A novel nucleic acid linker for multi-gene expression enhances plant and animal synthetic biology.

The production of compact vectors for gene stacking is hindered by a lack of effective linkers. Here, we report that a 26-nt nucleic acid linker, NAL1, from the fungus Glarea lozoyensis and its truncated derivatives could connect two genes as a bicistron, enabling independent translation in a maize protoplast transient expression system and human 293 T cells. The optimized 9-nt NAL10 linker was then used to connect four genes driven by a bidirectional promoter; this combination was successfully used to reconstruct the astaxanthin biosynthesis pathway in transgenic maize. The short and efficient nucleic acid linker NAL10 can be widely used in multi-gene expression and synthetic biology in animals and plants.

Identification and characterization of yellow stripe-like genes in maize suggest their roles in the uptake and transport of zinc and iron.

BACKGROUND: Yellow Stripe-Like (YSL) proteins are involved in the uptake and transport of metal ions. They play important roles in maintaining the zinc and iron homeostasis in Arabidopsis, rice (Oryza sativa), and barley (Hordeum vulgare). However, proteins in this family have not been fully identified and comprehensively analyzed in maize (Zea mays L.). RESULTS: In this study, we identified 19 ZmYSLs in the maize genome and analyzed their structural features. The results of a phylogenetic analysis showed that ZmYSLs are homologous to YSLs of Arabidopsis and rice, and these proteins are divided into four independent branches. Although their exons and introns have structural differences, the motif structure is relatively conserved. Analysis of the cis-regulatory elements in the promoters indicated that ZmYSLs might play a role in response to hypoxia and light. The results of RNA sequencing and quantitative real-time PCR analysis revealed that ZmYSLs are expressed in various tissues and respond differently to zinc and iron deficiency. The subcellular localization of ZmYSLs in the protoplast of maize mesophyll cells showed that they may function in the membrane system. CONCLUSIONS: This study provided important information for the further functional analysis of ZmYSL, especially in the spatio-temporal expression and adaptation to nutrient deficiency stress. Our findings provided important genes resources for the maize biofortification.

Perimeter monitoring of urban buried pipeline threated by construction activities based on distributed fiber optic sensing and real-time object detection.

Urban construction activities seriously jeopardize the security of buried pipeline. Distributed optical fiber vibration monitoring is one of the most promising ways to prevent third-party threats, of which the biggest challenge is to quickly and accurately detect rare abnormal events from extremely large amounts of time-space raw data. By analogy with image recognition, the task here is similar to object detection if considering the time-space optical signals as the grayscale images and the abnormal events as the objects. Given this, what we believe to be a novel monitoring method is proposed, which consists of two Faster R-CNN models, a max pooling layer and a monitoring strategy. In the field tests, the 86-hour optical vibration signals for 5.25 km distance are recognized within 6.6 minutes with the recognition rate of 98.85% for construction activities, and only two false alarms are issued. The proposed method can reduce the recognition time by 99.59% compared to the CNN-based method.

Co-expression of transcription factors ZmC1 and ZmR2 establishes an efficient and accurate haploid embryo identification system in maize.

Because of their high efficiency during chromosome doubling, immature haploid maize (Zea mays L.) embryos are useful for doubled haploid production. The R1-nj marker is commonly used in doubled haploid breeding and has improved the efficiency of haploid identification. However, its effectiveness is limited by genetic background and environmental factors. We addressed this technical challenge by developing an efficient and accurate haploid embryo identification marker through co-expression of two transcription factor genes (ZmC1 and ZmR2) driven by the embryo-aleurone-specific bidirectional promoter PZmBD1 ; these factors can activate anthocyanin biosynthesis in the embryo and aleurone layer during early seed development. We developed a new haploid inducer, Maize Anthocyanin Gene InduCer 1 (MAGIC1), by introducing the transgenes into the haploid inducer line CAU6. MAGIC1 could identify haploids at 12 days after pollination, which is nine days earlier than CAU6. Importantly, MAGIC1 increased haploid identification accuracy to 99.1%, compared with 88.3% for CAU6. In addition, MAGIC1 could effectively overcome the inhibition of anthocyanin synthesis in some germplasms. Furthermore, an upgraded anthocyanin marker was developed from ZmC1 and ZmR2 to generate MAGIC2, which could identify haploids from diploids due to differential anthocyanin accumulation in immature embryos, coleoptiles, sheaths, roots, leaves, and dry seeds. This haploid identification system is more efficient and accurate than the conventional R1-nj-based method, and it simplifies the haploid identification process. Therefore, this system provides technical support for large-scale doubled haploid line production.

Pentatricopeptide repeat protein CNS1 regulates maize mitochondrial complex III assembly and seed development.

Mitochondrial function relies on the assembly of electron transport chain complexes, which requires coordination between proteins encoded by the mitochondrion and those of the nucleus. Here, we cloned a maize (Zea mays) cytochrome c maturation FN stabilizer1 (CNS1) and found it encodes a pentatricopeptide repeat (PPR) protein. Members of the PPR family are widely distributed in plants and are associated with RNA metabolism in organelles. P-type PPR proteins play essential roles in stabilizing the 3'-end of RNA in mitochondria; whether a similar process exists for stabilizing the 5'-terminus of mitochondrial RNA remains unclear. The kernels of cns1 exhibited arrested embryo and endosperm development, whereas neither conventional splicing deficiency nor RNA editing difference in mitochondrial genes was observed. Instead, most of the ccmFN transcripts isolated from cns1 mutant plants were 5'-truncated and therefore lacked the start codon. Biochemical and molecular data demonstrated that CNS1 is a P-type PPR protein encoded by nuclear DNA and that it localizes to the mitochondrion. Also, one binding site of CNS1 located upstream of the start codon in the ccmFN transcript. Moreover, abnormal mitochondrial morphology and dramatic upregulation of alternative oxidase genes were observed in the mutant. Together, these results indicate that CNS1 is essential for reaching a suitable level of intact ccmFN transcripts through binding to the 5'-UTR of the RNAs and maintaining 5'-integrity, which is crucial for sustaining mitochondrial complex III function to ensure mitochondrial biogenesis and seed development in maize.

Synthesis of Substituted of Benzo[4,5]imidazo[1,2-a]pyrimidines through (3 + 2+1) Cyclization of 2-Aminobenzimidazole, Acetophenone, and N,N-Dimethylformamide as One-Carbon Synthon.

An efficient method for the construction of benzo[4,5]imidazo[1,2-a]pyrimidines using N,N-dimethylformamide as a one-carbon source and 2-aminobenzimidazoles and acetophenone as substrates through a one-pot, three-component cascade reaction is described. Spectra investigations indicated the fluorescent properties of selected products, exhibiting quantum yields 0.07-0.16 with maxima absorption at 266-294 nm and emission at 472-546 nm.

Mediation of Zinc and Iron Accumulation in Maize by ZmIRT2, a Novel Iron-Regulated Transporter.

Iron (Fe) is an essential micronutrient for plant growth. Iron-regulated transporters (IRTs) play important roles in Fe2+ uptake and transport in strategy I plants. Maize (Zea mays) belongs to a strategy II plant, in which mugineic acid (MA)-Fe3+ uptake is mainly carried out by Yellow Stripe 1 (YS1). However, ZmIRT1 was previously identified by our laboratory. In this study, we isolated a novel gene from maize (ZmIRT2), which is highly homologous to OsIRT2 and ZmIRT1. ZmIRT2 was expressed in roots and anther and was induced by Fe and zinc (Zn) deficiencies. ZmIRT2-GFP fusion protein localized to the plasma membrane and endoplasmic reticulum. ZmIRT2 reversed growth defects involving Zn and Fe uptake in mutant yeast. ZmIRT2 overexpression in maize led to elevated Zn and Fe levels in roots, shoots and seeds of transgenic plants. Transcript levels of ZmIRT1 were elevated in roots, while levels of YS1 were reduced in shoots of ZmIRT2 transgenic plants. Our results imply that ZmIRT2 may function solely with ZmIRT1 to mediate Fe uptake in roots. ZmIRT1, ZmIRT2 and ZmYS1 may function in a cooperative manner to maintain Zn and Fe homeostasis in ZmIRT2 overexpressing plants. Furthermore, ZmIRT2 could be used in fortification efforts to elevate Zn and Fe levels in crop plants.

Genome-wide analysis of the NAAT, DMAS, TOM, and ENA gene families in maize suggests their roles in mediating iron homeostasis.

BACKGROUND: Nicotianamine (NA), 2'-deoxymugineic acid (DMA), and mugineic acid (MA) are chelators required for iron uptake and transport in plants. Nicotianamine aminotransferase (NAAT), 2'-deoxymugineic acid synthase (DMAS), transporter of MAs (TOM), and efflux transporter of NA (ENA) are involved in iron uptake and transport in rice (Oryza sativa), wheat (Triticum aestivum), and barley (Hordeum vulgare); however, these families have not been fully identified and comprehensively analyzed in maize (Zea mays L.). RESULTS: Here, we identified 5 ZmNAAT, 9 ZmDMAS, 11 ZmTOM, and 2 ZmENA genes by genome mining. RNA-sequencing and quantitative real-time PCR analysis revealed that these genes are expressed in various tissues and respond differently to high and low iron conditions. In particular, iron deficiency stimulated the expression of ZmDMAS1, ZmTOM1, ZmTOM3, and ZmENA1. Furthermore, we determined protein subcellular localization by transient expression of green fluorescent protein fusions in maize mesophyll protoplasts. ZmNAAT1, ZmNAAT-L4, ZmDMAS1, and ZmDMAS-L1 localized in the cytoplasm, whereas ZmTOMs and ZmENAs targeted to plasma and tonoplast membranes, endomembranes, and vesicles. CONCLUSIONS: Our results suggest that the different gene expression profiles and subcellular localizations of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA family members may enable specific regulation of phytosiderophore metabolism in different tissues and under different external conditions, shedding light on iron homeostasis in maize and providing candidate genes for breeding iron-rich maize varieties.

A rapid construction of new boron heterocycles and evaluation of photophysical properties of iminoboronates.

A green and efficient method for the synthesis of oxadiazaborole, dioxazaborinine, and oxadiazaborinine from the reactions of phenylboronic acid with amidoxime, α-hydroxyl oxime and α-hydroxyl hydrazone, respectively, is described. The reactions were performed under catalyst-free and mild conditions. All products can be rapidly purified by filtration and washing. In addition, a set of iminoboronates were prepared following a one-pot multicomponent reaction procedure using α-hydroxyl hydrazone, salicylaldehyde and boronic acid derivatives as starting materials and their photophysical properties were assessed. Then, cross-coupling reactions can be carried out smoothly on some target compounds, which may help develop new boron masking strategies.

Metabolic engineering of astaxanthin-rich maize and its use in the production of biofortified eggs.

Production of the high-value carotenoid astaxanthin, which is widely used in food and feed due to its strong antioxidant activity and colour, is less efficient in cereals than in model plants. Here, we report a new strategy for expressing ß-carotene ketolase and hydroxylase genes from algae, yeasts and flowering plants in the whole seed using a seed-specific bidirectional promoter. Engineered maize events were backcrossed to inbred maize lines with yellow endosperm to generate progenies that accumulate astaxanthin from 47.76 to 111.82 mg/kg DW in seeds, and the maximum level is approximately sixfold higher than those in previous reports (16.2-16.8 mg/kg DW) in cereals. A feeding trial with laying hens indicated that they could take up astaxanthin from the maize and accumulate it in egg yolks (12.10-14.15 mg/kg) without affecting egg production and quality, as observed using astaxanthin from Haematococcus pluvialis. Storage stability evaluation analysis showed that the optimal conditions for long-term storage of astaxanthin-rich maize are at 4 °C in the dark. This study shows that co-expressing of functional genes driven by seed-specific bidirectional promoter could dramatically boost astaxanthin biosynthesis in every parts of kernel including embryo, aleurone layer and starch endosperm other than previous reports in the starch endosperm only. And the staple crop maize could serve as a cost-effective plant factory for reliably producing astaxanthin.

ZmGRAS11, transactivated by Opaque2, positively regulates kernel size in maize.

Although the genetic basis for endosperm development in maize (Zea mays) has been well studied, the mechanism for coordinating grain filling with increasing kernel size remains elusive. Here, we report that increased kernel size was selected during modern breeding and identify a novel DELLA-like transcriptional regulator, ZmGRAS11, which positively regulates kernel size and kernel weight in maize. We find that Opaque2, a core transcription factor for zein protein and starch accumulation, transactivates the expression of ZmGRAS11. Our data suggest that the Opaque2-ZmGRAS11 module mediates synergistic endosperm enlargement with grain filling.

Unambiguous Phosphosite Localization through the Combination of Trypsin and LysargiNase Mirror Spectra in a Large-Scale Phosphoproteome Study.

Understanding of the kinase-guided signaling pathways requires the identification and analysis of phosphosites. Mass spectrometry (MS)-based phosphoproteomics is a rapid and highly sensitive approach for high-throughput identification of phosphosites. However, phosphosite determination from MS data with a single protease is more likely to be ambiguous, regardless of the strategy used for phosphopeptide detection. Here, we explored the application of LysargiNase, which was recently reported to mirror trypsin in specificity to cleave arginine and lysine residues exclusively at the N-terminal side. We found that the combination of trypsin and LysargiNase mirror spectra resulted in higher ion coverage in MS2 spectra. The median ion coverage values of b ions in tryptic spectra, LysargiNase spectra, and combined spectra are 8.3, 20.5, and 25.0%, respectively. As for the median ion coverage of y ions, these values are 27.8, 10.0, and 32.3%. Higher ion coverage was helpful to pinpoint the precise phosphosites. Compared to trypsin alone, the combined use of trypsin and LysargiNase mirror spectra enabled 67.1% of mirror spectra with unreliable scores (confidence score <0.75) to become reliable (confidence score ≥ 0.75). Meanwhile, all of the mirror peptide-spectrum matches (PSMs) with multiple potential phosphosites from trypsin and LysargiNase digests could be assigned one precise phosphosite after applying the combination strategy. Besides, the combination strategy could identify more novel phosphosites than the union strategy did. We synthesized three phosphopeptides corresponding to the three novel phosphosites and validated the reliability of the identification. Taken together, our data demonstrated the distinctive potential of the combination strategy presented here for unambiguous phosphosite localization (Project accession PXD011178).

Laparoscopic resection of hepatic epithelioid hemangioendothelioma: report of eleven rare cases and literature review.

BACKGROUND: Hepatic epithelioid hemangioendothelioma (HEHE) is an extremely rare borderline tumor of vascular endothelial origin. Laparoscopic resection of HEHE has never been reported. METHODS: The clinical data of eleven patients with HEHE (4 women and 7 men) who were diagnosed and treated at the Union Hospital (Wuhan, China), and Wuhan Asia General Hospital (Wuhan, China), between March 2012 and July 2020 were analyzed retrospectively. RESULTS: The mean age of HEHE patients was 42.4 ± 13.9 years (range 22-67 years). All patients underwent laparoscopic surgery alone or in combination with radiofrequency ablation. Most tumors showed aggressive growth or metastasis. By immunohistochemistry, tumor cells were positive for CD31, CD34, ERG, PCK, FLi-1, TFE-3, and Ki-67 (labeling index range, 5-15%). In one of the patients, the tumor was accompanied by partial necrosis with a local appearance of epithelioid angiosarcoma. Postoperative adjuvant treatment included chemotherapy, sorafenib, and Huaier granule. As of July 2020, the median follow-up duration was 36 months (range, 9-60 months), with 2 (18.2%) patients experiencing tumor recurrence. CONCLUSIONS: This is the first report of laparoscopic hepatectomy of HEHE. Curative laparoscopic hepatectomy might be an acceptable treatment for appropriate HEHE patients.

Improving Zinc and Iron Accumulation in Maize Grains Using the Zinc and Iron Transporter ZmZIP5.

Zinc (Zn) and iron (Fe) are essential micronutrients for plant growth. Thus, it is important to understand the mechanisms of uptake, transport and accumulation of these micronutrients in maize to improve crop nutritional quality. Members of the zinc-regulated transporters, iron-regulated transporter-like protein (ZIP) family are responsible for the uptake and transport of divalent metal ions in plant. Previously, we showed that ZmZIP5 functionally complemented the Zn uptake double mutant zrt1zrt2, Fe-uptake double mutant fet3fet4 in yeast. In our ß-glucuronidase (GUS) assay, the germinated seeds, young sheaths, and stems of ZmZIP5-promoter-GUS transgenic plants were stained. We generated and compared two maize lines for this study: Ubi-ZmZIP5, in which ZmZIP5 was constitutively overexpressed, and ZmZIP5i, a RNAi line. At the seedling stage, high levels of Zn and Fe were found in the roots and shoots of Ubi-ZmZIP5 plants, whereas low levels were found in the ZmZIP5i plants. Zn and Fe contents decreased in the seeds of Ubi-ZmZIP5 plants and remained unchanged in the seeds of ZmZIP5i plants. The seeds of Leg-ZmZIP5 plants, in which ZmZIP5 overexpression is specific to the endosperm, had higher levels of Zn and Fe. Our results imply that ZmZIP5 may play a role in Zn and Fe uptake and root-to-shoot translocation. Endosperm-specific ZmZIP5 overexpression could be useful for Zn and Fe biofortification of cereal grains.

Synthesis of Seed-Specific Bidirectional Promoters for Metabolic Engineering of Anthocyanin-Rich Maize.

Tissue-specific promoters play an important role in plant molecular farming. Here, we describe a strategy to modify the tissue specificity of a maize embryo-specific bidirectional promoter PZmBD1. Six types of cis-elements, i.e. RY repeats (R), GCN4 (G), the prolamin box (P), Skn-1 (S), and the ACGT and AACA (A) motifs, were collected and fused to PZmBD1 to generate eight chimeric putative bidirectional promoters. Qualitative and quantitative analysis of reporter genes driven by the promoters showed that two promoters exhibited high seed-specific bidirectional activity in maize transient and stable transformed systems. The stronger one was chosen and fused to the intergenic region of two gene clusters consisting of four anthocyanin biosynthesis-related genes (ZmBz1, ZmBz2, ZmC1 and ZmR2) and seven reporter genes, resulting in the first embryo and endosperm anthocyanin-rich purple maize. Anthocyanin analysis showed that the total anthocyanin content reaches 2,910 mg kg-1 DW in transgenic maize and cyanidin is the major anthocyanin in transgenic maize, as in natural varieties. The expression profile analysis of endogenous genes showed that the anthocyanin biosynthesis pathway was activated by two transgenic transcription factor genes ZmC1 and ZmR2. Our results indicate that both the modification strategy and these functionally characterized tissue-specific bidirectional promoters generated could be used for genetic research and development of plant biotechnology products. The anthocyanin-rich purple maize could provide economic natural colorants for the food and beverage industry, and valuable germplasm for developing anthocyanin-rich fresh corn.

Identification of Missing Proteins in the Phosphoproteome of Kidney Cancer.

Identifying missing proteins (MPs) has been one of the critical missions of the Chromosome-Centric Human Proteome Project (C-HPP). Since 2012, over 30 research teams from 17 countries have been trying to search adequate and accurate evidence of MPs through various biochemical strategies. MPs mainly fall into the following classes: (1) low-molecular-weight (LMW) proteins, (2) membrane proteins, (3) proteins that contained various post-translational modifications (PTMs), (4) nucleic acid-associated proteins, (5) low abundance, and (6) unexpressed genes. In this study, kidney cancer and adjacent tissues were used for phosphoproteomics research, and 8962 proteins were identified, including 6415 phosphoproteins, and 44 728 phosphosites, of which 10 266 were unreported previously. In total, 75 candidate detections were found, including 45 phoshoproteins. GO analysis for these 75 candidate detections revealed that these proteins mainly clustered as membrane proteins and took part in nephron and kidney development. After rigorous screening and manual check, 9 of them were verified with the synthesized peptides. Finally, only one missing protein was confirmed. All mass spectrometry data from this study have been deposited in the PRIDE with identifier PXD006482.

The intergenic region of the maize defensin-like protein genes Def1 and Def2 functions as an embryo-specific asymmetric bidirectional promoter.

Bidirectional promoters are identified in diverse organisms with widely varied genome sizes, including bacteria, yeast, mammals, and plants. However, little research has been done on any individual endogenous bidirectional promoter from plants. Here, we describe a promoter positioned in the intergenic region of two defensin-like protein genes, Def1 and Def2 in maize (Zea mays). We examined the expression profiles of Def1 and Def2 in 14 maize tissues by qRT-PCR, and the results showed that this gene pair was expressed abundantly and specifically in seeds. When fused to either green fluorescent protein (GFP) or ß-glucuronidase (GUS) reporter genes, P ZmBD1 , P ZmDef1 , and P ZmDef2 were active and reproduced the expression patterns of both Def1 and Def2 genes in transformed immature maize embryos, as well as in developing seeds of transgenic maize. Comparative analysis revealed that PZmBD1 shared most of the expression characteristics of the two polar promoters, but displayed more stringent embryo specificity, delayed expression initiation, and asymmetric promoter activity. Moreover, a truncated promoter study revealed that the core promoters only exhibit basic bidirectional activity, while interacting with necessary cis-elements, which leads to polarity and different strengths. The sophisticated interaction or counteraction between the core promoter and cis-elements may potentially regulate bidirectional promoters.

Isolation of a maize ZmCI-1B promoter and characterization of its activity in transgenic maize and tobacco.

KEY MESSAGE: The 2-kb ZmCI - 1B promoter is active in the root and embryo and induced by wounding in maize and the 220-bp 5'-deleted segment maybe the minimal promoter. The subtilisin-chymotrypsin inhibitor gene, CI-1B of Zea mays (ZmCI-1B), has been suggested to induce the maize defense system to resist insect attack. Real-time RT-PCR showed that ZmCI-1B gene exhibited especially high expression in roots and embryos. The 2-kb full-length promoter of ZmCI-1B gene was isolated from the maize genome and used to drive expression of a beta-glucuronidase (GUS) reporter gene for transient expression and stable expression analysis in maize. The results of GUS histochemical staining in transgenic maize plants revealed that the ZmCI-1B promoter induced GUS expression preferentially in roots and embryos and in response to wounding. A series of 5'-deleted segments of the ZmCI-1B promoter were cloned individually to drive GUS expression for further analysis. Deletion analysis combined with the histochemical staining of transgenic tobacco plants revealed 220-bp segment could drive GUS in a tissue-specific and wounding-induced expression in tobacco; thus, it maybe the minimally active promoter of ZmCI-1B gene. Furthermore, it revealed that the ZmCI-1B promoter contained tissue-specific and wounding-induced elements.

Identification and functional characterization of bidirectional gene pairs and their intergenic regions in maize.

BACKGROUND: Bidirectional gene pairs exist as a specific form of gene organization in microorganisms and mammals as well as in model plant species, such as Arabidopsis and rice. Little is known about bidirectional gene pairs in maize, which has a large genome and is one of the most important grain crops. RESULTS: We conducted a genome-wide search in maize using genome sequencing results from the inbred line B73. In total, 1696 bidirectional transcript pairs were identified using a modified search model. We functionally characterized the promoter activity of the intergenic regions of most of the bidirectional transcript pairs that were expressed in embryos using a maize embryo transient expression system. A comparative study of bidirectional gene pairs performed for three monocot (Zea mays, Sorghum bicolor and Oryza sativa) and two dicot (Arabidopsis thaliana and Glycine max) plant genomes showed that bidirectional gene pairs were abundant in the five plant species. Orthologous bidirectional gene pairs were clearly distinguishable between the monocot and dicot species although the total numbers of orthologous bidirectional genes were similar. Analysis of the gene pairs using the Blast2GO software suite showed that the molecular functions (MF), cellular components (CC), and biological processes (BP) associated with the bidirectional transcripts were similar among the five plant species. CONCLUSIONS: The evolutionary analysis of the function and structure of orthologous bidirectional gene pairs in various plant species revealed a potential pathway of their origin, which may be required for the evolution of a new species.