. polymorpha can provide genes crucial to the production of C20-LCPUFAs in transgenic plants.

A front-end desaturase from Chlamydomonas reinhardtii produces pinolenic and coniferonic acids by omega13 desaturation in methylotrophic yeast and tobacco.

Pinolenic acid (PA; 18:3Delta(5,9,12)) and coniferonic acid (CA; 18:4Delta(5,9,12,15)) are Delta(5)-unsaturated bis-methylene-interrupted fatty acids (Delta(5)-UBIFAs) commonly found in pine seed oil. They are assumed to be synthesized from linoleic acid (LA; 18:2Delta(9,12)) and alpha-linolenic acid (ALA; 18:3Delta(9,12,15)), respectively, by Delta(5)-desaturation. A unicellular green microalga Chlamydomonas reinhardtii also accumulates PA and CA in a betain lipid. The expressed sequence tag (EST) resource of C. reinhardtii led to the isolation of a cDNA clone that encoded a putative fatty acid desaturase named as CrDES containing a cytochrome b5 domain at the N-terminus. When the coding sequence was expressed heterologously in the methylotrophic yeast Pichia pastoris, PA and CA were newly detected and comparable amounts of LA and ALA were reduced, demonstrating that CrDES has Delta(5)-desaturase activity for both LA and ALA. CrDES expressed in the yeast showed Delta(5)-desaturase activity on 18:1Delta(9) but not 18:1Delta(11). Unexpectedly, CrDES also showed Delta(7)-desaturase activity on 20:2Delta(11,14) and 20:3Delta(11,14,17) to produce 20:3Delta(7,11,14) and 20:4Delta(7,11,14,17), respectively. Since both the Delta(5) bond in C18 and the Delta(7) bond in C20 fatty acids are 'omega13' double bonds, these results indicate that CrDES has omega13 desaturase activity for omega9 unsaturated C18/C20 fatty acids, in contrast to the previously reported front-end desaturases. In order to evaluate the activity of CrDES in higher plants, transgenic tobacco plants expressing CrDES were created. PA and CA accumulated in the leaves of transgenic plants. The highest combined yield of PA and CA was 44.7% of total fatty acids, suggesting that PA and CA can be produced in higher plants on a large scale.

Transgene-triggered, epigenetically regulated ectopic expression of a flower homeotic gene pMADS3 in Petunia.

pMADS3 is a class C floral homeotic gene of Petunia that is specifically expressed in stamens and carpels of developing flowers. We previously reported that introduction of a part of the pMADS3 genomic sequence silenced endogenous pMADS3 (sil-pMADS3) in transgenic Petunia hybrida. Here we report that introduction of the same sequence triggers ectopic expression of endogenous pMADS3 in sepals, petals and leaves (ect-pMADS3), accompanied by homeotic conversion of the floral organs and altered leaf morphology similar to that of an Arabidopsis curly leaf mutant. The occurrence of the ect-pMADS3 phenotype depended on the presence of pMADS3 intron 2 in the transgenes. Occasionally, sil-pMADS3 and ect-pMADS3 phenotypes somatically interconverted. Some T1 progeny inherited their parent's pMADS3 expression pattern, while others switched from sil-pMADS3 to ect-pMADS3 and vice versa. Both phenotypes occasionally occurred even after the transgenes were segregated away. RT-PCR analyses of ectopically expressed pMADS3 transcripts indicated that two pMADS3 alleles were often differently regulated. Furthermore, reciprocal crosses with untransformed Petunia indicated that pMADS3 alleles other than the one ectopically expressed in T0 plants were sometimes expressed ectopically in T1 plants: the paramutation-like transmission of epigenetic regulation between alleles. We detected in the transformants aberrant transcripts, including sense and antisense pMADS3 intron 2 sequences of heterogeneous molecular sizes, irrespective of the pMADS3 phenotypes. We speculate on possible molecular mechanisms underlying these epigenetic phenomena.

The HALTED ROOT gene encoding the 26S proteasome subunit RPT2a is essential for the maintenance of Arabidopsis meristems.

In higher plants, post-embryonic development is dependent on the activity of the root and shoot apical meristem (RAM and SAM). The quiescent center (QC) in the RAM and the organizing center (OC) in the SAM are known to be essential for the maintenance of meristematic activity. To understand the mechanism that maintains post-embryonic meristems, we isolated an Arabidopsis mutant, halted root (hlr). In this mutant, the cellular organization was disrupted in post-embryonic meristems both in the root and in the shoot, and their meristematic activity was reduced or became abnormal. We showed that the mutant RAM lost its QC identity after germination, which was specified during embryogenesis, whereas the identity of differentiated tissues was maintained. In the post-embryonic SAM, the expression pattern of a typical OC marker gene, WUSCHEL, was disturbed in the mutant. These observations indicate that the HLR gene is essential to maintain the cellular organization and normal nature of the RAM and SAM. The HLR gene encodes RPT2a, which is a subunit of the 26S proteasome that degrades key proteins in diverse cellular processes. We showed that the HLR gene was expressed both in the RAM and in the SAM, including in the QC and the OC, respectively, and that the activity of proteasomes were reduced in the mutant. We propose that proteasome-dependent programmed proteolysis is required to maintain the meristem integrity both in the shoot and in the root.