SPL13 together with SPL9 redundantly regulates wax biosynthesis upon drought stress.

Wax biosynthesis is strictly regulated by many regulators under different environmental conditions. Our previous study showed that the regulation module miR156/SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9)/DEWAX is identified to be involved in the diurnal regulation of wax production, however, it was unknown if other SPLs are also involved in the wax synthesis. Here, we reported that SPL13 regulates drought-induced wax production as well. Moreover, its regulatory role directly or indirectly affects the expression of two wax biosynthesis genes CER1 and CER4. Further study showed that SPL13 together with SPL9 redundantly regulated the wax accumulation upon either normal conditions or drought stress, simultaneous mutation of both genes additively enhanced cuticle permeability and decreased the drought tolerance. However, different from SPL9, SPL13 seemed not to participate in the DEWAX-mediated diurnal regulation of wax production.

Acetyl-CoA Carboxylase1 Influences ECERIFERUM2 Activity to Mediate the Synthesis of Very-Long-Chain Fatty Acid Past C28.

Cuticular wax is a protective layer on the aerial surfaces of land plants. In Arabidopsis (Arabidopsis thaliana), cuticular wax is mainly constituted of compounds derived from very-long-chain fatty acids (VLCFAs) with chain lengths longer than C28. CER2-LIKE (ECERIFERUM2-LIKE) proteins interact with CER6/KCS6 (ECERIFERUM6/ß-Ketoacyl-CoA Synthase6), the key enzyme of the fatty acid elongase complex, to modify its substrate specificity for VLCFA elongation past C28. However, the molecular regulatory mechanism of CER2-LIKE proteins remains unclear. Arabidopsis eceriferum19 (cer19) mutants display wax-deficient stems caused by loss of waxes longer than C28, indicating that CER19 may participate in the CER2-LIKE-mediated VLCFA elongation past C28. Using positional cloning and genetic complementation, we showed that CER19 encodes Acetyl-CoA Carboxylase1 (ACC1), which catalyzes the synthesis of malonyl-CoA, the essential substrate for the CER6/KCS6-mediated condensation reaction in VLCFA synthesis. We demonstrated that ACC1 physically interacts with CER2-LIKE proteins via split-ubiquitin yeast two-hybrid (SUY2H) and firefly luciferase complementation imaging (LCI) analysis. Additionally, heterologous expression in yeast and genetic analysis in Arabidopsis revealed that ACC1 affects CER2 activity to influence VLCFA elongation past C28. These findings imply that CER2-LIKE proteins might function as a link between ACC1 and CER6/KCS6 and subsequently enhance CER6/KCS6 binding to malonyl-CoA for further utilization in VLCFA elongation past C28. This information deepens our understanding of the complex mechanism of cuticular wax biosynthesis.

The Arabidopsis cytochrome P450 enzyme CYP96A4 is involved in the wound-induced biosynthesis of cuticular wax and cutin monomers.

The plant cuticle is composed of cuticular wax and cutin polymers and plays an essential role in plant tolerance to diverse abiotic and biotic stresses. Several stresses, including water deficit and salinity, regulate the synthesis of cuticular wax and cutin monomers. However, the effect of wounding on wax and cutin monomer production and the associated molecular mechanisms remain unclear. In this study, we determined that the accumulation of wax and cutin monomers in Arabidopsis leaves is positively regulated by wounding primarily through the jasmonic acid (JA) signaling pathway. Moreover, we observed that a wound- and JA-responsive gene (CYP96A4) encoding an ER-localized cytochrome P450 enzyme was highly expressed in leaves. Further analyses indicated that wound-induced wax and cutin monomer production was severely inhibited in the cyp96a4 mutant. Furthermore, CYP96A4 interacted with CER1 and CER3, the core enzymes in the alkane-forming pathway associated with wax biosynthesis, and modulated CER3 activity to influence aldehyde production in wax synthesis. In addition, transcripts of MYC2 and JAZ1, key genes in JA signaling pathway, were significantly reduced in cyp96a4 mutant. Collectively, these findings demonstrate that CYP96A4 functions as a cofactor of the alkane synthesis complex or participates in JA signaling pathway that contributes to cuticular wax biosynthesis and cutin monomer formation in response to wounding.

Tetramerization of pyruvate kinase M2 attenuates graft-versus-host disease by inhibition of Th1 and Th17 differentiation.

Lethal graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic stem-cell transplantation (Allo-HSCT). Pyruvate kinase M2 (PKM2) is essential for CD4+ T-cell differentiation. Using the well-characterized mouse models of Allo-HSCT, we explored the effects of TEPP-46-induced PKM2 tetramerization on GVHD and graft-versus-leukemia (GVL) activity. TEPP-46 administration significantly improved the survival rate of GVHD. The severity of GVHD and histopathological damage of GVHD-targeted organs were obviously alleviated by PKM2 tetramerization. Additionally, tetramerized PKM2 inhibited the activation of NF-κB pathway and decreased the inflammation level of GVHD mice. PKM2 tetramerization blocked Th1 and Th17 cell differentiation and secretion of pro-inflammatory cytokine (IFN-γ, TNF-α, and IL-17). Meanwhile, differentiation of Treg cells and IL-10 secretion were promoted by tetramerized PKM2. These findings demonstrated that PKM2 enhanced the augment of Th1 and Th17 cells to accelerate the progression of GVHD, and allosteric activation of PKM2 targeted Th1 and Th17 cells attenuated GVHD. Furthermore, we also confirmed that TEPP-46 administration did not compromise GVL activity and resulted in slightly improvement of leukemia-free survive. Thus, targeting Th1 and Th17 cell response with PKM2 allosteric activator may be a promising therapeutic strategy for GVHD prevention while preserving the GVL activity in patients receiving Allo-HSCT.

Function identification of Arabidopsis GPAT4 and GPAT8 in the biosynthesis of suberin and cuticular wax.

Surface lipids in plants include cutin, cuticular wax and suberin. sn-Glycerol-3-phosphate acyltransferases (GPATs) facilitate the acylation of sn-glycerol-3-phosphate (G3P) utilizing a fatty acyl group from acyl-coenzyme A (acyl-CoA) or acyl-acyl carrier protein (acyl-ACP) as substrates for the biosynthesis of plant extracellular lipids such as suberin and cutin. Here we found that Arabidopsis GPAT4 and GPAT8 are specifically expressed in endodermis cells of roots where suberin was accumulated. GPAT4 mutation significantly decreased the amounts of the C16 and C18 ω-oxidized suberin monomers, whereas the mutation of GPAT8 had little effect on the suberin production, and the functions of both were not redundant. Root suberin phenotype analysis of gpat4-1 and gpat6-1 single or double mutant revealed that GPAT4 and GPAT6 play redundant functions. Interestingly, the gpat4-1 gpat8-1 double mutant displayed a glossy stem phenotype since fewer wax crystals were accumulated. This phenotype was not shown in either parent. Further study showed that the amounts of most wax components were significantly decreased. Taken together, our findings revealed that GPAT4 has an additive effect with GPAT6 in the root suberin biosynthesis, and plays a redundant role in wax production with GPAT8.

Single-cell transcriptional gene signature analysis identifies IL-17 signaling pathway as the key pathway in sepsis.

Sepsis is a multiple dysregulated systemic inflammatory response with high mortality and leads to public concern. This study was designed to identify possible critical pathways associated with sepsis clinical severity and outcome, which offer potential biomarkers and therapeutic targets for sepsis diagnosis and treatment. Single-cell transcriptome profiles of human peripheral blood mononuclear (PBMC) in the healthy control population and sepsis patients were downloaded from the sepsis database GSE167363 and performed quality control before subsequent analysis. The bulk-RNA sequencing of blood samples in the sepsis-associated databases GSE100159 and GSE133822 was also used to confirm the association between critical pathways and sepsis pathology after processing raw data. We found there was a total of 18 distinct clusters in PBMC of sepsis, which was identified by the t-SNE and UMAP dimension reduction analysis. Meanwhile, the main cell types including B, NK, T, and monocyte cells were identified via the cell maker website and the "Single R" package cell-type annotation analysis. Subsequently, GO and KEGG enrichment analysis of differential expression genes in each cluster found that DEGs between healthy control and sepsis patients were significantly enriched in the IL-17 signaling pathway in monocyte, NK, and T cells. Finally, GSE100159 and GSE133822 confirmed IL-17 signaling pathway-associated genes including IL-17R, TRAF6, RELB, TRAF5, CEBPB, JUNB, CXCL1, CXCL3, CXCL8, CXCR1, and CXCR2 were significantly up-regulated in sepsis blood samples compared with the age-matched healthy control population. Taken together, we concluded that the IL-17 signaling pathway serves as a significant potential mechanism of sepsis and provides a promising therapeutic target for sepsis treatment. This research will further deepen our understanding of sepsis development.

Fine-tuning the activities of ß-KETOACYL-COA SYNTHASE 3 (KCS3) and KCS12 in Arabidopsis is essential for maintaining cuticle integrity.

The plant cuticle, consisting of wax and cutin, is involved in adaptations to various environments. ß-Ketoacyl-CoA synthases (KCSs) usually serve as a component of the fatty acid elongation complex that participates in the production of very long-chain fatty acids and provides precursors for the synthesis of various lipids, including wax; however, we recently reported that KCS3 and KCS12 negatively regulate wax biosynthesis. In this current study, we observed that unlike KCS3-overexpressing (OE) lines, KCS12-OE lines had fused floral organs because of abnormal cuticle biosynthesis. This prompted us to compare the functions of KCS3 and KCS12 during cuticle formation. Mutation of KCS3 caused greater effects on wax production, whereas mutation of KCS12 exerted more severe effects on cutin synthesis. The double-mutant kcs3 kcs12 had significantly increased wax and cutin contents compared to either single-mutant, suggesting that KCS12 and KCS3 have additive effects on cuticle biosynthesis. Cuticle permeability was greater for the double-mutant than for the single mutants, which ultimately led to increased susceptibility to drought stress and floral-organ fusion. Taken together, our results demonstrate the regulatory roles of KCS3 and KCS12 during cuticle biosynthesis, and show that maintaining KCS3 and KCS12 expression at certain levels is essential for the formation of a functional cuticle layer.

An ancestral role for 3-KETOACYL-COA SYNTHASE3 as a negative regulator of plant cuticular wax synthesis.

The plant cuticle, a structure primarily composed of wax and cutin, forms a continuous coating over most aerial plant surfaces. The cuticle plays important roles in plant tolerance to environmental stress, including stress imposed by drought. Some members of the 3-KETOACYL-COA SYNTHASE (KCS) family are known to act as metabolic enzymes involved in cuticular wax production. Here we report that Arabidopsis (Arabidopsis thaliana) KCS3, which was previously shown to lack canonical catalytic activity, instead functions as a negative regulator of wax metabolism by reducing the enzymatic activity of KCS6, a key KCS involved in wax production. We demonstrate that the role of KCS3 in regulating KCS6 activity involves physical interactions between specific subunits of the fatty acid elongation complex and is essential for maintaining wax homeostasis. We also show that the role of the KCS3-KCS6 module in regulating wax synthesis is highly conserved across diverse plant taxa from Arabidopsis to the moss Physcomitrium patens, pointing to a critical ancient and basal function of this module in finely regulating wax synthesis.

Efficacy and safety of rituximab in elderly patients with membranous nephropathy.

Objectives: Advancing age is a risk factor for treatment-related side effects and mortality in membranous nephropathy (MN) patients treated with traditional immunosuppressive regimens. This study aimed to determine the efficacy and safety of rituximab (RTX) in the treatment of elderly patients with MN. Methods: We performed a single center retrospective review of 37 consecutive MN patients aged 70 and older at the time of RTX infusion. We also enrolled 76 young patients (<70 years old) with MN as the control group. We assessed clinical and laboratory indices, remission rates, and adverse events at RTX infusion, 3 months, and last visit. Results: A total of 37 elderly patients with MN were included, with a median follow-up period of 15.50 (10.00, 24.40) months. Of the 37 patients, 75.68% were male, and mean age was 71.89 ± 2.47 years. At last visit, 7 (18.92%) patients achieved complete remission, and 26 (70.27%) patients achieved complete or partial remission. There were no differences in the complete remission rate and complete or partial remission rate at last visit compared to young patients (26.32% vs. 18.92%, p = 0.387; 85.53% vs. 70.27%, p = 0.055). After RTX treatment, three of 6 elderly patients with pneumonia died due to ineffective treatment of the infection in RTX therapy courses. The results of multivariant regression analysis showed that elderly patients have an increased risk of serious infection, compared with patients younger than 70 years (OR = 32.874, 95% CI 1.300-831.490, p = 0.034). For each increase of 1 g/L in serum albumin, the risk of serious infection would decrease by 43.2% (OR = 0.568, 95% CI 0.334-0.969, p = 0.038). Conclusion: This study demonstrates that RTX is effective in the treatment of elderly patients with MN. However, we also observed a high incidence of infectious complications. Our experience was limited by its retrospective design and relatively small sample size, and further randomized controlled studies with large sample size are needed to confirm our preliminary findings.

The Plant Fatty Acyl Reductases.

Fatty acyl reductase (FAR) is a crucial enzyme that catalyzes the NADPH-dependent reduction of fatty acyl-CoA or acyl-ACP substrates to primary fatty alcohols, which in turn acts as intermediate metabolites or metabolic end products to participate in the formation of plant extracellular lipid protective barriers (e.g., cuticular wax, sporopollenin, suberin, and taproot wax). FARs are widely present across plant evolution processes and play conserved roles during lipid synthesis. In this review, we provide a comprehensive view of FAR family enzymes, including phylogenetic analysis, conserved structural domains, substrate specificity, subcellular localization, tissue-specific expression patterns, their varied functions in lipid biosynthesis, and the regulation mechanism of FAR activity. Finally, we pose several questions to be addressed, such as the roles of FARs in tryphine, the interactions between transcription factors (TFs) and FARs in various environments, and the identification of post-transcriptional, translational, and post-translational regulators.

Metformin Mitigates Sepsis-Related Neuroinflammation via Modulating Gut Microbiota and Metabolites.

Gut microbiota affects the functions of brains. However, its mechanism in sepsis remains unclear. This study evaluated the effect of metformin on ameliorating sepsis-related neurodamage by regulating gut microbiota and metabolites in septic rats. Cecal ligation and puncture (CLP) was used to establish the sepsis-related neurodamage animal models. Metformin therapy by gavage at 1 h after CLP administration was followed by fecal microbiota transplantation (FMT) to ensure the efficacy and safety of metformin on the sepsis-related neurodamage by regulating gut microbiota. The gut microbiota and metabolites were conducted by 16S rRNA sequencing and liquid chromatography-tandem mass spectrometry metabolomic analysis. The brain tissue inflammation response was analyzed by histopathology and reverse transcription-polymerase chain reaction (RT-PCR). This study reported brain inflammatory response, hemorrhage in sepsis-related neurodamage rats compared with the control group (C group). Surprisingly, the abundance of gut microbiota slightly increased in sepsis-related neurodamage rats than C group. The ratio of Firmicutes/Bacteroidetes was significantly increased in the CLP group than the C group. However, no difference was observed between the CLP and the metformin-treated rats (MET group). Interestingly, the abundance of Escherichia_Shigella increased in the MET group than the C and CLP groups, while Lactobacillaceae abundance decreased. Furthermore, Prevotella_9, Muribaculaceae, and Alloprevotella related to short-chain fatty acids production increased in the sepsis-related neurodamage of metformin-treated rats. Additionally, Prevotella_9 and Muribaculaceae correlated positively to 29 metabolites that might affect the inflammatory factors in the brain. The FMT assay showed that metformin improved sepsis-related neurodamage by regulating the gut microbiota and metabolites in septic rats. The findings suggest that metformin improves the sepsis-related neurodamage through modulating the gut microbiota and metabolites in septic rats, which may be an effective therapy for patients with sepsis-related neurodamage.

Fatty alcohol oxidase 3 (FAO3) and FAO4b connect the alcohol- and alkane-forming pathways in Arabidopsis stem wax biosynthesis.

The alcohol- and alkane-forming pathways in cuticular wax biosynthesis are well characterized in Arabidopsis. However, potential interactions between the two pathways remain unclear. Here, we reveal that mutation of CER4, the key gene in the alcohol-forming pathway, also led to a deficiency in the alkane-forming pathway in distal stems. To trace the connection between the two pathways, we characterized two homologs of fatty alcohol oxidase (FAO), FAO3 and FAO4b, which were highly expressed in distal stems and localized to the endoplasmic reticulum. The amounts of waxes from the alkane-forming pathway were significantly decreased in stems of fao4b and much lower in fao3 fao4b plants, indicative of an overlapping function for the two proteins in wax synthesis. Additionally, overexpression of FAO3 and FAO4b in Arabidopsis resulted in a dramatic reduction of primary alcohols and significant increases of aldehydes and related waxes. Moreover, expressing FAO3 or FAO4b led to significantly decreased amounts of C18-C26 alcohols in yeast co-expressing CER4 and FAR1. Collectively, these findings demonstrate that FAO3 and FAO4b are functionally redundant in suppressing accumulation of primary alcohols and contributing to aldehyde production, which provides a missing and long-sought-after link between these two pathways in wax biosynthesis.

ArabidopsisKCS5 and KCS6 Play Redundant Roles in Wax Synthesis.

3-ketoacyl-CoA synthases (KCSs), as components of a fatty acid elongase (FAE) complex, play key roles in determining the chain length of very-long-chain fatty acids (VLCFAs). KCS6, taking a predominate role during the elongation from C26 to C28, is well known to play an important role in wax synthesis. KCS5 is one paralog of KCS6 and its role in wax synthesis remains unknown. Wax phenotype analysis showed that in kcs5 mutants, the total amounts of wax components derived from carbon 32 (C32) and C34 were apparently decreased in leaves, and those of C26 to C32 derivatives were obviously decreased in flowers. Heterologous yeast expression analysis showed that KCS5 alone displayed specificity towards C24 to C28 acids, and its coordination with CER2 and CER26 catalyzed the elongation of acids exceeding C28, especially displaying higher activity towards C28 acids than KCS6. BiLC experiments identified that KCS5 physically interacts with CER2 and CER26. Wax phenotype analysis of different organs in kcs5 and kcs6 single or double mutants showed that KCS6 mutation causes greater effects on the wax synthesis than KCS5 mutation in the tested organs, and simultaneous repression of both protein activities caused additive effects, suggesting that during the wax biosynthesis process, KCS5 and KCS6 play redundant roles, among which KCS6 plays a major role. In addition, simultaneous mutations of two genes nearly block drought-induced wax production, indicating that the reactions catalyzed by KCS5 and KCS6 play a critical role in the wax biosynthesis in response to drought.

Alterations in the gut microbiome and metabolome profiles of septic rats treated with aminophylline.

The treatment of sepsis remains a major challenge worldwide. Aminophylline has been shown to have anti-inflammatory effects; however, the role of aminophylline in sepsis, a disease characterized by immune dysregulation, is unknown. In this study, we combined microbiome sequencing and metabolomic assays to investigate the effect of aminophylline administration on the intestinal flora and metabolites in septic rats. Sixty SD rats were randomly divided into three groups: a sham-operated (SC) group, a sepsis model (CLP) group and a CLP + aminophylline treatment (Amino) group. The intestinal flora and metabolic profile of rats in the CLP group were significantly different than those of the SC group, while aminophylline administration resulted in a return to a state similar to healthy rats. Differential abundance analysis showed that aminophylline significantly back-regulated the abundance of Firmicutes, unidentified_Bacteria, Proteobacteria, Lactobacillus, Escherichia-Shigella and other dominant bacteria (P < 0.05) and altered chenodeoxycholic acid, isolithocholic acid and a total of 26 metabolites (variable importance in the projection (VIP) > 1, P < 0.05). In addition, we found that there were significant correlations between differential metabolites and bacterial genera of the Amino and CLP groups. For example, Escherichia-Shigella was associated with 12 metabolites, and Lactobacillus was associated with two metabolites (P < 0.05), suggesting that differences in the metabolic profiles caused by aminophylline were partly dependent on its influence on the gutmicrobiome. In conclusion, this study identified a novel protective mechanism whereby aminophylline could regulate disordered intestinal flora and metabolites in septic rats.

A Chromosome-level Genome Assembly of Wild Castor Provides New Insights into its Adaptive Evolution in Tropical Desert.

Wild castor grows in the high-altitude tropical desert of the African Plateau, a region known for high ultraviolet radiation, strong light, and extremely dry condition. To investigate the potential genetic basis of adaptation to both highland and tropical deserts, we generated a chromosome-level genome sequence assembly of the wild castor accession WT05, with a genome size of 316 Mb, a scaffold N50 of 31.93 Mb, and a contig N50 of 8.96 Mb, respectively. Compared with cultivated castor and other Euphorbiaceae species, the wild castor exhibits positive selection and gene family expansion for genes involved in DNA repair, photosynthesis, and abiotic stress responses. Genetic variations associated with positive selection were identified in several key genes, such as LIG1, DDB2, and RECG1, involved in nucleotide excision repair. Moreover, a study of genomic diversity among wild and cultivated accessions revealed genomic regions containing selection signatures associated with the adaptation to extreme environments. The identification of the genes and alleles with selection signatures provides insights into the genetic mechanisms underlying the adaptation of wild castor to the high-altitude tropical desert and would facilitate direct improvement of modern castor varieties.

Arabidopsis ACYL-ACTIVATING ENZYME 9 (AAE9) encoding an isobutyl-CoA synthetase is a key factor connecting branched-chain amino acid catabolism with iso-branched wax biosynthesis.

Iso-branched wax compounds are well known in plants, but their biosynthetic pathways are still mostly unknown. It has been speculated that branched waxes are derived from branched-chain amino acid (BCAA) catabolism, but the evidence for this is very limited. Gas chromatography-flame ionisation detection (GC-FID) analysis revealed that mutations in two subunits of the branched-chain ketoacid dehydrogenase (BCKDH) complex, a key enzyme complex in the degradation of BCAAs, significantly decreased the amounts of branched wax compounds, indicating that BCAA degradation may be integral to the synthesis of iso-branched wax. Substrate feeding studies further revealed that the metabolic precursor of iso-branched wax compounds is isobutyric acid (iBA), which is derived from valine degradation in Arabidopsis. We also isolated a novel mutant and found that its branched wax deficient phenotype could not be rescued by iBA. Map-based cloning together with complementation analysis revealed that mutation in ACYL-ACTIVATING ENZYME 9 (AAE9) is responsible for this phenotype. Genetic and enzyme activity analysis demonstrated that AAE9 is located downstream of the BCAA degradation pathway, and that it activates iBA to isobutyryl-CoA for use on branched wax synthesis. Taken together, our study demonstrates that AAE9 is a key factor connecting BCAA catabolism with branched wax biosynthesis.

MiR-204 suppresses the progression of acute myeloid leukemia through HGF/c-Met pathway.

Acute myeloid leukemia (AML) was confirmed to be associated with hematopoietic insufficiency, as well as abnormal proliferation, differentiation or survival of myeloid progenitors. Multiple studies reported that microRNA-204 (miR-204) and Hepatocyte growth factor (HGF) played important roles in types of cancers. However, the potential molecular regulatory mechanism between miR-204 and HGF in AML remains to be further defined. Real-time PCR (RT-PCR) was adopted to detect the expression of miR-204 and HG. Relative protein levels were detected by western blot assay. The viability, cell cycle, apoptosis, migration, and invasion were analyzed by MTT, flow cytometry, and transwell assays. Moreover, the target relationship between miR-204 and HGF was predicted by MiRcode website and confirmed by luciferase reporter, RNA pull-down, and western blot assays. Our data suggested that miR-204 was downregulated in AML serum samples and cells. MiR-204 overexpression repressed cell proliferation, migration, invasion, and induced cell apoptosis in AML cells. HGF was upregulated in AML samples and cells, and HGF knockdown inhibited the malignancy of AML cells. In addition, HGF was directly targeted by miR-204. HGF overexpression reversed the effects of miR-204 mimic on AML cell proliferation, apoptosis, migration, and invasion. Besides, miR-204 regulated the c-Met signaling by targeting HGF, thereby regulating the downstream protein levels related to cell proliferation, apoptosis, migration, and invasion in AML cells. In conclusion, miR-204 could regulate AML progression through regulating the HGF/c-Met pathway.

Molecular Cloning and Functional Analysis of GmLACS2-3 Reveals Its Involvement in Cutin and Suberin Biosynthesis along with Abiotic Stress Tolerance.

Cutin and wax are the main precursors of the cuticle that covers the aerial parts of plants and provide protection against biotic and abiotic stresses. Long-chain acyl-CoA synthetases (LACSs) play diversified roles in the synthesis of cutin, wax, and triacylglycerol (TAG). Most of the information concerned with LACS functions is obtained from model plants, whereas the roles of LACS genes in Glycine max are less known. Here, we have identified 19 LACS genes in Glycine max, an important crop plant, and further focused our attention on 4 LACS2 genes (named as GmLACS2-1, 2, 3, 4, respectively). These GmLACS2 genes display different expression patterns in various organs and also show different responses to abiotic stresses, implying that these genes might play diversified functions during plant growth and against stresses. To further identify the role of GmLACS2-3, greatly induced by abiotic stresses, we transformed a construct containing its full length of coding sequence into Arabidopsis. The expression of GmLACS2-3 in an Arabidopsis atlacs2 mutant greatly suppressed its phenotype, suggesting it plays conserved roles with that of AtLACS2. The overexpression of GmLACS2-3 in wild-type plants significantly increased the amounts of cutin and suberin but had little effect on wax amounts, indicating the specific role of GmLACS2-3 in the synthesis of cutin and suberin. In addition, these GmLACS2-3 overexpressing plants showed enhanced drought tolerance. Taken together, our study deepens our understanding of the functions of LACS genes in different plants and also provides a clue for cultivating crops with strong drought resistance.

Insight into the roles of the ER-associated degradation E3 ubiquitin ligase HRD1 in plant cuticular lipid biosynthesis.

Cuticular lipids consisting of cutin and wax coat aerial plant surfaces providing protection against biotic and abiotic stresses. Although much progress has been made on comprehending the regulation of plant cuticular lipid biosynthesis, their functional relevance in plant protection merits further investigation of potential regulators of their synthesis. HRD1 and DOA10 mediate two major Endoplasmic Reticulum-Associated Degradation (ERAD) pathways in yeast and also regulate common pathways during lipid metabolism. However, their roles in plant lipid metabolism are not well studied. CER9, an Arabidopsis homolog of DOA10, is known to play important roles in cuticular lipid biosynthesis. This prompted us to determine if HRD1 also plays a role in regulation cuticular lipid biosynthesis. Here we report that an Arabidopsis hrd1a hrd1b double mutant is impacted in the accumulation of both cutin and cuticular waxes including a large increase in total stem cutin with a concomitant decrease in stem wax content. We further investigated genetic relationship between HRD1A/1B- and CER9-mediated ERAD pathways with regard to cuticular lipid synthesis. Surprisingly, simultaneous mutation of HRD1 and CER9 revealed additive effects on stem wax synthesis, but not stem cutin synthesis. Collectively, our study advances our understanding of the ERAD regulatory roles in cuticular lipid synthesis identifying HRD1 as an important player in the regulated deposition of Arabidopsis stem cuticular lipids.

Lupeol Accumulation Correlates with Auxin in the Epidermis of Castor.

Lupeol, a natural lupane-type pentacyclic triterpene, possesses various pharmacological properties, and its production attracts attention. Significant quantities of lupeol are deposited on the castor aerial organ surface and are easily extractable as a predominant wax constituent. Thus, castor might be considered as a potential bioreactor for the production of lupeol. The lupeol biosynthesis pathway is well known, but how it is regulated remains largely unknown. Among large numbers of castor cultivars, we targeted one accession line (337) with high levels of lupeol on its stem surface and low levels thereof on its hypocotyl surface, implicating that lupeol synthesis is differentially regulated in the two organs. To explore the underlying mechanisms, we did comparative transcriptome analysis of the first internode of 337 stem and the upper hypocotyl. Our results show that large amounts of auxin-related genes are differentially expressed in both parts, implying some possible interactions between auxin and lupeol production. We also found that several auxin-responsive cis-elements are present in promoter regions of HMGR and LUS genes encoding two key enzymes involved in lupeol production. Furthermore, auxin treatments apparently induced the expression levels of RcHMGR and RcLUS. Furthermore, we observed that auxin treatment significantly increased lupeol contents, whereas inhibiting auxin transport led to an opposite phenotype. Our study reveals some relationships between hormone activity and lupeol synthesis and might provide a promising way for improving lupeol yields in castor.