Overexpression of the maize γ-tocopherol methyltransferase gene (ZmTMT) increases α-tocopherol content in transgenic Arabidopsis and maize seeds.

The vitamin E family includes tocopherols and tocotrienols, which are essential lipid-soluble antioxidants necessary for human and livestock health. The seeds of many plant species, including maize, have high gamma (γ)-tocopherol but low alpha (α)-tocopherol contents; however, α-tocopherol is the most effective antioxidant. Therefore, it is necessary to optimize the tocopherol composition in plants. α-Tocopherol is synthesized from γ-tocopherol by γ-tocopherol methyltransferase (γ-TMT, VTE4) in the final step of the tocopherol biosynthetic pathway. In the present study, the full-length coding sequence (CDS) of γ-TMT was isolated from Zea mays, named ZmTMT. The ZmTMT CDS was 1059 bp in size, encoding 352 amino acids. Recombinant ZmTMT was expressed in Escherichia coli and the purified protein effectively converted γ-tocopherol into α-tocopherol in vitro. A comparison of enzyme activities showed that the activity of ZmTMT was higher than that of GmTMT2a (Glycine max) and AtTMT (Arabidopsis thaliana). Overexpression of ZmTMT increased the α-tocopherol content 4-5-fold in transgenic Arabidopsis and around 6.5-fold in transgenic maize kernels, and increased the α-/γ-tocopherol ratio to approximately 15 and 17, respectively. These results show that it is feasible to overexpress ZmTMT to optimize the tocopherol composition in maize; such a corn product might be useful in the feed industry in the near future.

Interactions of Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN reveal a novel intracellular auxin transport mechanism.

Little is known about the transport mechanism of intracellular auxin. Here, we report two vacuole-localized proteins, Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN (OsCLIP), that regulate intracellular auxin transport and homoeostasis. Overexpression of OsCOLE1 markedly increased the internode length and auxin content of the stem base, whereas these parameters were decreased in RNA interference (RNAi) plants. OsCOLE1 was localized on the tonoplast and preferentially expressed in mature tissues. We further identified its interacting protein OsCLIP, which was co-localized on the tonoplast. Protein-protein binding assays demonstrated that the N-terminus of OsCOLE1 directly interacted with OsCLIP in yeast cells and the rice protoplast. Furthermore, (3) H-indole-3-acetic acid ((3) H-IAA) transport assays revealed that OsCLIP transported IAA into yeast cells, which was promoted by OsCOLE1. The results indicate that OsCOLE1 affects rice development by regulating intracellular auxin transport through interaction with OsCLIP, which provides a new insight into the regulatory mechanism of intracellular transport of auxin and the roles of vacuoles in plant development.

ZmSOC1, a MADS-box transcription factor from Zea mays, promotes flowering in Arabidopsis.

Zea mays is an economically important crop, but its molecular mechanism of flowering remains largely uncharacterized. The gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), integrates multiple flowering signals to regulate floral transition in Arabidopsis. In this study, ZmSOC1 was isolated from Zea mays. Sequence alignment and phylogenetic analysis demonstrated that the ZmSOC1 protein contained a highly conserved MADS domain and a typical SOC1 motif. ZmSOC1 protein was localized in the nucleus in protoplasts and showed no transcriptional activation activity in yeast cells. ZmSOC1 was highly expressed in maize reproductive organs, including filaments, ear and endosperm, but expression was very low in embryos; on the other hand, the abiotic stresses could repress ZmSOC1 expression. Overexpression of ZmSOC1 resulted in early flowering in Arabidopsis through increasing the expression of AtLFY and AtAP1. Overall, these results suggest that ZmSOC1 is a flowering promoter in Arabidopsis.

The Evolution of Curve Patterns in Adolescent Idiopathic Scoliosis during Chêneau Brace Treatment.

STUDY DESIGN/SETTING: This retrospective study analyzed bracing outcomes in AIS patients, focusing on curve pattern changes and brace efficacy. OBJECTIVE: To analyze the effectiveness of the Chêneau brace across different curve patterns and to evaluate the tendencies in curve evolution during treatment. SUMMARY OF BACKGROUND DATA: Adolescent idiopathic scoliosis (AIS) presents diverse curve patterns, each responding differently to bracing. Understanding these variations is crucial for optimizing treatment strategies. METHODS: The study included 177 AIS patients treated with Chêneau orthoses, categorized based on curve patterns as per the main curve and modified Lenke (mLenke) classifications. We compared patients according to curve patterns and assessed changes in curve magnitude and pattern before and after treatment. RESULTS: Over an average follow-up of 28.1±10.7 months, the primary curve magnitude decreased from 28.8±6.6° to 25.9±10.5°. Significant reductions were observed in mLenke V and VI patients (P<0.05). Patients with main lumbar curves showed better initial in-brace correction and curve control compared with those with main thoracic curves (P<0.05). In single-curve patterns, binary logistic regression indicated that mLenke V patients demonstrated higher rates of curve control compared with mLenke I patients (P<0.05). No significant differences were found in double-curve patterns between mLenke III and VI (P>0.05). At the final follow-up, thoracolumbar/lumbar curves improved significantly in mLenke III and VI patients (P<0.05), while thoracic curves did not (P>0.05). Furthermore, at the last follow-up, the proportions of mLenke I, II, and IV increased, while mLenke III, V, and VI decreased. CONCLUSIONS: Bracing outcomes were more favorable in patients with main lumbar curves than those with main thoracic curves. However, no significant differences were found in patients with double-curve patterns. Thoracic curves exhibited a higher progression risk compared with thoracolumbar/lumbar curves within the same curve pattern. During bracing, a tendency for primary curves to shift proximally was noted.

Construction of a genomewide RNAi mutant library in rice.

Long hairpin RNA (hpRNA) transgenes are a powerful tool for gene function studies in plants, but a genomewide RNAi mutant library using hpRNA transgenes has not been reported for plants. Here, we report the construction of a hpRNA library for the genomewide identification of gene function in rice using an improved rolling circle amplification-mediated hpRNA (RMHR) method. Transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function. The target mRNA was down-regulated in the hpRNA lines, and this was correlated with the accumulation of siRNAs corresponding to the double-stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of such cross-silencing depended on the level of sequence homology between the members as well as the abundance of matching siRNAs. The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted. Deep sequencing analysis of small RNAs showed that the hpRNA-derived siRNAs were characteristic of Argonaute-binding small RNAs. Our results indicate that RNAi mutant library is a high-efficient approach for genomewide gene identification in plants.

GmTMT2a from soybean elevates the α-tocopherol content in corn and Arabidopsis.

Tocochromanol, or vitamin E, plays a crucial role in human and animal nutrition and is synthesized only by photosynthetic organisms. γ-Tocopherol methyltransferase (γ-TMT), one of the key enzymes in the tocopherol biosynthetic pathway in plants, converts γ, δ-tocopherols into α-, ß-tocopherols. Tocopherol content was investigated in 15 soybean cultivars and GmTMT2 was isolated from five varieties based on tocopherol content. GmTMT2a was expressed in E. coli and the purified protein effectively converted γ-tocopherol into α-tocopherol in vitro. Overexpression of GmTMT2a enhanced α-tocopherol content 4-6-fold in transgenic Arabidopsis, and α-tocopherol content increased 3-4.5-fold in transgenic maize seed, which correlated with the accumulation of GmTMT2a. Transgenic corn that is α-tocopherol-rich may be beneficial for animal health and growth.

ZmmiR169q/ZmNF-YA8 is a module that homeostatically regulates primary root growth and salt tolerance in maize.

In response to salt stress, plants alter the expression of manifold gene networks, enabling them to survive and thrive in the face of adversity. As a result, the growth and development of plant roots could be drastically altered, with significant inhibition of the growth of root meristematic zones. Although it is known that root growth is primarily regulated by auxins and cytokinins, the molecular regulatory mechanism by which salt stress stunts root meristems remains obscure. In this study, we found that the ZmmiR169q/ZmNF-YA8 module regulates the growth of maize taproots in response to salt stress. Salt stress downregulates ZmmiR169q expression, allowing for significant upregulation of ZmNF-YA8, which, in turn, activates ZmERF1B, triggering the upregulation of ASA1 and ASA2, two rate-limiting enzymes in the biosynthesis of tryptophan (Trp), leading to the accumulation of auxin in the root tip, thereby inhibiting root growth. The development of the maize root is stymied as meristem cell division and meristematic zone expansion are both stifled. This study reveals the ZmmiR169q/ZmNF-YA8 module's involvement in maintaining an equilibrium in bestowing plant salt tolerance and root growth and development under salt stress, providing new insights into the molecular mechanism underlying the homeostatic regulation of plant development in response to salt stress.

Molecular Characterization and Efficacy Evaluation of Transgenic Maize Harboring cry2Ab-vip3A-cp4epsps for Insect Resistance and Herbicide Tolerance.

Insect infestation and weed interference have a seriously negative impact on the growth, yield, and grain quality of maize. In this study, transgenic maize plants harboring three exogenous genes, cry2Ab, vip3A, and cp4epsps, that were constructed into a single T-DNA were developed for protection against insects and weeds. The transgene integration sites on the chromosomes in two transgenic maize events, CVC-1 and CVC-2, were determined using whole genome sequencing and specific PCR detection. As revealed by laboratory insect bioassays, these two transgenic events exhibited strong insecticidal toxicity against three major species of Lepidoptera insects, including Mythimna separata, Helicoverpa armigera, and Spodoptera frugiperda, with mortality rates exceeding 96%, 100%, and 100%, respectively, after six days of infestation. In addition, CVC-1 exhibited a high tolerance to glyphosate under field conditions. The successful expressions of cry2Ab, vip3A, and cp4epsps in various tissues at different developmental stages of CVC-1 were validated at the transcriptional and translational levels using quantitative real-time reverse transcription PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. These findings demonstrated that the transgenic maize CVC-1 developed using this triple gene construct has excellent insect resistance and herbicide tolerance, which may provide a valuable germplasm resource and data support for future maize breeding of insect and weed control.

Scoliosis Secondary to Neurofibromatosis Type I Combined With Moyamoya Syndrome: A Case Report.

CASE: A 13-year-old girl presenting with scoliosis accompanied by type I neurofibromatosis (NF1) underwent deformity correction surgery. On the second postoperative day, she had sudden-onset acute cerebral infarctions and was diagnosed with moyamoya syndrome. After neurological conservative treatment, at the sixth month after surgery, her symptoms and signs were significantly improved and the daily life was not affected by herself. CONCLUSION: NF1 scoliosis with moyamoya syndrome is an uncommon inherited disorder. Intracranial vasculopathy is hardly detected from routine preoperative examinations. The specific intracranial vascular examination should be performed for patients with NF1 scoliosis, especially those who have a positive history of cerebral ischemic or hemorrhagic symptoms.

Functional analysis of BnMAR element in transgenic tobacco plants.

Scaffold/matrix attachment regions (S/MARs) are defined as genomic DNA sequences, located at the physical boundaries of chromatin loops. Previous reports suggest that S/MARs elements may increase and stabilize the expression of transgene. In this study, DNA sequence with MAR characteristics has been isolated from B. napus . The BnMARs sequence was used to flank the CaMV35S-GUS-NOS expression cassette within the T-DNA of the plant expression vector pPZP212. These constructs were introduced into tobacco plants, respectively and the GUS reporter gene expression was investigated in stably transformed plants. When the forward BnMARs sequence was inserted into the upstream of CaMV35S promoter, the average GUS activities were much higher than those without BnMARs in transgenic tobacco. The GUS expression of M(+)35S:GUS, M(+)35S:GUSM(+) and M(+)35S:GUSM(-) constructs increased average 1.0-fold, with or without BnMARs located downstream of NOS. The GUS expression would not be affected when reverse BnMARs sequence inserted whether upstream of CaMV35S promoter or downstream of NOS. The GUS expression was affected a little when reverse BnMARs sequence was inserted the downstream of NOS and BnMARs could not act by serving as of promoter. The results showed that the presence of forward BnMARs sequence does have an obvious impact on enhancing downstream gene expression and its effect is unidirectional.

A simple artificial microRNA vector based on ath-miR169d precursor from Arabidopsis.

Artificial microRNA (amiRNA) is becoming a powerful tool for silencing genes in plants, and several amiRNA vectors have recently been developed based on the natural precursor structures of ath-miR159a, ath-miR164b, ath-miR172a, ath-miR319a and osa-miR528. In this study we generated a simple amiRNA vector (pAmiR169d) based on the structure of Arabidopsis miR169d precursor (pre-miR169d). Two unique restriction sites were created inside the stem region of pre-miR169d, which allows for the artificial miRNA sequences to be cloned as either ~80 bp synthetic oligonucleotides or PCR products. A beta-glucuronidase:green florescent protein fusion gene (GUS-GFP) was efficiently silenced in transient assays using a pAmiR169d-derived construct targeting the GUS-GFP sequence. 5'-RACE showed that the target GUS-GFP transcript was cleaved precisely at the expected position across nucleotides 10 and 11 of the artificial miRNA. Thus, pAmiR169d allows for both easy construction of artificial miRNA constructs and efficient silencing of target genes in plants.

Rolling circle amplification-mediated hairpin RNA (RMHR) library construction in plants.

Long hairpin RNA (lhRNA) construct-induced gene silencing facilitates the study of gene function in plants and animals, but constructing multiple lhRNA vectors using traditional approaches is both time-consuming and costly. Also, most of the existing approaches are based on sequence-specific cloning of individual sequences, and are therefore not suitable for preparing hpRNA libraries from a pool of mixed target sequences. Here we describe a rolling-circle amplification (RCA)-mediated hpRNA (RMHR) construction system suitable for generating libraries of lhRNA constructs from any gene of interest or pool of genes. Using RMHR we successfully generated a lhRNA library from a Arabidopsis cDNA population containing known and unknown genes, with an average size of 500-800 bp for the inverted-repeat inserts. To validate the RMHR system, lhRNA constructs targeting the beta-glucuronidase (GUS) gene were tested using Agrobacterium infiltration and shown to be effective at inducing GUS silencing in tobacco leaves. Our results indicate that the RMHR technique permits rapid, efficient and low-cost preparation of genome-wide lhRNA expression libraries.

A novel fluoroscopy-based robot system for pedicle screw fixation surgery.

BACKGROUND: Robot-assisted pedicle screw insertion has gained popularity in the spinal surgery field. Due to high cost, these spinal robots are not extensively applied in clinical surgeries. Developing an effective robot system with low cost and high clinical acceptability is one of the future trends. METHODS: We developed a novel fluoroscopy-based robot system for pedicle screw insertion. Four live pigs were conducted with percutaneous pedicle screw insertion. Robot-assisted surgery was performed on the left side of pedicle, while the right opposite side is placed by freehand. The respect accuracy, surgical time and fluoroscopy time were recorded. RESULTS: Robot-assisted group achieved 100% (23/23) accuracy. The average times (6.4 ± 1.7) for intraoperative fluoroscopy usage per procedure were lesser than freehand group (12.5 ± 3.6), and the surgical time (6.8 ± 2.1 min) per screw was reduced compared with freehand group (12.1 ± 4.8 min). CONCLUSIONS: Our robot system is cost-effective and feasible for pedicle screw placement. Low economic cost makes it easier for extensive application in primary hospitals.

HS1 Is Involved in Hygromycin Resistance Through Facilitating Hygromycin Phosphotransferase Transportation From Cytosol to Chloroplast.

The transportation of proteins encoded by nuclear genes from plant cytosol to chloroplast is essential for chloroplast functions. Proteins that have a chloroplast transit peptide (cTP) are imported into chloroplasts via translocases on the outer and inner chloroplast envelope. How proteins lacking transit sequence are imported into chloroplast remains largely unknown. During screening of an Arabidopsis population transformed with a hairpin RNA gene-silencing library, we identified some transgenic plants that had active expression of the selectable marker gene, hygromycin phosphotransferase (HPT), but were sensitive to the selection agent, hygromycin B (HyB). Mutant and complementation analysis showed that this HyB sensitivity of transgenic plants was due to silencing of the HS1 (Hygromycin-Sensitive 1) gene. HS1 is localized in the chloroplast and interacts physically with HPT in yeast cells and in planta. Fluorescence and immunoblotting analysis showed that HPT could not be transported effectively into chloroplasts in Aths1, which resulted in Aths1 is sensitivity to hygromycin on higher HyB-containing medium. These data revealed that HS1 is involved in HyB resistance in transgenic Arabidopsis through facilitating cytosol-chloroplast transportation of HPT. Our findings provide novel insights on transportation of chloroplast cTP-less proteins.

Isolation and characterization of a C-repeat binding transcription factor from maize.

C-repeat binding proteins (CBFs) are a group of transcription factors that have been proven to be important for stress tolerance in plants. Many of these transcription factors transactivate the promoters of cold-regulated genes via binding to low temperature- or dehydration-responsive cis-elements, thus conferring plants cold acclimation. In the present study, we isolated a C-repeat binding transcription factor from maize using the yeast one-hybrid system with the C-repeat motif from the promoter of the Arabidopsis COR15a gene as bait. The isolated transcription factor is highly similar to the Arabidopsis CBF3 in their predicted amino acid sequences, and is therefore designated ZmCBF3. Point mutation analyses of the ZmCBF3-binding cis-element revealed (A/G)(C/T)CGAC as the core binding sequence. Expression analyses showed that ZmCBF3 was upregulated by both abscisic acid and low temperature, and was actively expressed during embryogenesis, suggesting that ZmCBF3 plays a role in stress response in maize.