Phosphorus deficiency-induced cell wall pectin demethylesterification enhances cadmium accumulation in roots of Salix caprea.

Regions affected by heavy metal contamination frequently encounter phosphorus (P) deficiency. Numerous studies highlight crucial role of P in facilitating cadmium (Cd) accumulation in woody plants. However, the regulatory mechanism by which P affects Cd accumulation in roots remains ambiguous. This study aims to investigate the effects of phosphorus (P) deficiency on Cd accumulation, Cd subcellular distribution, and cell wall components in the roots of Salix caprea under Cd stress. The results revealed that under P deficiency conditions, there was a 35.4% elevation in Cd content in roots, coupled with a 60.1% reduction in Cd content in shoots, compared to the P sufficiency conditions. Under deficient P conditions, the predominant response of roots to Cd exposure was the increased sequestration of Cd in root cell walls. The sequestration of Cd in root cell walls increased from 37.1% under sufficient P conditions to 66.7% under P deficiency, with pectin identified as the primary Cd binding site under both P conditions. Among cell wall components, P deficiency led to a significant 31.7% increase in Cd content within pectin compared to P sufficiency conditions, but did not change the pectin content. Notably, P deficiency significantly increased pectin methylesterase (PME) activity by regulating the expression of PME and PMEI genes, leading to a 10.4% reduction in the degree of pectin methylesterification. This may elucidate the absence of significant changes in pectin content under P deficiency conditions and the concurrent increase in Cd accumulation in pectin. Fourier transform infrared spectroscopy (FTIR) results indicated an increase in carboxyl groups in the root cell walls under P deficiency compared to sufficient P treatment. The results provide deep insights into the mechanisms of higher Cd accumulation in root mediated by P deficiency.

CRISPR/Cas9-based genome editing of 14 lipid metabolic genes reveals a sporopollenin metabolon ZmPKSB-ZmTKPR1-1/-2 required for pollen exine formation in maize.

Lipid biosynthesis and transport are essential for plant male reproduction. Compared with Arabidopsis and rice, relatively fewer maize lipid metabolic genic male-sterility (GMS) genes have been identified, and the sporopollenin metabolon in maize anther remains unknown. Here, we identified two maize GMS genes, ZmTKPR1-1 and ZmTKPR1-2, by CRISPR/Cas9 mutagenesis of 14 lipid metabolic genes with anther stage-specific expression patterns. Among them, tkpr1-1/-2 double mutants displayed complete male sterility with delayed tapetum degradation and abortive pollen. ZmTKPR1-1 and ZmTKPR1-2 encode tetraketide α-pyrone reductases and have catalytic activities in reducing tetraketide α-pyrone produced by ZmPKSB (polyketide synthase B). Several conserved catalytic sites (S128/130, Y164/166 and K168/170 in ZmTKPR1-1/-2) are essential for their enzymatic activities. Both ZmTKPR1-1 and ZmTKPR1-2 are directly activated by ZmMYB84, and their encoded proteins are localized in both the endoplasmic reticulum and nuclei. Based on protein structure prediction, molecular docking, site-directed mutagenesis and biochemical assays, the sporopollenin biosynthetic metabolon ZmPKSB-ZmTKPR1-1/-2 was identified to control pollen exine formation in maize anther. Although ZmTKPR1-1/-2 and ZmPKSB formed a protein complex, their mutants showed different, even opposite, defective phenotypes of anther cuticle and pollen exine. Our findings discover new maize GMS genes that can contribute to male-sterility line-assisted maize breeding and also provide new insights into the metabolon-regulated sporopollenin biosynthesis in maize anther.

[Effect of acupuncture at "Houxi" (SI3) and"Huantiao" (GB30) on high mobility group box 1 protein in spinal nerve trunk of rats with lumbar disc herniation].

OBJECTIVE: To observe the effect of acupuncture at "Houxi"(SI3) and "Huantiao"(GB30) on high mobility group box 1(HMGB1) protein and mRNA in spinal nerve trunk(SNT) of rats with lumbar disc herniation(LDH), so as to explore the mechanisms of acupuncture at this paired points on the treatment for LDH. METHODS: SD rats were randomly divided into sham operation, model, conventional acupuncture(CA) and paired points(PP) groups (with 8 rats in each group). The LDH model was established by injection of autologous suspension made from rats' own nucleus pulsus into the epidural space. Rats in the CA group received acupuncture treatment at bilateral "Weizhong"(BL40), "Dachangshu"(BL25) and "Shenshu"(BL23), while rats in the PP group received acupuncture at bilateral SI3 and GB30, 30 min each time, once daily for 14 consecutive days. The thermal pain threshold of bilateral hind feet of rats was detected by thermal pain stimulator. The contents of serum IL-1ß, IL-6 and IL-8 of rats were detected by ELISA. Western blot and immunofluorescence were used to detect the expression of HMGB1 protein in the lumbar(L)5 SNT of rats. The relative expression of HMGB1 mRNA in L5 SNT was determined by qPCR. HE staining was used to observe the morphological changes of L5 SNT. RESULTS: Compared with the sham operation group, the thermal pain threshold of bilateral hind feet in the model group was decreased (P<0.05); compared with the model group, the thermal pain threshold of bilateral hind feet in the CA group and the PP group were increased (P<0.05). The expressions of HMGB1 protein and mRNA in L5 SNT, and the contents of serum IL-1ß, IL-6 and IL-8 of rats in the model group were significantly increased(P<0.000 1, P<0.001) in contrast to the sham operation group. The expressions of HMGB1 protein and mRNA in L5 SNT, and the levels of serum IL-1ß, IL-6 and IL-8 were significantly decreased (P<0.01, P<0.000 1, P<0.001, P<0.05) in the CA and PP group, in comparison with those of the model group. Compared with the CA group, the above indexes of rats in the PP group recovered more significantly (P<0.05,P<0.001, P<0.01,P<0.000 1). The histomorphological results showed scattered and various-sized nerve fibers, vacuolation, a large number of disintegrating myelin sheath and lysed Schwann cells in the model group. Myelin sheaths regeneration, regularly-arranged nerve fibers were seen in the CA group and the PP group, with more obvious histopathological recovery observed in the PP group than the CA group. CONCLUSION: Acupuncture intervention inhibites the expressions of HMGB1 protein and mRNA in rats with LDH, and further reduces the production of IL-1ß, IL-6 and IL-8, which is beneficial to inflammatory response inhibition and pain alleviation. The therapeutic effect of the PP group is more obvious than that of the CA group.

Rich Organic Nitrogen Impacts Clavulanic Acid Biosynthesis through the Arginine Metabolic Pathway in Streptomyces clavuligerus F613-1.

Clavulanic acid (CA) is the preferred clinical drug for the treatment of infections by ß-lactam antibiotic-resistant bacteria. CA is produced by Streptomyces clavuligerus, and although there have been many reports on the effects of carbon and nitrogen sources on CA production, the mechanisms involved remain unclear. In this study, we found that CA accumulation in S. clavuligerus F613-1 was increased significantly in MH medium, which is rich in organic nitrogen, compared with that in ML medium, which contains half the amount of organic nitrogen present in MH medium. Transcriptome analysis revealed that genes involved in CA biosynthesis, such as ceas1, ceas2, bls1, bls2, cas2, pah2, gcaS, and cad, and arginine biosynthesis, such as argB, argC, argD, argG, argH, argJ, and argR, were upregulated under rich organic nitrogen. Metabolome data revealed notable differences between cultures of F613-1 grown in MH and ML media with regard to levels of key intracellular metabolites, most of which are involved in arginine metabolic pathways, including arginine, glutamine, and glutamic acid. Additionally, supplementation of ML medium with arginine, glutamine, or glutamic acid resulted in increased CA production by S. clavuligerus F613-1. Our results indicate that rich organic nitrogen mainly affects CA biosynthesis by increasing the levels of amino acids associated with the arginine metabolic pathway and activating the expression of the CA biosynthetic gene cluster. These findings provide important insights for improving medium optimization and engineering of S. clavuligerus F613-1 for high-yield production of CA. IMPORTANCE The bacterium Streptomyces clavuligerus is used for the industrial production of the broad-spectrum ß-lactamase inhibitor clavulanic acid (CA). However, much remains unknown about the factors which affect CA yields. We investigated the effects of different levels of organic nitrogen on CA production. Our analyses indicate that higher organic nitrogen levels were associated with increased CA yields and increased levels of arginine biosynthesis. Further analyses supported the relationship between arginine metabolism and CA production and demonstrated that increasing the levels of arginine or associated amino acids could boost CA yields. These findings suggest approaches for improving the production of this clinically important antibiotic.

A Comparative Study on the Clinical Efficacy of Stereotaxic Catheter Drainage and Conservative Treatment for Small and Medium Amount Intracerebral Hemorrhage in the Basal Ganglia.

The incidence rate and fatal disability rate of cerebral hemorrhage increase year by year. At present, most patients with a hematoma volume of ≤20 mL are treated conservatively by internal medicine. With the development of the stereotactic technique, it has been widely used for the treatment of cerebral hemorrhage in clinics. This study compared the clinical differences between stereotactic surgery and conservative treatment for small- and medium-sized cerebral hemorrhages. The results show that stereotactic hematoma evacuation is more effective than conservative treatment in the treatment of medium and small intracerebral hemorrhages in the basal ganglia. It can accelerate the resolution of hematoma and improve the neurological function and quality of life of patients, which is worthy of clinical promotion and application.

The ZmMYB84-ZmPKSB regulatory module controls male fertility through modulating anther cuticle-pollen exine trade-off in maize anthers.

Anther cuticle and pollen exine are two crucial lipid layers that ensure normal pollen development and pollen-stigma interaction for successful fertilization and seed production in plants. Their formation processes share certain common pathways of lipid biosynthesis and transport across four anther wall layers. However, molecular mechanism underlying a trade-off of lipid-metabolic products to promote the proper formation of the two lipid layers remains elusive. Here, we identified and characterized a maize male-sterility mutant pksb, which displayed denser anther cuticle but thinner pollen exine as well as delayed tapetal degeneration compared with its wild type. Based on map-based cloning and CRISPR/Cas9 mutagenesis, we found that the causal gene (ZmPKSB) of pksb mutant encoded an endoplasmic reticulum (ER)-localized polyketide synthase (PKS) with catalytic activities to malonyl-CoA and midchain-fatty acyl-CoA to generate triketide and tetraketide α-pyrone. A conserved catalytic triad (C171, H320 and N353) was essential for its enzymatic activity. ZmPKSB was specifically expressed in maize anthers from stages S8b to S9-10 with its peak at S9 and was directly activated by a transcription factor ZmMYB84. Moreover, loss function of ZmMYB84 resulted in denser anther cuticle but thinner pollen exine similar to the pksb mutant. The ZmMYB84-ZmPKSB regulatory module controlled a trade-off between anther cuticle and pollen exine formation by altering expression of a series of genes related to biosynthesis and transport of sporopollenin, cutin and wax. These findings provide new insights into the fine-tuning regulation of lipid-metabolic balance to precisely promote anther cuticle and pollen exine formation in plants.

Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize.

Male sterility represents an important trait for hybrid breeding and seed production in crops. Although the genes required for male fertility have been widely studied and characterized in many plant species, most of them are single genic male-sterility (GMS) genes. To investigate the role of multiple homologous genes in anther and pollen developments of maize, we established the CRISPR/Cas9-based gene editing method to simultaneously mutate the homologs in several putative GMS gene families. By using the integrated strategies of multi-gene editing vectors, maize genetic transformation, mutation-site analysis of T0 and F1 plants, and genotyping and phenotyping of F2 progenies, we further confirmed gene functions of every member in ZmTGA9-1/-2/-3 family, and identified the functions of ZmDFR1, ZmDFR2, ZmACOS5-1, and ZmACOS5-2 in controlling maize male fertility. Single and double homozygous gene mutants of ZmTGA9-1/-2/-3 did not affect anther and pollen development, while triple homozygous gene mutant resulted in complete male sterility. Two single-gene mutants of ZmDFR1/2 displayed partial male sterility, but the double-gene mutant showed complete male sterility. Additionally, only the ZmACOS5-2 single gene was required for anther and pollen development, while ZmACOS5-1 had no effect on male fertility. Our results show that the CRISPR/Cas9 gene editing system is a highly efficient and convenient tool for identifying multiple homologous GMS genes. These findings enrich GMS genes and mutant resources for breeding of maize GMS lines and promote deep understanding of the gene family underlying pollen development and male fertility in maize.

ZmFAR1 and ZmABCG26 Regulated by microRNA Are Essential for Lipid Metabolism in Maize Anther.

The function and regulation of lipid metabolic genes are essential for plant male reproduction. However, expression regulation of lipid metabolic genic male sterility (GMS) genes by noncoding RNAs is largely unclear. Here, we systematically predicted the microRNA regulators of 34 maize white brown complex members in ATP-binding cassette transporter G subfamily (WBC/ABCG) genes using transcriptome analysis. Results indicate that the ZmABCG26 transcript was predicted to be targeted by zma-miR164h-5p, and their expression levels were negatively correlated in maize B73 and Oh43 genetic backgrounds based on both transcriptome data and qRT-PCR experiments. CRISPR/Cas9-induced gene mutagenesis was performed on ZmABCG26 and another lipid metabolic gene, ZmFAR1. DNA sequencing, phenotypic, and cytological observations demonstrated that both ZmABCG26 and ZmFAR1 are GMS genes in maize. Notably, ZmABCG26 proteins are localized in the endoplasmic reticulum (ER), chloroplast/plastid, and plasma membrane. Furthermore, ZmFAR1 shows catalytic activities to three CoA substrates in vitro with the activity order of C12:0-CoA > C16:0-CoA > C18:0-CoA, and its four key amino acid sites were critical to its catalytic activities. Lipidomics analysis revealed decreased cutin amounts and increased wax contents in anthers of both zmabcg26 and zmfar1 GMS mutants. A more detailed analysis exhibited differential changes in 54 monomer contents between wild type and mutants, as well as between zmabcg26 and zmfar1. These findings will promote a deeper understanding of miRNA-regulated lipid metabolic genes and the functional diversity of lipid metabolic genes, contributing to lipid biosynthesis in maize anthers. Additionally, cosegregating molecular markers for ZmABCG26 and ZmFAR1 were developed to facilitate the breeding of male sterile lines.