Banana MaNAC1 activates secondary cell wall cellulose biosynthesis to enhance chilling resistance in fruit.

Chilling injury has a negative impact on the quantity and quality of crops, especially subtropical and tropical plants. The plant cell wall is not only the main source of biomass production, but also the first barrier to various stresses. Therefore, improving the understanding of the alterations in cell wall architecture is of great significance for both biomass production and stress adaptation. Herein, we demonstrated that the cell wall principal component cellulose accumulated during chilling stress, which was caused by the activation of MaCESA proteins. The sequence-multiple comparisons show that a cold-inducible NAC transcriptional factor MaNAC1, a homologue of Secondary Wall NAC transcription factors, has high sequence similarity with Arabidopsis SND3. An increase in cell wall thickness and cellulosic glucan content was observed in MaNAC1-overexpressing Arabidopsis lines, indicating that MaNAC1 participates in cellulose biosynthesis. Over-expression of MaNAC1 in Arabidopsis mutant snd3 restored the defective secondary growth of thinner cell walls and increased cellulosic glucan content. Furthermore, the activation of MaCESA7 and MaCESA6B cellulose biosynthesis genes can be directly induced by MaNAC1 through binding to SNBE motifs within their promoters, leading to enhanced cellulose content during low-temperature stress. Ultimately, tomato fruit showed greater cold resistance in MaNAC1 overexpression lines with thickened cell walls and increased cellulosic glucan content. Our findings revealed that MaNAC1 performs a vital role as a positive modulator in modulating cell wall cellulose metabolism within banana fruit under chilling stress.

The Class II KNOX family members KNAT3 and KNAT7 redundantly participate in Arabidopsis seed coat mucilage biosynthesis.

The production of Arabidopsis seed mucilage involves complex polysaccharide biosynthetic pathways and developmental processes in seed epidermal cells. Although the polysaccharide components of Arabidopsis seed mucilage have been identified, their regulatory mechanism requires further investigation. Here, we show that Class II KNOX gene family members KNAT3 and KNAT7 play an essential role in regulating mucilage production in the early developmental stages of Arabidopsis seeds. Double mutant knat3knat7 resulted in defective seed mucilage production and columellae formation, whereas knat3 showed a normal phenotype compared with wild type, and the mucilage thickness in knat7 was slightly disturbed. Rhamnogalacturonan I (RG-I) and its biosynthetic substrates galacturonic acid and rhamnose were reduced in both the adherent and soluble mucilage of knat3knat7. Comparative transcriptome analysis on whole seeds suggested that polysaccharide, glucosinolate and anthocyanin biosynthetic pathways were specifically repressed in knat3knat7. Transient co-expression of KNAT3 and KNAT7 with promoter regions of candidate genes in Arabidopsis protoplasts revealed that both KNAT3 and KNAT7 act as positive regulators of the RG-I biosynthetic gene MUCILAGE-MODIFIED 4 (MUM4, AT1G53500). Collectively, our results demonstrate that KNAT3 and KNAT7 are multifunctional transcription factors in secondary cell wall development and redundantly modulate mucilage biosynthesis in Arabidopsis seeds.

Transcriptomic Evidence Reveals Low Gelatinous Layer Biosynthesis in Neolamarckia cadamba after Gravistimulation.

Trees can control their shape and resist gravity by producing tension wood (TW), which is a special wood that results from trees being put under stress. TW is characterized by the presence of a gelatinous layer (G layer) and the differential distribution of cell wall polymers. In this study, we investigated whether or not gravistimulation in N. cadamba resulted in TW with an obvious G layer. The results revealed an absence of an obvious G layer in samples of the upper side of a leaning stem (UW), as well as an accumulation of cellulose and a decrease in lignin content. A negligible change in the content of these polymers was recorded and compared to untreated plant (NW) samples, revealing the presence of a G layer either in much lower concentrations or in a lignified form. A transcriptomic investigation demonstrated a higher expression of cell wall esterase- and hydrolase-related genes in the UW, suggesting an accumulation of noncellulosic sugars in the UW, similar to the spectroscopy results. Furthermore, several G-layer-specific genes were also downregulated, including fasciclin-like arabinogalactan proteins (FLA), beta-galactosidase (BGAL) and chitinase-like proteins (CTL). The gene coexpression network revealed a strong correlation between cell-wall-synthesis-related genes and G-layer-synthesis-specific genes, suggesting their probable antagonistic role during G layer formation. In brief, the G layer in N. cadamba was either synthesized in a very low amount or was lignified during an early stage of growth; further experimental validation is required to understand the exact mechanism and stage of G layer formation in N. cadamba during gravistimulation.

The class II KNOX transcription factors KNAT3 and KNAT7 synergistically regulate monolignol biosynthesis in Arabidopsis.

The function of the transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is still unclear since it appears to be either a negative or a positive regulator for secondary cell wall deposition with its loss-of-function mutant displaying thicker interfascicular and xylary fiber cell walls but thinner vessel cell walls in inflorescence stems. To explore the exact function of KNAT7, class II KNOTTED1-LIKE HOMEOBOX (KNOX II) genes in Arabidopsis including KNAT3, KNAT4, and KNAT5 were studied together. By chimeric repressor technology, we found that both KNAT3 and KNAT7 repressors exhibited a similar dwarf phenotype. Both KNAT3 and KNAT7 genes were expressed in the inflorescence stems and the knat3 knat7 double mutant exhibited a dwarf phenotype similar to the repressor lines. A stem cross-section of knat3 knat7 displayed an enhanced irregular xylem phenotype as compared with the single mutants, and its cell wall thickness in xylem vessels and interfascicular fibers was significantly reduced. Analysis of cell wall chemical composition revealed that syringyl lignin was significantly decreased while guaiacyl lignin was increased in the knat3 knat7 double mutant. Coincidently, the knat3 knat7 transcriptome showed that most lignin pathway genes were activated, whereas the syringyl lignin-related gene Ferulate 5-Hydroxylase (F5H) was down-regulated. Protein interaction analysis revealed that KNAT3 and KNAT7 can form a heterodimer, and KNAT3, but not KNAT7, can interact with the key secondary cell wall formation transcription factors NST1/2, which suggests that the KNAT3-NST1/2 heterodimer complex regulates F5H to promote syringyl lignin synthesis. These results indicate that KNAT3 and KNAT7 synergistically work together to promote secondary cell wall biosynthesis.

The existence of intravertebral cleft impact on outcomes of nonacute osteoporotic vertebral compression fractures patients treated by percutaneous kyphoplasty: a comparative study.

STUDY DESIGN: A retrospective comparative study. OBJECTIVE: The purpose of this study is to assess radiologic features of intravertebral cleft (IVC) in nonacute osteoporotic vertebral compression fractures (OVCFs) patients, and analyze the existence of IVC impact on outcomes of percutaneous kyphoplasty (PKP). SUMMARY OF BACKGROUND DATA: The IVC sign is regarded as vertebral instability and the cause of persisting pain. It is more likely to happen at nonacute OVCFs patients. Patients with IVC sign have different outcomes from these without IVC treated by percutaneous vertebroplasty. There were rare reports about the outcomes of patients with IVC sign treated by PKP. MATERIALS AND METHODS: We divided 92 nonacute OVCFs patients (total of 113 vertebrae) into 2 groups according to the existence of IVC. Preoperative and postoperative Visual Analogue Scales, Oswestry Disability Index, kyphotic angulation (KA), and anterior vertebral height were recorded; the incidence and radiologic features of IVC were analyzed. RESULTS: The diagnostic sensitivity of IVC on plain radiograph, computed tomography, and magnetic resonance imaging were 35.4%, 89.3%, and 83.3%, respectively. The IVC group had an average correction KA of 9.14 degrees and reduction of ratio of compression of 20.09%, and the non-IVC group was 8.76 degrees and 20.23%, respectively. Cleft pattern of cement accounted for 64.6% in IVC group and 27.7% in non-IVC group. Five/7 of cement leakage in IVC group was intradiscal leakage, whereas 7/9 of cement leakage in non-IVC group was perivertebral leakage. CONCLUSIONS: Computed tomography and magnetic resonance imaging were more sensitivity to diagnose IVC sign than X-ray. PKP could improve pain, functional activity, KA, and anterior height of both IVC and non-IVC groups, however, there was more cleft pattern of cement and higher intradiscal cement leakage in the IVC group.

Woody plant cell walls: Fundamentals and utilization.

Cell walls in plants, particularly forest trees, are the major carbon sink of the terrestrial ecosystem. Chemical and biosynthetic features of plant cell walls were revealed early on, focusing mostly on herbaceous model species. Recent developments in genomics, transcriptomics, epigenomics, transgenesis, and associated analytical techniques are enabling novel insights into formation of woody cell walls. Here, we review multilevel regulation of cell wall biosynthesis in forest tree species. We highlight current approaches to engineering cell walls as potential feedstock for materials and energy and survey reported field tests of such engineered transgenic trees. We outline opportunities and challenges in future research to better understand cell type biogenesis for more efficient wood cell wall modification and utilization for biomaterials or for enhanced carbon capture and storage.

CRISPR/Cas9 mutated p-coumaroyl shikimate 3'-hydroxylase 3 gene in Populus tomentosa reveals lignin functioning on supporting tree upright.

The lignin plays one of the most important roles in plant secondary metabolism. However, it is still unclear how lignin can contribute to the impressive height of wood growth. In this study, C3'H, a rate-limiting enzyme of the lignin pathway, was used as the target gene. C3'H3 was knocked out by CRISPR/Cas9 in Populus tomentosa. Compared with wild-type popular trees, c3'h3 mutants exhibited dwarf phenotypes, collapsed xylem vessels, weakened phloem thickening, decreased hydraulic conductivity and photosynthetic efficiency, and reduced auxin content, except for reduced total lignin content and significantly increased H-subunit lignin. In the c3'h3 mutant, the flavonoid biosynthesis genes CHS, CHI, F3H, DFR, ANR, and LAR were upregulated, and flavonoid metabolite accumulations were detected, indicating that decreasing the lignin biosynthesis pathway enhanced flavonoid metabolic flux. Furthermore, flavonoid metabolites, such as naringenin and hesperetin, were largely increased, while higher hesperetin content suppressed plant cell division. Thus, studying the c3'h3 mutant allows us to deduce that lignin deficiency suppresses tree growth and leads to the dwarf phenotype due to collapsed xylem and thickened phloem, limiting material exchanges and transport.

The physiological and transcriptomic study of secondary growth in Neolamarckia cadamba stimulated by the ethylene precursor ACC.

Though many biological roles of ethylene have been investigated intensively, the molecular mechanism of ethylene's action in woody plants remains unclear. In this study, we investigated the effects of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, on the growth of Neolamarckia cadamba seedlings, a fast-growing tropical tree. After 14 days of ACC treatment, the plants showed a reduced physiological morphology while stem diameter increased; however, this did not occur after the addition of 1-MCP. Meanwhile, the lignin content of N. cadamba also increased. Transcriptome analysis revealed that the expression of the ethylene biosynthesis and signaling genes ACC oxidase (ACO) and ethylene insensitive 3 (EIN3) were up-regulated mainly at the 6th hour and the 3rd day of the ACC treatment, respectively. The transcription levels of transcription factors, mainly in the basic helix-loop-helix (bHLH), ethylene response factor (ERF), WRKY and v-myb avian myeloblastosis viral oncogene homolog (MYB) families, involved in the ethylene signaling and secondary growth also increased significantly. Furthermore, in accordance to the increased lignification of the stem, the transcriptional level of key enzymes in the phenylalanine pathway were elevated after the ACC treatment. Our results revealed the physiological and molecular mechanisms underlying the secondary growth stimulated by exogenous ACC treatment on N. cadamba seedlings.

Characterization of hemicelluloses in sugarcane (Saccharum spp. hybrids) culm during xylogenesis.

Hemicelluloses are effective renewable biopolymers that can be used in many different industrial processes and preparations. In plants, the content of hemicellulose might change with different developmental stages and/or tissues. Thus, in here chemical and structural differences in hemicellulose isolated from the apical, middle and basal segments of sugarcane stem were characterized using chemical techniques. Further, difference in expression levels of genes related to synthesis of hemicelluloses from these three segments were studied by RNA-seq and qRT-PCR etc. The sugarcane hemicellulose backbone was xylose residues connected via ß-1,4 glycosidic linkages which was further substituted with arabinose, acetyl and glucuronic acid side chains. Hemicellulose content was higher in the middle and basal segments with less backbone substitutions compared to apical segments. In terms of gene expression, hemicellulose synthesis and modification genes were intensely expressed in middle and basal segments. Taken together, our research describes differences in hemicellulose content and substitutions in sugarcane during xylogenesis, which will increase our knowledge for finding more refined use of sugarcane bagasse.

Acceptable Chin-Brow Vertical Angle for Neutral Position Radiography: Preliminary Analyses Based on Parameters of the Whole Sagittal Spine of an Asymptomatic Chinese Population.

OBJECTIVE: We sought to acquire the whole sagittal spine parameters and investigated the acceptable chin-brow vertical angle (CBVA) for neutral position radiography in an asymptomatic Chinese population. METHODS: The parameters measured in 257 asymptomatic volunteers included CBVA, occipital slope, orbital tilt, occipital incidence, C0-C2 Cobb angle, C2-C7 Cobb angle, C1-C7 Cobb angle, C2-C7 sagittal vertical axis and absolute rotation angle, cervical tilt, cranial tilt, T1 slope, and thoracic kyphosis, and others. We used Pearson correlation analyses to find relationships between CBVA and other variables. The subjects were divided into 5 groups according to the CBVA percentile: group A, 0%-20% CBVA; group B, 20%-40% CBVA; group C, 40%-60% CBVA; group D, 60%-80% CBVA; and group E, 80%-100% CBVA. We used analysis of variance to analyze differences among the 5 groups. RESULTS: Orbital tilt, Occipital incidence, C1-C7 Cobb angle, C2-C7 sagittal vertical axis, and cranial tilt all increased with increasing CBVA (P < 0.001). The occipital slope, C2-C7 Cobb angle, C2-C7 absolute rotation angle, cervical tilt, T1 slope, and thoracic kyphosis decreased with decreasing CBVA (P < 0.05). No correlations between other sagittal parameters and the CBVA were found. A slight deviation was found in groups B-D, with a greater deviation in groups A, C, and E. CONCLUSIONS: An acceptable range of -1.5° to 5.8° is recommended for the CBVA for cervical radiography in the neutral position. When spinal surgeons evaluate the cervical plane, the effects of the CBVA deviation on cervical curvature must be considered.

Effective extraction of Arabidopsis adherent seed mucilage by ultrasonic treatment.

The Arabidopsis seed coat is composed of two layers of mucilage, a water-soluble non-adherent outer layer and an adherent inner layer. The non-adherent mucilage can easily be extracted by gentle shaking. However, adherent mucilage is extremely difficult to dissociate from the seed coat. Despite various treatments to extract the adherent mucilage, including EDTA, ammonium oxalate, dilute alkali or acid washes, most of it remains on the seed coat. Here, we show for the first time the extraction of almost all of the adherent mucilage from the Arabidopsis seed coat. Our results demonstrate that ultrasonic treatment was able to extract the adherent mucilage effectively within 20 seconds. Adherent mucilage, like non-adherent mucilage, is mainly composed of rhamnogalacturonan I (RG I). The crystalline cellulose content in adherent mucilage was measured as 3.7 mg g-1 of dry seed. Compared with non-adherent mucilage, the adherent mucilage exhibits relatively stable levels of sugar under various environmental conditions. In all cases, adherent mucilage showed higher levels of sugar than non-adherent mucilage. The cell wall remnant could associate with the adherent mucilage, which could prevent the extraction of the adherent mucilage. Our results show that ultrasonic treatment is an effective method for the quick extraction of Arabidopsis adherent mucilage with little effort.

Percutaneous anterior C1/2 transarticular screw fixation: salvage of failed percutaneous odontoid screw fixation for odontoid fracture.

BACKGROUND: The objective of this study is to investigate the outcomes and safety of using percutaneous anterior C1/2 transarticular screw fixation as a salvage technique for odontoid fracture if percutaneous odontoid screw fixation fails. METHODS: Fifteen in 108 odontoid fracture patients (planned to be treated by percutaneous anterior odontoid screw fixation) were failed to introduce satisfactory odontoid screw trajectory. To salvage this problem, we chose the percutaneous anterior C1/2 transarticular screw fixation technique in treatment of these patients. The visual analogue score (VAS) of neck pain and Neck Disability Index (NDI) of all patients were scored at pre-operation, 3 months after operation, and final follow-up. Additional, technique-related complications were recorded and collected. RESULTS: Percutaneous C1/2 transarticular screw fixation was performed successfully in all 15 patients whose odontoid screw fixation failed. No technique-related complications (such as nerve injury, spinal cord injury, and esophageal injury) occurred. The VAS of neck pain and NDI score improved significantly (P = 0.000) after operation, and no significant differences were found when compared to 93 non-salvage patients who successfully performed the percutaneous anterior odontoid screw fixation. No screw loose or breakage occurred, all of the odontoid fractures achieve radiographic fusion, bony fusion bridge could be observed at the C1/2 lateral articular facet on 9/15 patients. CONCLUSIONS: We suggest that percutaneous anterior C1/2 transarticular screw fixation is a good alternative salvage technique if percutaneous odontoid screw fixation failed, and it is a minimally invasive, feasible, and safe technique.

Percutaneous atlantoaxial anterior transarticular screw fixation combined with mini-open posterior C1/2 wire fusion for patients with a high-riding vertebral artery.

CONTEXT/OBJECTIVE: To describe the technique and clinical results of percutaneous atlantoaxial anterior transarticular fixation combined with limited exposure posterior C1/2 arthrodesis in patients with a high-riding vertebral artery. DESIGN SETTING: Zhejiang Spine Center, China. PARTICIPANTS: Five patients with a high-riding vertebral artery and an upper cervical fracture. INTERVENTIONS: Percutaneous atlantoaxial anterior transarticular screw fixation combined with limited exposure posterior C1/2 wire fusion. OUTCOME MEASURES: Computed tomography scans were used to assess the high-riding vertebral artery and feasibility of anterior transarticular screw fixation preoperatively. A Philadelphia collar was used to immobilize the neck postoperatively. Anteroposterior (open-mouth) and lateral views were obtained at pre/postoperation and at the follow-up. RESULTS: The operation was performed successfully on all of the patients, and no intraoperative operation-related complications such as nerve injury, vertebral artery, and soft tissue complications occurred. The mean follow-up period was 33.8 months (range: 24 to 58 months). No screw breakage, loosening, pullout, or cutout was observed. Bone union was achieved in all patients at the last follow-up. CONCLUSIONS: Our small case series results suggested that percutaneous anterior transarticular screw fixation combined with mini-open posterior C1/2 wire fusion is a technically minimally invasive, safe, feasible, and useful method to treat patients with a high-riding vertebral artery.

Use of Supplemental Short Pre-Contoured Accessory Rods and Cobalt Chrome Alloy Posterior Rods Reduces Primary Rod Strain and Range of Motion Across the Pedicle Subtraction Osteotomy Level: An In Vitro Biomechanical Study.

STUDY DESIGN: In vitro cadaveric biomechanical study. OBJECTIVE: To assess effects of 4-rod reconstruction, rod material, and anterior column support on motion and surface rod strain in a pedicle subtraction osteotomy model. SUMMARY OF BACKGROUND DATA: Pedicle subtraction osteotomy (PSO) can correct significant sagittal deformity of the lumbar spine; however, revision rates are high. To reduce rod strain and the incidence of rod fracture, clinical use of multi-rod construction, cobalt chrome (CoCr) alloy rods, and interbody spacers adjacent to PSO has been proposed. Investigating both motion and rod strain is necessary to determine the biomechanical efficacy of these techniques. METHODS: Five specimens (T12-S1) underwent PSO at L3 with pedicle screw stabilization at L1-S1. Pedicle subtraction was adjusted to achieve a final lordosis of 70°. Flexion-extension (FE), lateral bending, and axial rotation were applied. Linear strain gauges measured surface rod strain during FE on primary and accessory rods at PSO level. Testing evaluated (1) accessory rods (4-Rod) added at PSO level versus primary rods (2-Rod); (2) Ti versus CoCr rods; and (3) lateral interbody spacers (S) inserted adjacent to PSO. One-way and three-way analysis of variance was performed (P ≤ 0.05). RESULTS: All constructs significantly reduced FE and lateral bending motion relative to intact (P < 0.001). The main effect of accessory rods in reducing FE motion was significant (P = 0.021). Accessory and CoCr rods reduced relative surface strain on the primary rod, irrespective of construct (P < 0.001). CoCr 4-Rod + S provided the greatest reduction in strain (76% decrease; P = 0.003). CONCLUSION: Accessory and CoCr rods provided greatest reduction in motion and rod strain at PSO level. Interbody devices minimally affected motion-induced strain and might act primarily to maintain disc height. Clinicians must assess whether surface strain and motion reduction minimize the incidence of rod fracture. LEVEL OF EVIDENCE: N/A.

A novel technique of two-hole guide tube for percutaneous anterior odontoid screw fixation.

BACKGROUND CONTEXT: Surgical stabilization is recommended for odontoid fractures with mechanical instability. Compared with C1-C2 fusion, percutaneous anterior odontoid screw fixation has the advantages of preserving C1/C2 motion and being a minimally invasive procedure. However, determining the optimal screw trajectory is often difficult. When an initial suboptimal K-wire hole is drilled, it is especially difficult to drill a second optimal K-wire trajectory because the initial hole will be entered inadvertently. PURPOSE: To design a novel device, two-hole guide tube, to make drilling a second optimal K-wire trajectory easier, and thus, avoid unnecessary additional surgical time and reduce the likelihood of needing to change the procedure to traditional open surgery. STUDY DESIGN: A technical report. METHODS: Fifty-three patients with odontoid fractures were treated by percutaneous anterior odontoid screw fixation in our hospital, and the initial K-wire trajectories of 16 cases (12 men and 4 women) among the 53 patients were imperfect. The two-hole guide tube was applied for drilling the second trajectory in each of these 16 cases. RESULTS: No complications associated with this technique occurred. Satisfactory results and good screw placement was achieved in all patients. Radiographic fusion was confirmed for 15 of 16 patients. None of the patients experienced clinical symptoms or screw loosening or breakage in this study. CONCLUSIONS: Our novel device, two-hole guide tube, can be used to reduce the difficulty associated with redrilling an optimal K-wire trajectory if the initial trajectory is imperfect during percutaneous anterior odontoid screw fixation. Moreover, by referring to the initial misplaced K-wire, a more accurate trajectory for the second K-wire can be achieved.