ATF3 is a neuron-specific biomarker for spinal cord injury and ischaemic stroke.

BACKGROUND: Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions. METHODS: In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. RESULTS: Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function. CONCLUSIONS: ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties. HIGHLIGHTS: ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.

Exploring the role of the LkABCG36 transporter in lignin accumulation.

Lignin is a complex biopolymer formed through the condensation of three monomeric precursors known as monolignols. However, the mechanism underlying lignin precursor transport remains elusive, with uncertainty over whether it occurs through passive diffusion or an active energized process. ATP-binding cassette 36 (ABCG36) plays important roles in abiotic stress resistance. In this study, we investigated the transport functions of LkABCG36 (Larix kaempferi) for lignin precursors and the potential effects of LkABCG36 overexpression in plants. LkABCG36 enhanced the ability of tobacco (Nicotiana tabacum) bright yellow-2 (BY-2) cells to resist monolignol alcohol stress. Furthermore, LkABCG36 overexpression promoted lignin deposition in tobacco plant stem tissue. To understand the underlying mechanism, we measured the BY-2 cell ability to export lignin monomers and the uptake of monolignol precursors in inside-out (inverted) plasma membrane vesicles. We found that the transport of coniferyl and sinapyl alcohols is an ATP-dependent process. Our data suggest that LkABCG36 contributes to lignin accumulation in tobacco stem tissues through a mechanism involving the active transport of lignin precursors to the cell wall. These findings shed light on the lignin biosynthesis process, with important implications for enhancing lignin deposition in plants, potentially leading to improved stress tolerance and biomass production.

Hypothermia Treatment after Hypoxia-Ischemia in Glutathione Peroxidase-1 Overexpressing Mice.

The developing brain is uniquely susceptible to oxidative stress, and endogenous antioxidant mechanisms are not sufficient to prevent injury from a hypoxic-ischemic challenge. Glutathione peroxidase (GPX1) activity reduces hypoxic-ischemic injury. Therapeutic hypothermia (HT) also reduces hypoxic-ischemic injury, in the rodent and the human brain, but the benefit is limited. Here, we combined GPX1 overexpression with HT in a P9 mouse model of hypoxia-ischemia (HI) to test the effectiveness of both treatments together. Histological analysis showed that wild-type (WT) mice with HT were less injured than WT with normothermia. In the GPX1-tg mice, however, despite a lower median score in the HT-treated mice, there was no significant difference between HT and normothermia. GPX1 protein expression was higher in the cortex of all transgenic groups at 30 min and 24 h, as well as in WT 30 min after HI, with and without HT. GPX1 was higher in the hippocampus of all transgenic groups and WT with HI and normothermia, at 24 h, but not at 30 min. Spectrin 150 was higher in all groups with HI, while spectrin 120 was higher in HI groups only at 24 h. There was reduced ERK1/2 activation in both WT and GPX1-tg HI at 30 min. Thus, with a relatively moderate insult, we see a benefit with cooling in the WT but not the GPX1-tg mouse brain. The fact that we see no benefit with increased GPx1 here in the P9 model (unlike in the P7 model) may indicate that oxidative stress in these older mice is elevated to an extent that increased GPx1 is insufficient for reducing injury. The lack of benefit of overexpressing GPX1 in conjunction with HT after HI indicates that pathways triggered by GPX1 overexpression may interfere with the neuroprotective mechanisms provided by HT.

Large-Scale Quantitative Proteomic Analysis during Different Stages of Somatic Embryogenesis in Larix olgensis.

Larix olgensis is an economically important tree species native to northeastern China. The use of somatic embryogenesis (SE) is efficient and enables the rapid production of varieties with desirable qualities. Here, isobaric labeling via tandem mass tags was used to conduct a large-scale quantitative proteomic analysis of proteins in three critically important stages of SE in L. olgensis: the primary embryogenic callus, the single embryo, and the cotyledon embryo. We identified 6269 proteins, including 176 shared differentially expressed proteins across the three groups. Many of these proteins are involved in glycolipid metabolism, hormone response/signal transduction, cell synthesis and differentiation, and water transport; proteins involved in stress resistance and secondary metabolism, as well as transcription factors, play key regulatory roles in SE. The results of this study provide new insights into the key pathways and proteins involved in SE in Larix. Our findings have implications for the expression of totipotency, the preparation of synthetic seeds, and genetic transformation.

Fusion gene 4CL-CCR promotes lignification in tobacco suspension cells.

KEY MESSAGE: The fusion gene 4CL-CCR promotes lignification and activates lignin-related MYB expression in tobacco but inhibits auxin-related gene expression and hinders the auxin absorption of cells. Given the importance of lignin polymers in plant growth and their industrial value, it is necessary to investigate how plants synthesize monolignols and regulate the level of lignin in cell walls. In our previous study, expression of the Populus tomentosa fusion gene 4CL-CCR significantly promoted the production of 4-hydroxycinnamyl alcohols. However, the function of 4CL-CCR in organisms remains poorly understood. In this study, the fusion gene 4CL-CCR was heterologously expressed in tobacco suspension cells. We found that the transgenic suspension cells exhibited lignification earlier. Furthermore, 4CL-CCR significantly reduced the content of phenolic acids and increased the content of aldehydes in the medium, which led to an increase in lignin deposition. Moreover, transcriptome results showed that the genes related to lignin synthesis, such as PAL, 4CL, CCoAOMT and CAD, were significantly upregulated in the 4CL-CCR group. The expression of genes related to auxin, such as ARF3, ARF5 and ARF6, was significantly downregulated. The downregulation of auxin affected the expression of transcription factor MYBs. We hypothesize that the upregulated genes MYB306 and MYB315 are involved in the regulation of cell morphogenesis and lignin biosynthesis and eventually enhance lignification in tobacco suspension cells. Our findings provide insight into the function of 4CL-CCR in lignification and how secondary cell walls are formed in plants.

Transcriptomic Insights into Functions of LkABCG36 and LkABCG40 in Nicotiana tabacum.

ATP-binding cassette transporters (ABC transporters) play crucial physiological roles in plants, such as being involved in the growth and development of organs, nutrient acquisition, response to biotic and abiotic stress, disease resistance, and the interaction of the plant with its environment. The ABCG subfamily of proteins are involved in the process of plant vegetative organ development. In contrast, the functions of the ABCG36 and ABCG40 transporters have received considerably less attention. Here, we investigated changes in the transcriptomic data of the stem tissue of transgenic tobacco (Nicotiana tabacum) with LkABCG36 and LkABCG40 (Larix kaempferi) overexpression, and compared them with those of the wild type (WT). Compared with the WT, we identified 1120 and 318 differentially expressed genes (DEGs) in the LkABCG36 and LkABCG40 groups, respectively. We then annotated the function of the DEGs against the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The results showed enrichment in cell wall biogenesis and hormone signal transduction functional classes in transgenic LkABCG36 tobacco. In transgenic LkABCG40 tobacco, the enrichment was involved in metabolic and biosynthetic processes, mainly those related to environmental adaptation. In addition, among these DEGs, many auxin-related genes were significantly upregulated in the LkABCG36 group, and we found key genes involved in environmental adaptation in the LkABCG40 group, including an encoding resistance protein and a WRKY transcription factor. These results suggest that LkABCG36 and LkABCG40 play important roles in plant development and environmental adaptation. LkABCG36 may promote plant organ growth and development by increasing auxin transport, whereas LkABCG40 may inhibit the expression of WRKY to improve the resistance of transgenic tobacco. Our results are beneficial to researchers pursuing further study of the functions of ABCG36 and ABCG40.

Integrated multi-omics analysis uncovers roles of mdm-miR164b-MdORE1 in strigolactone-mediated inhibition of adventitious root formation in apple.

Apple is one of the most important fruit crops in temperate regions and largely relies on cutting propagation. Adventitious root formation is crucial for the success of cutting propagation. Strigolactones have been reported to function in rooting of woody plants. In this study, we determined that strigolactones have inhibitory effects on adventitious root formation in apple. Transcriptome analysis identified 12 051 differentially expressed genes over the course of adventitious root initiation, with functions related to organogenesis, cell wall biogenesis or plant development. Further analysis indicated that strigolactones might inhibit adventitious root formation through repressing two core hub genes, MdLAC3 and MdORE1. Combining small RNA and degradome sequencing, as well as dual-luciferase sensor assays, we identified and validated three negatively correlated miRNA-mRNA pairs, including mdm-miR397-MdLAC3 and mdm-miR164a/b-MdORE1. Overexpression of mdm-miR164b and silencing MdORE1 exhibited enhanced adventitious root formation in tobacco and apple, respectively. Finally, we verified the role of mdm-miR164b-MdORE1 in strigolactone-mediated repression of rooting ability. Overall, the identified comprehensive regulatory network in apple not only provides insight into strigolactone-mediated adventitious root formation in other woody plants, but also points to a potential strategy for genetic improvement of rooting capacity in woody plants.

Cholesterol in Brain Development and Perinatal Brain Injury: More than a Building Block.

The central nervous system (CNS) is enriched with important classes of lipids, in which cholesterol is known to make up a major portion of myelin sheaths, besides being a structural and functional unit of CNS cell membranes. Unlike in the adult brain, where the cholesterol pool is relatively stable, cholesterol is synthesized and accumulated at the highest rate in the developing brain to meet the needs of rapid brain growth at this stage, which is also a critical period for neuroplasticity. In addition to its biophysical role in membrane organization, cholesterol is crucial for brain development due to its involvement in brain patterning, myelination, neuronal differentiation, and synaptogenesis. Thus any injuries to the immature brain that affect cholesterol homeostasis may have long-term adverse neurological consequences. In this review, we describe the unique features of brain cholesterol biosynthesis and metabolism, cholesterol trafficking between different cell types, and highlight cholesterol-dependent biological processes during brain maturation. We also discuss the association of impaired cholesterol homeostasis with several forms of perinatal brain disorders in term and preterm newborns, including hypoxic-ischemic encephalopathy. Strategies targeting the cholesterol pathways may open new avenues for the diagnosis and treatment of developmental brain injury.

Systematic Analysis and Biochemical Characterization of the Caffeoyl Shikimate Esterase Gene Family in Poplar.

Caffeoyl shikimate esterase (CSE) hydrolyzes caffeoyl shikimate into caffeate and shikimate in the phenylpropanoid pathway. In this study, we performed a systematic analysis of the CSE gene family and investigated the possible roles of CSE and CSE-like genes in Populus. We conducted a genome-wide analysis of the CSE gene family, including functional and phylogenetic analyses of CSE and CSE-like genes, using the poplar (Populus trichocarpa) genome. Eighteen CSE and CSE-like genes were identified in the Populus genome, and five phylogenetic groups were identified from phylogenetic analysis. CSEs in Group Ia, which were proposed as bona fide CSEs, have probably been lost in most monocots except Oryza sativa. Primary functional classification showed that PoptrCSE1 and PoptrCSE2 had putative function in lignin biosynthesis. In addition, PoptrCSE2, along with PoptrCSE12, might also respond to stress with a function in cell wall biosynthesis. Enzymatic assay of PoptoCSE1 (Populus tomentosa), -2 and -12 showed that PoptoCSE1 and -2 maintained CSE activity. PoptoCSE1 and 2 had similar biochemical properties, tissue expression patterns and subcellular localization. Most of the PoptrCSE-like genes are homologs of AtMAGL (monoacylglycerol lipase) genes in Arabidopsis and may function as MAG lipase in poplar. Our study provides a systematic understanding of this novel gene family and suggests the function of CSE in monolignol biosynthesis in Populus.

Neuronal deficiency of hypoxia-inducible factor 2α increases hypoxic-ischemic brain injury in neonatal mice.

The cellular responses to hypoxia or hypoxia-ischemia (HI) are governed largely by the hypoxia-inducible factor (HIF) family of transcription factors. Our previous studies show that HIF-1α induction is an important factor that mediates protective effects in the brain after neonatal HI. In the present study, we investigated the contribution of another closely related HIF α isoform, HIF-2α, specifically the neuronal HIF-2α, to brain HI injury. Homozygous transgenic mice with a floxed exon 2 of HIF-2α were bred with CaMKIIα-Cre mice to generate a mouse line with selective deletion of HIF-2α in forebrain neurons. These mice, along with their wildtype littermates, were subjected to HI at postnatal day 9. Brain injury at different ages was evaluated by the levels of cleaved caspase-3 and spectrin breakdown products at 24 hr; and histologically at 6 days or 3 months after HI. Multiple behavioral tests were performed at 3 months, prior to sacrifice. Loss of neuronal HIF-2α exacerbated brain injury during the acute (24 hr) and subacute phases (6 days), with a trend toward more severe volume loss in the adult brain. The long-term brain function for coordinated movement and recognition memory, however, were not impacted in the neuronal HIF-2α deficient mice. Our data suggest that, similar to HIF-1α, neuronal HIF-2α promotes cell survival in the immature mouse brain. The two HIF alpha isoforms may act through partially overlapping or distinct transcriptional targets to mediate their intrinsic protective responses against neonatal HI brain injury.

Molecular and physiological characterization of the effects of auxin-enriched rootstock on grafting.

Grafting is a highly useful technique, and its success largely depends on graft union formation. In this study, we found that root-specific expression of the auxin biosynthetic gene iaaM in tobacco, when used as rootstock, resulted in more rapid callus formation and faster graft healing. However, overexpression of the auxin-inactivating iaaL gene in rootstocks delayed graft healing. We observed increased endogenous auxin levels and auxin-responsive DR5::GUS expression in scions of WT/iaaM grafts compared with those found in WT/WT grafts, which suggested that auxin is transported upward from rootstock to scion tissues. A transcriptome analysis showed that auxin enhanced graft union formation through increases in the expression of genes involved in graft healing in both rootstock and scion tissues. We also observed that the ethylene biosynthetic gene ACS1 and the ethylene-responsive gene ERF5 were upregulated in both scions and rootstocks of the WT/iaaM grafts. Furthermore, exogenous applications of the ethylene precursor ACC to the junction of WT/WT grafts promoted graft union formation, whereas application of the ethylene biosynthesis inhibitor AVG delayed graft healing in WT/WT grafts, and the observed delay was less pronounced in the WT/iaaM grafts. These results demonstrated that elevated auxin levels in the iaaM rootstock in combination with the increased auxin levels in scions caused by upward transport/diffusion enhanced graft union formation and that ethylene was partially responsible for the effects of auxin on grafting. Our findings showed that grafting success can be enhanced by increasing the auxin levels in rootstocks using transgenic or gene-editing techniques.

Characterization and functional analysis of the Hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT) gene family in poplar.

Hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT) divides the mass flux to H, G and S units in monolignol biosynthesis and affects lignin content. Ten HCT homologs were identified in the Populus trichocarpa (Torr. & Gray) genome. Both genome duplication and tandem duplication resulted in the expansion of HCT orthologs in Populus. Comprehensive analysis including motif analysis, phylogenetic analysis, expression profiles and co-expression analysis revealed the divergence and putative function of these candidate PoptrHCTs. PoptrHCT1 and 2 were identified as likely involved in lignin biosynthesis. PoptrHCT9 and 10- are likely to be involved in plant development and the response to cold stress. Similar functional divergence was also identified in Populus tomentosa Carr. Enzymatic assay of PtoHCT1 showed that PtoHCT1 was able to synthesize caffeoyl shikimate using caffeoyl-CoA and shikimic acid as substrates.

Functional Characteristics Analysis of Dehydrins in Larix kaempferi under Osmotic Stress.

Dehydrins (DHN) belong to the late embryogenesis abundant II family and have been found to enhance plant tolerance to abiotic stress. In the present study, we reported four DHNs in Larix kaempferi (LkDHN) which were identified from the published transcriptome. Alignment analysis showed that these four LkDHNs shared close relationships and belonged to SK3-type DHNs. The electrophoretic mobility shift assay indicated that these four LkDHNs all possess sequence-independent binding capacity for double-strands DNAs. The subcellular localizations of the four LkDHNs were in both the nucleus and cytoplasm, indicating that these LkDHNs enter the nucleus to exert the ability to bind DNA. The preparation of tobacco protoplasts with different concentrations of mannitol showed that LkDHNs enhanced the tolerance of plant cells under osmotic stress. The overexpression of LkDHNs in yeasts enhanced their tolerance to osmotic stress and helped the yeasts to survive severe stress. In addition, LkDHNs in the nucleus of salt treated tobacco increased. All of these results indicated that the four LkDHNs help plants survive from heavy stress by participating in DNA protection. These four LKDHNs played similar roles in the response to osmotic stress and assisted in the adaptation of L. kaempferi to the arid and cold winter of northern China.

Serum 24S-hydroxycholesterol predicts long-term brain structural and functional outcomes after hypoxia-ischemia in neonatal mice.

The major pathway of brain cholesterol turnover relies on its hydroxylation into 24S-hydroxycholesterol (24S-HC) using brain-specific cytochrome P450 46A1 (CYP46A1). 24S-HC produced exclusively in the brain normally traverses the blood-brain barrier to enter the circulation to the liver for excretion; therefore, the serum 24S-HC level is an indication of cholesterol metabolism in the brain. We recently reported an upregulation of CYP46A1 following hypoxia-ischemia (HI) in the neonatal mouse brain and a correlation between serum 24S-HC levels and acute brain damage. Here, we performed a longitudinal study to investigate whether the serum 24S-HC concentrations predict long-term brain structural and functional outcomes. In postnatal day 9 mice subjected to HI, the serum 24S-HC levels increased at 6 h and 24 h after HI and correlated with the infarct volumes measured histologically or by T2-weighted MRI. The 24 h levels were associated with white matter volume loss quantified by MBP immunostaining and luxol fast blue staining. The animals with higher serum 24S-HC at 6 h and 24 h corresponded to those with more severe motor and cognitive deficits at 35-40 days after HI. These data suggest that 24S-HC could be a novel and early blood biomarker for severity of neonatal HI brain damage and associated functional impairments.

Distinct Residential and Infiltrated Macrophage Populations and Their Phagocytic Function in Mild and Severe Neonatal Hypoxic-Ischemic Brain Damage.

Neonatal brain injury, especially severe injury induced by hypoxia-ischemia (HI), causes mortality and long-term neurological impairments. Our previous study demonstrated activation of CD11b+ myeloid cells, including residential microglial cells (MGs) and infiltrating monocyte-derived macrophages (MDMs) in a murine model of hypoxic-ischemic brain damage (HIBD), with unknown functions. Here, we study the differences in the phagocytic function of MGs and MDMs to clarify their potential roles after HIBD. HI was induced in 9-10-day postnatal mice. On days 1 and 3 after injury, pathological and neurobehavioral tests were performed to categorize the brain damage as mild or severe. Flow cytometry was applied to quantify the dynamic change in the numbers of MGs and MDMs according to the relative expression level of CD45 in CD11b+ cells. CX3CR1 GFPCCR2 RFP double-transformed mice were used to identify MGs and MDMs in the brain parenchyma after HIBD. Lysosome-associated membrane protein 1 (LAMP1), toll-like receptor 2 (TLR2), CD36, and transforming growth factor (TGF-ß) expression levels were measured to assess the underlying function of phagocytes and neuroprotective factors in these cells. The FITC-dextran 40 phagocytosis assay was applied to examine the change in phagocytic function under oxygen-glucose deprivation (OGD) in vitro. We found that neonatal HI induced a different degree of brain damage: mild or severe injury. Compared with mildly injured animals, mice with severe injury had lower weight, worse neurobehavioral scores, and abnormal brain morphology. In a severely injured brain, CD11b+ cells remarkably increased, including an increase in the MDM population and a decrease in the MG population. Furthermore, MDM infiltration into the brain parenchyma was evident in CX3CR1 GFPCCR2 RFP double-transformed mice. Mild and severe brain injury caused different phagocytosis-related responses and neuroprotective functions of MDMs and MGs at 1 and 3 days following HI. The phagocytic function was activated in BV2 cells but downregulated in Raw264.7 cells under OGD in vitro. These observations indicate that neonatal HI induced different degrees of brain injury. The proportion of infiltrated macrophage MDMs was increased and they were recruited into the injured brain parenchyma in severe brain injury. The resident macrophage MGs proportion decreased and maintained activated phagocytic function in both mild and severe brain injury, and restored neuroprotective function in severe brain injury.

[Value of Serum Free Light Chain Kappa/Lambda Ratio Detection Combined with Immunofixation Electrophoresis in the Prognosis Evaluation of Patients with Multiple Myeloma].

OBJECTIVE: To analyzed the prognostic value of serum free light chain kappa/lambda ratio detection combined with immunofixation electrophoresis in multiple myeloma (MM) patients. METHODS: 72 patients with MM treated in our hospital from January 2017 to December 2018 were selected. Serum free light chain kappa/lambda ratio (sFLCR) and immune typing were detected respectively. The clinical characteristics and survival time were compared among patients. COX regression was used to analyze the factors influencing prognosis. RESULTS: 38 patients showed high sFLCR, and 34 showed low sFLCR. Compared with the low sFLCR group, the DS stage of patients in high sFLCR group elevated, the levels of ß2-MG and Scrwere increased, and Hb decreased, all the differences were statistically significant (P＜0.05). Among 72 patients, there were 40 cases of IgG type (55.56%), 27 cases of IgA type (37.50%) and 5 cases of IgM type (6.94%). Compared with IgG and IgA patients, the serum calcium and creatinine in IgM patients were increased significantly, while Hb decreased significantly (P＜0.05). The median survival time was 19.2 months in 21 patients with IgG type and high sFLCR; 24.0 months in 19 patients with IgG type and low sFLCR; 15.0 months in 12 patients with IgA type and high sFLCR; 16.7 months in 15 patients with IgA type and low sFLCR; 6.0 months in 5 patients with IgM type and high sFLCR,respectively. DS stage, M protein typing and sFLCR correlated with prognosis of patients (P＜0.05). CONCLUSION: The serum free light chain kappa/lambda ratio combined with immunofixation electrophoresis is valuable for the prognostic evaluation of patients with multiple myeloma.

Isolation and characterization of Populus xyloglucan endotransglycosylase/hydrolase (XTH) involved in osmotic stress responses.

Xyloglucan endotransglycosylase/hydrolase (XTH) belongs to the GH16 subfamily of the glycoside hydrolases of carbohydrate active enzymes and plays an important role in the structure and function of plant cell walls. In this study, 11 members of the XTH gene family were cloned from Populus tomentosa. A bioinformatics analysis revealed that 11 PtoXTHs could be classified into three groups, where PtoXTH27 and PtoXTH34 were most likely to exhibit XTH activity. Biochemical analyses of purified PtoXTHs demonstrated that PtoXTH27 and PtoXTH34 had detectable xyloglucan endotransglucosylase (XET) activity, while the others did not exhibit XET or XEH activity. Moreover, enzymatic assays revealed that the optimum reaction temperature of both PtoXTH27 and PtoXTH34 was 37 °C, while their optimum pH values differed, such that PtoXTH27 was 6.0 and PtoXTH34 was 5.0. Enzyme kinetic parameters indicated that PtoXTH34 had higher affinity for the receptor substrate, XXXG, implying that PtoXTH34 and PtoXTH27 in plants have different substrate structure specificity. Finally, heterologous expression of XTH significantly increased intracellular total sugar content and osmotolerance of yeast cells, indicating that PtoXTH27 and PtoXTH34 are potentially involved in osmotic stress responses. These results clearly demonstrate the enzymatic characteristics and putative role of XTH in osmotic stress responses.

Functional Characteristics of Caffeoyl Shikimate Esterase in Larix Kaempferi and Monolignol Biosynthesis in Gymnosperms.

Caffeoyl shikimate esterase (CSE) has been reported to be involved in lignin biosynthesis; however, studies of CSE in gymnosperms are lacking. In this study, CSE was successfully cloned from Larix kaempferi (LkCSE) based on Larix laricina transcriptome screening. LkCSE was likely to have catalytic activity based on homologous sequence alignment and phylogenetic analyses of CSEs from different species. In vitro assays with the recombinant enzyme validated the catalytic activity of LkCSE, indicating its function in converting caffeoyl shikimate into caffeate and shikimate. Additionally, the optimum reaction pH and temperature of LkCSE were determined to be 6.0 and 30 °C, respectively. The values of Km and Vmax of CSE for caffeoyl shikimate were 98.11 µM and 14.44 nM min-1, respectively. Moreover, LkCSE was observed to have tissue expression specificity and was abundantly expressed in stems and leaves, especially stems, which was 50 times higher than the expression levels of roots. Lastly, translational fusion assays using LkCSE fused with green fluorescent proteins (GFP) in tobacco leaves indicated that LkCSE was localized in the plasma membrane and endoplasmic reticulum (ER). These results revealed that CSE clearly functions in gymnosperms and it is possible for LkCSE to interact with other ER-resident proteins and regulate mass flux in the monolignol biosynthesis pathway.

Rapid, simplified microscale quantitative analysis of lignin H/G/S composition with GC-MS in glass ampules and glass capillaries.

The characterization of monolignols H-, G- and S- units composition Vanholme et al. (2010) of lignin is important in agriculture, forestry, herb medicine, livestock, and health care research Vanholme et al. (2008) and Sticklen (2008). The conventional methods often require a great deal of samples and reagents and are time-consuming. Here, we present a newly developed method with fewer operations. The optimized method is suitable for detecting and characterizing lignin composition of cell wall in different plant species and has the advantages of: •Avoiding the influence of plasticizer by plasticware and enhancing the accuracy of monolignols analysis.•Lowering the required samples from grams to milligrams, and organic reagents from milliliters to microliters.•Reducing the time required from a few days to 6 h.

Novel motif is capable of determining CCR and CCR-like proteins based on the divergence of CCRs in plants.

Cinnamoyl-coenzyme A reductases (CCRs) have been reported as key enzymes involved in monolignol biosynthesis. In this study, a motif-aware workflow based on a new signature motif effectively distinguished CCRs from CCR-like proteins. The divergence of CCRs and CCR-like sequences in Populus tomentosa Carr, Panicum virgatum L, Oryza sativa L and Selaginella moellendorffii Hieron suggests that NWYCY is not efficient for CCR recognition. The novel motif H202(X)2K205 (CCR-SBM or CCR substrate binding motif) was introduced to distinguish between CCRs and CCR-like proteins. The site-directed mutant R205K in Os(I)CCR-like and H202 in PtoCCR7 resulted in the rescue and loss of activity, respectively, further validating the fact that CCR-SBM is critical for maintaining CCR activity. The molecular docking using feruloyl-cinnamoyl-coenzyme A (CoA) as the ligand and binary PhCCR-NADP structures as receptors indicated an interaction between H202 and K205 with CoA moiety. The genuine CCRs and CCR-like proteins from several angiosperms and gymnosperms were screened using a motif-aware workflow and were validated using a biochemical assay. Our results suggest that the motif-aware workflow is efficient and effective for the identification of CCRs and CCR-like proteins in land plants and can be used as a more accurate way of identifying genuine CCRs among land plants.