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.

Transcription factors KNAT3 and KNAT4 are essential for integument and ovule formation in Arabidopsis.

Integuments form important protective cell layers surrounding the developing ovules in gymno- and angiosperms. Although several genes have been shown to influence the development of integuments, the transcriptional regulatory mechanism is still poorly understood. In this work, we report that the Class II KNOTTED1-LIKE HOMEOBOX (KNOX II) transcription factors KNOTTED1-LIKE HOMEBOX GENE 3 (KNAT3) and KNAT4 regulate integument development in Arabidopsis (Arabidopsis thaliana). KNAT3 and KNAT4 were co-expressed in inflorescences and especially in young developing ovules. The loss-of-function double mutant knat3 knat4 showed an infertility phenotype, in which both inner and outer integuments of the ovule are arrested at an early stage and form an amorphous structure as in the bell1 (bel1) mutant. The expression of chimeric KNAT3- and KNAT4-EAR motif repression domain (SRDX repressors) resulted in severe seed abortion. Protein-protein interaction assays demonstrated that KNAT3 and KNAT4 interact with each other and also with INNER NO OUTER (INO), a key transcription factor required for the outer integument formation. Transcriptome analysis showed that the expression of genes related with integument development is influenced in the knat3 knat4 mutant. The knat3 knat4 mutant also had a lower indole-3-acetic acid (IAA) content, and some auxin signaling pathway genes were downregulated. Moreover, transactivation analysis indicated that KNAT3/4 and INO activate the auxin signaling gene IAA INDUCIBLE 14 (IAA14). Taken together, our study identified KNAT3 and KNAT4 as key factors in integument development in Arabidopsis.

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.

UDP-Api/UDP-Xyl synthases affect plant development by controlling the content of UDP-Api to regulate the RG-II-borate complex.

Rhamnogalacturonan-II (RG-II) is structurally the most complex glycan in higher plants, containing 13 different sugars and 21 distinct glycosidic linkages. Two monomeric RG-II molecules can form an RG-II-borate diester dimer through the two apiosyl (Api) residues of side chain A to regulate cross-linking of pectin in the cell wall. But the relationship of Api biosynthesis and RG-II dimer is still unclear. In this study we investigated the two homologous UDP-D-apiose/UDP-D-xylose synthases (AXSs) in Arabidopsis thaliana that synthesize UDP-D-apiose (UDP-Api). Both AXSs are ubiquitously expressed, while AXS2 has higher overall expression than AXS1 in the tissues analyzed. The homozygous axs double mutant is lethal, while heterozygous axs1/+ axs2 and axs1 axs2/+ mutants display intermediate phenotypes. The axs1/+ axs2 mutant plants are unable to set seed and die. By contrast, the axs1 axs2/+ mutant plants exhibit loss of shoot and root apical dominance. UDP-Api content in axs1 axs2/+ mutants is decreased by 83%. The cell wall of axs1 axs2/+ mutant plants is thicker and contains less RG-II-borate complex than wild-type Col-0 plants. Taken together, these results provide direct evidence of the importance of AXSs for UDP-Api and RG-II-borate complex formation in plant growth and development.

MicroRNAs play important roles in regulating the rapid growth of the Phyllostachys edulis culm internode.

Moso bamboo (Phyllostachys edulis) is a fast-growing species with uneven growth and lignification from lower to upper segments within one internode. MicroRNAs (miRNAs) play a vital role in post-transcriptional regulation in plants. However, how miRNAs regulate fast growth in bamboo internodes is poorly understood. In this study, one moso bamboo internode was divided during early rapid growth into four segments called F4 (bottom) to F1 (upper) and these were then analysed for transcriptomes, miRNAs and degradomes. The F4 segment had a higher number of actively dividing cells as well as a higher content of auxin (IAA), cytokinin (CK) and gibberellin (GA) compared with the F1 segment. RNA-seq analysis showed DNA replication and cell division-associated genes highly expressed in F4 rather than in F1. In total, 63 miRNAs (DEMs) were identified as differentially expressed between F4 and F1. The degradome and the transcriptome indicated that many downstream transcription factors and hormonal responses genes were modulated by DEMs. Several miR-target interactions were further validated by tobacco co-infiltration. Our findings give new insights into miRNA-mediated regulatory pathways in bamboo, and will contribute to a comprehensive understanding of the molecular mechanisms governing rapid growth.

The transcription factor PagLBD3 contributes to the regulation of secondary growth in Populus.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) genes encode plant-specific transcription factors that participate in regulating various developmental processes. In this study, we genetically characterized PagLBD3 encoding an important regulator of secondary growth in poplar (Populus alba × Populus glandulosa). Overexpression of PagLBD3 increased stem secondary growth in Populus with a significantly higher rate of cambial cell differentiation into phloem, while dominant repression of PagLBD3 significantly decreased the rate of cambial cell differentiation into phloem. Furthermore, we identified 1756 PagLBD3 genome-wide putative direct target genes (DTGs) through RNA sequencing (RNA-seq)-coupled DNA affinity purification followed by sequencing (DAP-seq) assays. Gene Ontology analysis revealed that genes regulated by PagLBD3 were enriched in biological pathways regulating meristem development, xylem development, and auxin transport. Several central regulator genes for vascular development, including PHLOEM INTERCALATED WITH XYLEM (PXY), WUSCHEL RELATED HOMEOBOX4 (WOX4), Secondary Wall-Associated NAC Domain 1s (SND1-B2), and Vascular-Related NAC-Domain 6s (VND6-B1), were identified as PagLBD3 DTGs. Together, our results indicate that PagLBD3 and its DTGs form a complex transcriptional network to modulate cambium activity and phloem/xylem differentiation.

De novo transcriptome assembly for the five major organs of Zanthoxylum armatum and the identification of genes involved in terpenoid compound and fatty acid metabolism.

BACKGROUND: Zanthoxylum armatum (Z. armatum) is a highly economically important tree that presents a special numbing taste. However, the underlying regulatory mechanism of the numbing taste remains poorly understood. Thus, the elucidation of the key genes associated with numbing taste biosynthesis pathways is critical for providing genetic information on Z. armatumand the breeding of high-quality germplasms of this species. RESULTS: Here, de novo transcriptome assembly was performed for the five major organs of Z. armatum, including the roots, stems, leaf buds, mature leaves and fruits. A total of 111,318 unigenes were generated with an average length of 1014 bp. Additionally, a large number of SSRs were obtained to improve our understanding of the phylogeny and genetics of Z. armatum. The organ-specific unigenes of the five major samples were screened and annotated via GO and KEGG enrichment analysis. A total of 53 and 34 unigenes that were exclusively upregulated in fruit samples were identified as candidate unigenes for terpenoid biosynthesis or fatty acid biosynthesis, elongation and degradation pathways, respectively. Moreover, 40 days after fertilization (Fr4 stage) could be an important period for the accumulation of terpenoid compounds during the fruit development and maturation of Z. armatum. The Fr4 stage could be a key point at which the first few steps of the fatty acid biosynthesis process are promoted, and the catalysis of subsequent reactions could be significantly induced at 62 days after fertilization (Fr6 stage). CONCLUSIONS: The present study realized de novo transcriptome assembly for the five major organs of Z. armatum. To the best of our knowledge, this study provides the first comprehensive analysis revealing the genes underlying the special numbing taste of Z. armatum. The assembled transcriptome profiles expand the available genetic information on this species and will contribute to gene functional studies, which will aid in the engineering of high-quality cultivars of Z. armatum.

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.

Balanced Xylan Acetylation is the Key Regulator of Plant Growth and Development, and Cell Wall Structure and for Industrial Utilization.

Xylan is the most abundant hemicellulose, constitutes about 25-35% of the dry biomass of woody and lignified tissues, and occurs up to 50% in some cereal grains. The accurate degree and position of xylan acetylation is necessary for xylan function and for plant growth and development. The post synthetic acetylation of cell wall xylan, mainly regulated by Reduced Wall Acetylation (RWA), Trichome Birefringence-Like (TBL), and Altered Xyloglucan 9 (AXY9) genes, is essential for effective bonding of xylan with cellulose. Recent studies have proven that not only xylan acetylation but also its deacetylation is vital for various plant functions. Thus, the present review focuses on the latest advances in understanding xylan acetylation and deacetylation and explores their effects on plant growth and development. Baseline knowledge about precise regulation of xylan acetylation and deacetylation is pivotal to developing plant biomass better suited for second-generation liquid biofuel production.

Physiological, Biochemical, and Transcriptomic Responses of Neolamarckia cadamba to Aluminum Stress.

Aluminum is the most abundant metal of the Earth's crust accounting for 7% of its mass, and release of toxic Al3+ in acid soils restricts plant growth. Neolamarckia cadamba, a fast-growing tree, only grows in tropical regions with acidic soils. In this study, N. cadamba was treated with high concentrations of aluminum under acidic condition (pH 4.5) to study its physiological, biochemical, and molecular response mechanisms against high aluminum stress. High aluminum concentration resulted in significant inhibition of root growth with time in N. cadamba. The concentration of Al3+ ions in the root tip increased significantly and the distribution of absorbed Al3+ was observed in the root tip after Al stress. Meanwhile, the concentration of Ca, Mg, Mn, and Fe was significantly decreased, but P concentration increased. Aluminum stress increased activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase from micrococcus lysodeiktic (CAT), and peroxidase (POD) in the root tip, while the content of MDA was decreased. Transcriptome analysis showed 37,478 differential expression genes (DEGs) and 4096 GOs terms significantly associated with treatments. The expression of genes regulating aluminum transport and abscisic acid synthesis was significantly upregulated; however, the genes involved in auxin synthesis were downregulated. Of note, the transcripts of several key enzymes affecting lignin monomer synthesis in phenylalanine pathway were upregulated. Our results shed light on the physiological and molecular mechanisms of aluminum stress tolerance in N. cadamba.

Genome-wide transcriptional adaptation to salt stress in Populus.

BACKGROUND: Adaptation to abiotic stresses is crucial for the survival of perennial plants in a natural environment. However, very little is known about the underlying mechanisms. Here, we adopted a liquid culture system to investigate plant adaptation to repeated salt stress in Populus trees. RESULTS: We first evaluated phenotypic responses and found that plants exhibit better stress tolerance after pre-treatment of salt stress. Time-course RNA sequencing (RNA-seq) was then performed to profile changes in gene expression over 12 h of salt treatments. Analysis of differentially expressed genes (DEGs) indicated that significant transcriptional reprogramming and adaptation to repeated salt treatment occurred. Clustering analysis identified two modules of co-expressed genes that were potentially critical for repeated salt stress adaptation, and one key module for salt stress response in general. Gene Ontology (GO) enrichment analysis identified pathways including hormone signaling, cell wall biosynthesis and modification, negative regulation of growth, and epigenetic regulation to be highly enriched in these gene modules. CONCLUSIONS: This study illustrates phenotypic and transcriptional adaptation of Populus trees to salt stress, revealing novel gene modules which are potentially critical for responding and adapting to salt stress.

Two novel parameters to evaluate the influence of the age and gender on the anatomic relationship of the atlas and axis in children no more than 8 years old: imaging study.

PURPOSE: Because of the complex cervical vertebral embryology and some normal variations, the atlantoadental interval (ADI) was not suitable for the evaluation of the anatomic relationship between the atlas and axial in children less than 2 years old. And the influence of the age and gender on the anatomic relationship between atlas and axial in children was still unclear. Two novel parameters, atlas-axis anteroposterior distance (AAAD) and atlas-axis lateral distance (AALD), were invented to evaluate the anatomic relationship between the atlas and axis in the children no more than 8 years old with different age and gender. METHODS: Cross-sectional computed tomography (CT) scans of the atlantoaxial joint for 140 randomly selected pediatric patients no more than 8 years old were analyzed. On the ideal CT reconstruction images, AAAD, AALD, atlantoaxial lateral bending angle (AALB), and atlantoaxial rotation angle (AARA) were measured. RESULTS: There was no statistically significant difference between the mean AAAD in different age and gender groups. The 99% confidence interval for AAAD was 7.12-7.82 mm. There was no significant correlation between AAAD and AALB/AARA and AALD and AALB/AARA. CONCLUSION: The AAAD was less than 7.12 mm or much than 7.82 mm that suggested a possible instability in the atlantoaxial joint and could help the diagnosis of the atlantoaxial instability in children no more than 8 years old. There was no difference between the mean AAAD of pediatric patients no more than 8 years old in different age and gender groups.

Pertussis detection in children with cough of any duration.

BACKGROUND: The diagnosis of pertussis in clinical practice continues to be a challenge worldwide as the symptoms are variable. We aimed to determine the prevalence of pertussis in Chinese children irrespective of cough duration and explore the clinical characteristics of children with pertussis with different cough durations. METHODS: This was a prospective study of children 1 month to 11 years of age with different cough durations in one large Chinese hospital. Bilateral deep posterior nasopharyngeal swabs and venepuncture for full blood count, CRP and serology and sputum were obtained when possible for investigation. E-test strips were used for testing the susceptibility of the B.pertussis isolates against erythromycin, azithromycin, sulphamethoxazole/trimethoprim, levofloxacin, amoxicillin and doxycycline. Demographic, clinical and laboratory information on culture and antimicrobial susceptibility testing was collected from children, and analyzed using SAS v.10 (SAS Institute Inc., USA). RESULTS: After exclusions we analyzed 312 children. Ninety-seven (31.1%) children had laboratory evidence of pertussis. When grouped by cough duration, few characteristics were significant between children with and without pertussis. Of the 36 isolates, 72.2% (26/36)could not be inhibited by erythromycin and azithromycin at all. The MIC50 and MIC90 to amoxicillin were 0.75 mg/L and 1 mg/L respectively, sensitive to amoxicillin by the EUCAST points. CONCLUSIONS: The "one-size-fits-all" clinical pertussis case definition is no longer optimal to recognize this disease. A large comprehensive study of children with all types of cough is required to make substantial inroads into increasing both the sensitivity and specificity in pertussis diagnosis, which will have a beneficial impact on public health. Amoxicillin maybe an alternative for children with marolide-resistant B.pertussis infection; however, local sensitivities are required to inform clinical practice.

Three UDP-xylose transporters participate in xylan biosynthesis by conveying cytosolic UDP-xylose into the Golgi lumen in Arabidopsis.

UDP-xylose (UDP-Xyl) is synthesized by UDP-glucuronic acid decarboxylases, also termed UDP-Xyl synthases (UXSs). The Arabidopsis genome encodes six UXSs, which fall into two groups based upon their subcellular location: the Golgi lumen and the cytosol. The latter group appears to play an important role in xylan biosynthesis. Cytosolic UDP-Xyl is transported into the Golgi lumen by three UDP-Xyl transporters (UXT1, 2, and 3). However, while single mutants affected in the UDP-Xyl transporter 1 (UXT1) showed a substantial reduction in cell wall xylose content, a double mutant affected in UXT2 and UXT3 had no obvious effect on cell wall xylose deposition. This prompted us to further investigate redundancy among the members of the UXT family. Multiple uxt mutants were generated, including a triple mutant, which exhibited collapsed vessels and reduced cell wall thickness in interfascicular fiber cells. Monosaccharide composition, molecular weight, nuclear magnetic resonance, and immunolabeling studies demonstrated that both xylan biosynthesis (content) and fine structure were significantly affected in the uxt triple mutant, leading to phenotypes resembling those of the irx mutants. Pollination was also impaired in the uxt triple mutant, likely due to reduced filament growth and anther dehiscence caused by alterations in the composition of the cell walls. Moreover, analysis of the nucleotide sugar composition of the uxt mutants indicated that nucleotide sugar interconversion is influenced by the cytosolic UDP-Xyl pool within the cell. Taken together, our results underpin the physiological roles of the UXT family in xylan biosynthesis and provide novel insights into the nucleotide sugar metabolism and trafficking in plants.

Comparison of combined anterior-posterior approach versus posterior-only approach in neuromuscular scoliosis: a systematic review and meta-analysis.

PURPOSE: Neuromuscular scoliosis (NS) is a complicated spinal disorder, and it could be treated through posterior-only approach (POA) or combined anterior-posterior approach (APA), which one is better and how to choose the surgical tactic is still in controversy. So comparing POA with APA parameters in the treatment of NS is meaningful. METHODS: Database of PubMed, Embase and Cochrane Library was systematically searched, and the studies, which focus on the comparisons of POA and APA in the treatment of NS, were included. The meta-analysis was performed by RevMan 5.3. RESULTS: Seven retrospective studies with 602 patients were included in meta-analysis. In previous analysis, statistically significant differences were observed in the major parameters between APA and POA. However, the results of subgroup meta-analysis, which focused on the correction angle and loss angle to eliminate the influence of different preoperative angles, were tend to no difference between two groups, except loss angle of scoliosis (MD, 6.4; 95% CI - 0.19 to 13) and correction angle of pelvic obliquity (MD, - 3.44; 95% CI - 6.71 to - 0.17). CONCLUSIONS: Our meta-analysis suggested that POA was similar to APA in the correction of scoliosis in coronal and sagittal planes. However, APA had advantages in the correction of pelvic obliquity and decreasing the loss of angle between postoperation and follow-up in main scoliosis, whereas POA had advantages in operative time, blood loss, duration of hospital stay and complications. LEVEL OF EVIDENCE: Level II. These slides can be retrieved under Electronic Supplementary Material.

KNAT7 positively regulates xylan biosynthesis by directly activating IRX9 expression in Arabidopsis.

Xylan is the major plant hemicellulosic polysaccharide in the secondary cell wall. The transcription factor KNOTTED-LIKE HOMEOBOX OF ARABIDOPSIS THALIANA 7 (KNAT7) regulates secondary cell wall biosynthesis, but its exact role in regulating xylan biosynthesis remains unclear. Using transactivation analyses, we demonstrate that KNAT7 activates the promoters of the xylan biosynthetic genes, IRREGULAR XYLEM 9 (IRX9), IRX10, IRREGULAR XYLEM 14-LIKE (IRX14L), and FRAGILE FIBER 8 (FRA8). The knat7 T-DNA insertion mutants have thinner vessel element walls and xylary fibers, and thicker interfascicular fiber walls in inflorescence stems, relative to wild-type (WT). KNAT7 overexpression plants exhibited opposite effects. Glycosyl linkage and sugar composition analyses revealed lower xylan levels in knat7 inflorescence stems, relative to WT; a finding supported by labeling of inflorescence walls with xylan-specific antibodies. The knat7 loss-of-function mutants had lower transcript levels of the xylan biosynthetic genes IRX9, IRX10, and FRA8, whereas KNAT7 overexpression plants had higher mRNA levels for IRX9, IRX10, IRX14L, and FRA8. Electrophoretic mobility shift assays indicated that KNAT7 binds to the IRX9 promoter. These results support the hypothesis that KNAT7 positively regulates xylan biosynthesis.

ETHYLENE RESPONSE FACTOR 74 (ERF74) plays an essential role in controlling a respiratory burst oxidase homolog D (RbohD)-dependent mechanism in response to different stresses in Arabidopsis.

Recent studies indicate that the ETHYLENE RESPONSE FACTOR VII (ERF-VII) transcription factor is an important regulator of osmotic and hypoxic stress responses in plants. However, the molecular mechanism of ERF-VII-mediated transcriptional regulation remains unclear. Here, we investigated the role of ERF74 (a member of the ERF-VII protein family) by examining the abiotic stress tolerance of an ERF74 overexpression line and a T-DNA insertion mutant using flow cytometry, transactivation and electrophoretic mobility shift assays. 35S::ERF74 showed enhanced tolerance to drought, high light, heat and aluminum stresses, whereas the T-DNA insertion mutant erf74 and the erf74;erf75 double mutant displayed higher sensitivity. Using flow cytometry analysis, we found that erf74 and erf74;erf75 lines lack the reactive oxygen species (ROS) burst in the early stages of various stresses, as a result of the lower expression level of RESPIRATORY BURST OXIDASE HOMOLOG D (RbohD). Furthermore, ERF74 directly binds to the promoter of RbohD and activates its expression under different abiotic stresses. Moreover, induction of stress marker genes and ROS-scavenging enzyme genes under various stress conditions is dependent on the ERF74-RbohD-ROS signal pathway. We propose a pathway that involves ERF74 acting as an on-off switch controlling an RbohD-dependent mechanism in response to different stresses, subsequently maintaining hydrogen peroxide (H2 O2 ) homeostasis in Arabidopsis.

Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus.

Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis.

Biomechanical properties of posterior transpedicular-transdiscal oblique lumbar screw fixation with novel trapezoidal lateral interbody spacer: an in vitro human cadaveric model.

PURPOSE: To investigate biomechanical properties of posterior transpedicular-transdiscal (TPTD) oblique lumbar screw fixation whereby the screw traverses the inferior pedicle across the posterior disc space into the super-adjacent body and lateral trapezoidal interbody spacer. METHODS: Eight fresh-frozen osteoligamentous human cadaveric spines (L1-S1) were tested in flexion-extension (FE), lateral bending (LB), and axial rotation (AR), with pure bending moment set at 7.5 Nm. Surgical constructs included (1) intact spine; (2) bilateral pedicle screw (BPS) fixation at L3-L4; (3) TPTD screw fixation at L3-L4; (4) lateral L3-L4 discectomy; (5) TPTD screw fixation with lateral interbody spacer (TPTD+S); and (6) BPS fixation with lateral interbody spacer (BPS+S). Peak range of motion (ROM) at L3-L4 was normalized to intact for statistical analysis. RESULTS: In FE and LB, all posterior fixation with or without interbody spacers significantly reduced motion compared with intact and discectomy. BPS and BPS+S provided increased fixation in all planes of motion; significantly reducing FE and LB motion relative to TPTD (p = 0.005, p = 0.002 and p = 0.020, p = 0.004, respectively). In AR, only BPS significantly reduced normalized ROM to intact (p = 0.034); BPS+S provided greater fixation compared with TPTD+S (p = 0.005). CONCLUSIONS: Investigators found less stiffness with TPTD screw fixation than with BPS regardless of immediate stabilization with lateral discectomy and spacer. Clinical use should be decided by required biomechanical performance, difficulty of installation, and extent of paraspinal tissue disruption.