Proteomic profiling of polyglucosan bodies associated with glycogenin-1 deficiency in skeletal muscle.

AIMS: Polyglucosan storage disorders represent an emerging field within neurodegenerative and neuromuscular conditions, including Lafora disease (EPM2A, EPM2B), adult polyglucosan body disease (APBD, GBE1), polyglucosan body myopathies associated with RBCK1 deficiency (PGBM1, RBCK1) or glycogenin-1 deficiency (PGBM2, GYG1). While the storage material primarily comprises glycans, this study aimed to gain deeper insights into the protein components by proteomic profiling of the storage material in glycogenin-1 deficiency. METHODS: We employed molecular genetic analyses, quantitative mass spectrometry of laser micro-dissected polyglucosan bodies and muscle homogenate, immunohistochemistry and western blot analyses in muscle tissue from a 45-year-old patient with proximal muscle weakness from late teenage years due to polyglucosan storage myopathy. RESULTS: The muscle tissue exhibited a complete absence of glycogenin-1 due to a novel homozygous deep intronic variant in GYG1 (c.7+992T>G), introducing a pseudo-exon causing frameshift and a premature stop codon. Accumulated proteins in the polyglucosan bodies constituted components of glycogen metabolism, protein quality control pathways and desmin. Muscle fibres containing polyglucosan bodies frequently exhibited depletion of normal glycogen. CONCLUSIONS: The absence of glycogenin-1, a protein important for glycogen synthesis initiation, causes storage of polyglucosan that displays accumulation of several proteins, including those essential for glycogen synthesis, sequestosome 1/p62 and desmin, mirroring findings in RBCK1 deficiency. These results suggest shared pathogenic pathways across different diseases exhibiting polyglucosan storage. Such insights have implications for therapy in these rare yet devastating and presently untreatable disorders.

Cryptococcus neoformans trehalose-6-phosphate synthase (tps1) promotes organ-specific virulence and fungal protection against multiple lines of host defenses.

Trehalose-6-phosphate synthase (TPS1) was identified as a virulence factor for Cryptococcus neoformans and a promising therapeutic target. This study reveals previously unknown roles of TPS1 in evasion of host defenses during pulmonary and disseminated phases of infection. In the pulmonary infection model, TPS1-deleted (tps1Δ) Cryptococci are rapidly cleared by mouse lungs whereas TPS1-sufficent WT (H99) and revertant (tps1Δ:TPS1) strains expand in the lungs and disseminate, causing 100% mortality. Rapid pulmonary clearance of tps1Δ mutant is T-cell independent and relies on its susceptibility to lung resident factors and innate immune factors, exemplified by tps1Δ but not H99 inhibition in a coculture with dispersed lung cells and its rapid clearance coinciding with innate leukocyte infiltration. In the disseminated model of infection, which bypasses initial lung-fungus interactions, tps1Δ strain remains highly attenuated. Specifically, tps1Δ mutant is unable to colonize the lungs from the bloodstream or expand in spleens but is capable of crossing into the brain, where it remains controlled even in the absence of T cells. In contrast, strains H99 and tps1Δ:TPS1 rapidly expand in all studied organs, leading to rapid death of the infected mice. Since the rapid pulmonary clearance of tps1Δ mutant resembles a response to acapsular strains, the effect of tps1 deletion on capsule formation in vitro and in vivo was examined. Tps1Δ cryptococci form capsules but with a substantially reduced size. In conclusion, TPS1 is an important virulence factor, allowing C. neoformans evasion of resident pulmonary and innate defense mechanisms, most likely via its role in cryptococcal capsule formation.

Identification and analysis of UGT genes associated with triterpenoid saponin in soapberry (Sapindus mukorossi Gaertn.).

BACKGROUND: Soapberry (Sapindus mukorossi) is an economically important multifunctional tree species. Triterpenoid saponins have many functions in soapberry. However, the types of uridine diphosphate (UDP) glucosyltransferases (UGTs) involved in the synthesis of triterpenoid saponins in soapberry have not been clarified. RESULTS: In this study, 42 SmUGTs were identified in soapberry, which were unevenly distributed on 12 chromosomes and had sequence lengths of 450 bp to 1638 bp, with an average of 1388 bp. The number of amino acids in SmUGTs was 149 to 545, with an average of 462. Most SmUGTs were acidic and hydrophilic unstable proteins, and their secondary structures were mainly α-helices and random coils. All had conserved UDPGT and PSPG-box domains. Phylogenetic analysis divided them into four subclasses, which glycosylated different carbon atoms. Prediction of cis-acting elements suggested roles of SmUGTs in plant development and responses to environmental stresses. The expression patterns of SmUGTs differed according to the developmental stage of fruits, as determined by transcriptomics and RT-qPCR. Co-expression network analysis of SmUGTs and related genes/transcription factors in the triterpenoid saponin synthesis pathway was also performed. The results indicated potential roles for many transcription factors, such as SmERFs, SmGATAs and SmMYBs. A correlation analysis showed that 42 SmUGTs were crucial in saponin synthesis in soapberry. CONCLUSIONS: Our findings suggest optimal targets for manipulating glycosylation in soapberry triterpenoid saponin biosynthesis; they also provide a theoretical foundation for further evaluation of the functions of SmUGTs and analyses of their biosynthetic mechanisms.

[Molecular modification of cyclodextrin glucosyltransferase and its application in the synthesis of α-arbutin].

α-arbutin has important applications in cosmetics and medicine. However, the extraction yield from plant tissues is relatively low, which restricts its application value. In this study, we investigated the synthesis of α-arbutin using maltodextrin as the donor and hydroquinone as the acceptor, using a cyclodextrin glucosyltransferase (CGTase) from Anaerobranca gottschalkii. We performed site-saturated and site-directed mutagenesis on AgCGTase. The activity of the variant AgCGTase-F235G-N166H was 3.48 times higher than that of the wild type. Moreover, we achieved a conversion rate of 63% by optimizing the reaction pH, temperature, and hydroquinone addition amount. Overall, this study successfully constructed a strain with improved conversion rate for the synthetic production of α-arbutin and hydroquinone. These findings have significant implications for reducing the industrial production cost of α-arbutin and enhancing the conversion rate of the product.

[Analysis of enzyme activity and substrate specificity of dolichyl-phosphate ß-glucosyltransferase].

Protein folding and quality control processes primarily occur in the endoplasmic reticulum (ER). ER-resident molecular chaperones play a crucial role in guiding nascent polypeptides towards their correct tertiary structures. Some of these chaperones specifically recognize glucosylated N-glycan moieties on peptide. It is of great significance to study the N-glycan biosynthetic pathway and glycoprotein quality control system by analyzing the sugar donor of ER luminal glucosyltransferases, known as dolichol phosphate glucose (Dol-P-Glc), or its analogues in vitro. In this study, we investigated a range of dolichol analogues to synthesize lipid phosphate glucose, which served as substrates for dolichyl-phosphate ß-glucosyltransferase E (Alg5E) derived from Trichomonas vaginalis. The results demonstrated that the recombinant Alg5E, expressed in Escherichia coli, exhibited strong catalytic activity and the ability to recognize lipid phosphate glucose with varying chain lengths. Interestingly, the enzyme's catalytic reaction was found to be faster with longer carbon chains in the substrate. Additionally, Alg5E showed a preference for branched chain methyl groups in the lipid structure. Furthermore, our study confirmed the importance of divalent metal ions in the binding of the crucial DXD motif, which is essential for the enzyme's catalytic function. These findings lay the groundwork for future research on glucosyltransferases Alg6, Alg8, and Alg10 in the synthesis pathway of dolichol-linked oligosaccharide (DLO).

Five amino acid mismatches in the zinc-finger domains of Cellulose Synthase 5 and Cellulose Synthase 6 cooperatively modulate their functional properties by controlling homodimerization in Arabidopsis.

Cellulose synthase 5 (CESA5) and CESA6 are known to share substantial functional overlap. In the zinc-finger domain (ZN) of CESA5, there are five amino acid (AA) mismatches when compared to CESA6. These mismatches in CESA5 were replaced with their CESA6 counterparts one by one until all were replaced, generating nine engineered CESA5s. Each N-terminal enhanced yellow fluorescent protein-tagged engineered CESA5 was introduced to prc1-1, a cesa6 null mutant, and resulting mutants were subjected to phenotypic analyses. We found that five single AA-replaced CESA5 proteins partially rescue the prc1-1 mutant phenotypes to different extents. Multi-AA replaced CESA5s further rescued the mutant phenotypes in an additive manner, culminating in full recovery by CESA5G43R + S49T+S54P+S80A+Y88F. Investigations in cellulose content, cellulose synthase complex (CSC) motility, and cellulose microfibril organization in the same mutants support the results of the phenotypic analyses. Bimolecular fluorescence complementation assays demonstrated that the level of homodimerization in every engineered CESA5 is substantially higher than CESA5. The mean fluorescence intensity of CSCs carrying each engineered CESA5 fluctuates with the degree to which the prc1-1 mutant phenotypes are rescued by introducing a corresponding engineered CESA5. Taken together, these five AA mismatches in the ZNs of CESA5 and CESA6 cooperatively modulate the functional properties of these CESAs by controlling their homodimerization capacity, which in turn imposes proportional changes on the incorporation of these CESAs into CSCs.

Mechanism Analysis of Antimicrobial Peptide NoPv1 Related to Potato Late Blight through a Computer-Aided Study.

Phytophthora infestans (Mont.) de Bary, the oomycotic pathogen responsible for potato late blight, is the most devastating disease of potato production. The primary pesticides used to control oomycosis are phenyl amide fungicides, which cause environmental pollution and toxic residues harmful to both human and animal health. To address this, an antimicrobial peptide, NoPv1, has been screened to target Plasmopara viticola cellulose synthase 2 (PvCesA2) to inhibit the growth of Phytophthora infestans (P. infestans). In this study, we employed AlphaFold2 to predict the three-dimensional structure of PvCesA2 along with NoPv peptides. Subsequently, utilizing computational methods, we dissected the interaction mechanism between PvCesA2 and these peptides. Based on this analysis, we performed a saturation mutation of NoPv1 and successfully obtained the double mutants DP1 and DP2 with a higher affinity for PvCesA2. Meanwhile, dynamics simulations revealed that both DP1 and DP2 utilize a mechanism akin to the barrel-stave model for penetrating the cell membrane. Furthermore, the predicted results showed that the antimicrobial activity of DP1 was superior to that of NoPv1 without being toxic to human cells. These findings may offer insights for advancing the development of eco-friendly pesticides targeting various oomycete diseases, including late blight.

Elevated CO2 concentration enhance carbon and nitrogen metabolism and biomass accumulation of Ormosiahosiei.

Elevated CO2 concentrations may inhibit photosynthesis due to nitrogen deficiency, but legumes may be able to overcome this limitation and continue to grow. Our study confirms this conjecture well. First, we placed the two-year-old potted saplings of Ormosia hosiei (O. hosiei) (a leguminous tree species) in the open-top chamber (OTC) with three CO2 concentrations of 400 (CK), 600 (E1), and 800 µmol·mol-1 (E2) to simulate the elevated CO2 concentration environment. After 146 days, the light saturation point (LSP), light compensation point (LCP), apparent quantum efficiency (AQE), and dark respiration rate (Rd) of O. hosiei were increased under increasing CO2 concentration and obtain the maximum ribulose diphosphate (RuBP) carboxylation rate (Vc max) and RuBP regenerated photosynthetic electron transfer rate (Jmax) were also significantly increased under E2 treatment (P < 0.05). This results in a significant increase of the maximum assimilation rate (Amax) under elevated CO2 concentrations. Sucrose phosphate synthase (SPS) activity in sucrose metabolism increased in the leaves, more soluble sugars, starches, and sucrose was produced, but sucrose content only in leaves increased at E2, and more carbon flows to the roots. The activity of the NH4+ assimilating enzymes glutamine synthetase (GS), glutamate synthetase (GOGAT), and glutamate dehydrogenase (GDH) in the leaves of O. hosiei increases under elevated CO2 concentrations to promote nitrogen synthesis that reduces the content of ammonium nitrogen and increases the content of nitrate nitrogen. In addition, under E1 conditions, sucrose synthase (SS), direction of synthesis activity was highest and sucrose invertase (INV) activity was lowest, this means that the balance of C and N metabolism is maintained. While under E2 conditions SS activity decreased and INV activity increased, this increased C/N and nitrogen use efficiency. So, the elevated CO2 concentration promotes the accumulation of O. hosiei biomass, especially in the aboveground part, but did not have a significant effect on the accumulation of root biomass. This means that O. hosiei is able to cope under the elevated CO2 concentration without showing photosynthetic adaptation during the experimental period.

SA and NHP glucosyltransferase UGT76B1 affects plant defense in both SID2- and NPR1-dependent and independent manner.

KEY MESSAGE: The small-molecule glucosyltransferase loss-of-function mutant ugt76b1 exhibits both SID2- or NPR1-dependent and independent facets of enhanced plant immunity, whereupon FMO1 is required for the SID2 and NPR1 independence. The small-molecule glucosyltransferase UGT76B1 inactivates salicylic acid (SA), isoleucic acid (ILA), and N-hydroxypipecolic acid (NHP). ugt76b1 loss-of-function plants manifest an enhanced defense status. Thus, we were interested how UGT76B1 genetically integrates in defense pathways and whether all impacts depend on SA and NHP. We study the integration of UGT76B1 by transcriptome analyses of ugt76b1. The comparison of transcripts altered by the loss of UGT76B1 with public transcriptome data reveals both SA-responsive, ISOCHORISMATE SYNTHASE 1/SALICYLIC ACID INDUCTION DEFICIENT 2 (ICS1/SID2)- and NON EXPRESSOR OF PR GENES 1 (NPR1)-dependent, consistent with the role of UGT76B1 in glucosylating SA, and SA-non-responsive, SID2/NPR1-independent genes. We also discovered that UGT76B1 impacts on a group of genes showing non-SA-responsiveness and regulation by infections independent from SID2/NPR1. Enhanced resistance of ugt76b1 against Pseudomonas syringae is partially independent from SID2 and NPR1. In contrast, the ugt76b1-activated resistance is completely dependent on FMO1 encoding the NHP-synthesizing FLAVIN-DEPENDENT MONOOXYGENASE 1). Moreover, FMO1 ranks top among the ugt76b1-induced SID2- and NPR1-independent pathogen responsive genes, suggesting that FMO1 determines the SID2- and NPR1-independent effect of ugt76b1. Furthermore, the genetic study revealed that FMO1, ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), SID2, and NPR1 are required for the SA-JA crosstalk and senescence development of ugt76b1, indicating that EDS1 and FMO1 have a similar effect like stress-induced SA biosynthesis (SID2) or the key SA signaling regulator NPR1. Thus, UGT76B1 influences both SID2/NPR1-dependent and independent plant immunity, and the SID2/NPR1 independence is relying on FMO1 and its product NHP, another substrate of UGT76B1.

Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of Oral AL01211 in Healthy Chinese Volunteers.

BACKGROUND AND OBJECTIVE: Aberrant accumulation of glycosphingolipids (GSLs) in the lysosome leads to GSL storage diseases. Glucosylceramide synthase inhibitors (GCSi) have the potential to treat several GSL storage diseases by reducing the synthesis of the disease-causing GSLs. AL01211 is a potent oral GCSi under investigation for Type 1 Gaucher disease and Fabry disease. Here, we evaluate the pharmacokinetics, pharmacodynamics, safety, and tolerability of AL01211 in healthy Chinese volunteers. METHODS: AL01211 was tested in a Phase 1, single-center, randomized, double-blind, placebo-controlled study with single-dose (15 and 60 mg) and multiple-dose (30 mg) arms. RESULTS: Results of AL01211 demonstrated dose-dependent pharmacokinetics, rapid absorption (median time to maximum plasma concentration [tmax] 2.5-4 hours), relatively slow clearance rate (mean apparent total clearance from plasma [CL/F] 88.3-200 L/h) and the mean terminal half-life above 30 hours. Repeated once-daily oral administration of AL01211 for 14 days had an approximately 2-fold accumulation, reaching steady-state levels between 7 and 10 days, and led to a 73% reduction in plasma glucosylceramide (GL1) on Day 14. AL01211 was safe and well tolerated, with no identified serious adverse events. CONCLUSION: AL01211 showed a favorable pharmacokinetic, pharmacodynamics, safety, and tolerability profile in healthy Chinese volunteers. These data support the further clinical development of AL01211 as a therapy for GSL storage diseases. CLINICAL TRIAL REGISTRY: Clinical Trial Registry no. CTR20221202 ( http://www.chinadrugtrials.org.cn ) registered on 6 June 2022 and ChiCTR2200061431 ( http://www.chictr.org.cn ) registered on 24 June 2022.

Tailor-Made α-Glucans by Engineering the Processivity of α-Glucanotransferases via Tunnel-Cleft Active Center Interconversions.

The function of polysaccharides is intimately associated with their size, which is largely determined by the processivity of transferases responsible for their synthesis. A tunnel active center architecture has been recognized as a key factor that governs processivity of several glycoside hydrolases (GHs), e.g., cellulases and chitinases. Similar tunnel architecture is also observed in the Limosilactobacillus reuteri 121 GtfB (Lr121 GtfB) α-glucanotransferase from the GH70 family. The molecular element underpinning processivity of these transglucosylases remains underexplored. Here, we report the synthesis of the smallest (α1 → 4)-α-glucan interspersed with linear and branched (α1 → 6) linkages by a novel 4,6-α-glucanotransferase from L. reuteri N1 (LrN1 GtfB) with an open-clefted active center instead of the tunnel structure. Notably, the loop swapping engineering of LrN1 GtfB and Lr121 GtfB based on their crystal structures clarified the impact of the loop-mediated tunnel/cleft structure at the donor subsites -2 to -3 on processivity of these α-glucanotransferases, enabling the tailoring of both product sizes and substrate preferences. This study provides unprecedented insights into the processivity determinants and evolutionary diversification of GH70 α-glucanotransferases and offers a simple route for engineering starch-converting α-glucanotransferases to generate diverse α-glucans for different biotechnological applications.

α,ß-trehalose, an intracellular substance in resting cyst of colpodid ciliates as a key to environmental tolerances.

α,α-trehalose is a well-known sugar that plays a key role in establishing tolerance to environmental stresses in many organisms, except unicellular eukaryotes. However, almost nothing is known about α,ß-trehalose, including their synthesis, function, and even presence in living organisms. In this study, we identified α,ß-trehalose in the resting cyst, a dormancy cell form characterized by extreme tolerance to environmental stresses, of the ciliated protist Colpoda cucullus, using high-performance liquid chromatography (HPLC), and a proton nuclear magnetic resonance (1H NMR). Gene expression analysis revealed that the expression of trehalose-6-phosphate synthase (TPS), glycosyltransferase (GT), alpha-amylase (AMY), and trehalose transporter 1 (TRET1), were up-regulated in encystment, while the expression of α-glucosidase 2 (AG2) and trehalase (TREH) was up-regulated in excystment. These results suggest that α,ß-trehalose is synthesized during encystment process, while and contributes to extreme tolerances to environmental stressors, stored carbohydrates, and energy reserve during resting cyst and/or during excystment.

Cell wall-mediated root development is targeted by a soil-borne bacterial pathogen to promote infection.

Plant pathogens manipulate host development, facilitating colonization and proliferation. Ralstonia solanacearum is a soil-borne bacterial pathogen that penetrates roots and colonizes plants through the vascular system, causing wilting and death. Here, we find that RipAC, an effector protein from R. solanacearum, alters root development in Arabidopsis, promoting the formation of lateral roots and root hairs. RipAC interacts with CELLULOSE SYNTHASE (CESA)-INTERACTIVE PROTEIN 1 (CSI1), which regulates the activity of CESA complexes at the plasma membrane. RipAC disrupts CESA-CSI1 interaction, leading to a reduction in cellulose content, root developmental alterations, and a promotion of bacterial pathogenicity. We find that CSI1 also associates with the receptor kinase FERONIA, forming a complex that negatively regulates immunity in roots; this interaction, however, is not affected by RipAC. Our work reveals a bacterial virulence strategy that selectively affects the activities of a host target, promoting anatomical alterations that facilitate infection without causing activation of immunity.

Development of an engineered Bacillus subtilis strain for antibiotic-free sucrose isomerase production.

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, a functional sugar extensively used in the food industry. However, the lack of safe and efficient heterologous expression systems for SIase has constrained its production and application. In this study, an engineered Bacillus subtilis strain for antibiotic-free SIase production was developed via a food-grade expression system. First, the B. subtilis strain TEA was modified through the CRISPR/Cas9 system, resulting in a mutant strain TEA4, which exhibited enhanced capabilities for recombinant protein expression. For efficient and safe production of SIase, different constitutive and inducible promoters were evaluated. The maltose-inducible promoter Poglv was found to have an extracellular SIase activity of 21.7 U mL-1 in engineered strain TEA4. Subsequent optimization of the culture medium further increased SIase activity to 26.4 U mL-1 during shake flask cultivation. Eventually, using the crude enzyme solution of the engineered strain in biotransformation reactions resulted in a high yield of isomaltulose under high concentrations sucrose, achieving a maximum yield of 83.1%. These findings demonstrated an engineered B. subtilis strain for antibiotic-free SIase production, paving the way for its scale-up industrial production and application.

Emmer wheat (Triticum dicoccum Schuebl.) favours high photosynthetic capacity adaptation to low nitrogen stress.

Although tetraploid wheat has rich genetic variability for cultivar improvement, its physiological mechanisms associated with photosynthetic productivity and resilience under nitrogen (N) deficit stress have not been investigated. In this study, we selected emmer wheat (Kronos, tetraploid), Yangmai 25 (YM25, hexaploid), and Chinese Spring (CS, hexaploid) as materials and investigated the differences in net photosynthetic rate (Pn), carboxylation capacity, electron transfer capacity, photosynthetic product output, and photosynthetic N allocation under normal N (CK) and low N (LN) through hydroponic experiments. Tetraploid emmer wheat (Kronos) had a stronger photosynthetic capacity than hexaploid wheat (YM25, CS) under low N stress, which mainly associated with the higher degree of PSII opening, electron transfer rate, Rubisco content and activity, ATP/ADP ratio, Rubisco activase (Rca) activity and Rubisco activation state, and more leaves N allocation to the photosynthetic apparatus, especially the proportion of N allocation to carboxylation under low N stress. Moreover, Kronos reduced the feedback inhibition of photosynthesis by sucrose accumulation through higher sucrose phosphate synthetase (SPS) activity and triose phosphate utilization rate (VTPU). Overall, Kronos could allocate more N to the photosynthetic components to improve Rubisco content and activity to maintain photosynthetic capacity under low N stress while enhancing triose phosphate output to reduce feedback inhibition of photosynthesis. This study reveals the physiological mechanisms of emmer wheat that maintain the photosynthetic capacity under low N stress, which will provide indispensable germplasm resources for elite low-N-tolerant wheat improvement and breeding.

A fungal protein organizes both glycogen and cell wall glucans.

Glycogen is a glucose storage molecule composed of branched α-1,4-glucan chains, best known as an energy reserve that can be broken down to fuel central metabolism. Because fungal cells have a specialized need for glucose in building cell wall glucans, we investigated whether glycogen is used for this process. For these studies, we focused on the pathogenic yeast Cryptococcus neoformans, which causes ~150,000 deaths per year worldwide. We identified two proteins that influence formation of both glycogen and the cell wall: glycogenin (Glg1), which initiates glycogen synthesis, and a protein that we call Glucan organizing enzyme 1 (Goe1). We found that cells missing Glg1 lack α-1,4-glucan in their walls, indicating that this material is derived from glycogen. Without Goe1, glycogen rosettes are mislocalized and ß-1,3-glucan in the cell wall is reduced. Altogether, our results provide mechanisms for a close association between glycogen and cell wall.

Possible Roles of Carbohydrate Management and Cytokinin in the Process of Defoliation-Regrowth Cycles in Rice.

Defoliation is an inevitable abiotic stress for forage and turf grasses because harvesting, grazing, and mowing are general processes for their production and management. Vegetative regrowth occurs upon defoliation, a crucial trait determining the productivity and persistence of these grasses. However, the information about the molecular regulation of this trait is limited because it is still challenging to perform molecular analyses in forage and turf grasses. Here, we used rice as a model to investigate vegetative regrowth upon defoliation at physiological and molecular levels. This study analyzed stubble and regrown leaves following periodic defoliation using two rice varieties with contrasting regrowth vigor. Vigorous regrowth was associated with maintained chlorophyll content and photosystem II performance; a restricted and promoted mRNA accumulation of sucrose synthase (SUS) I and III subfamilies, respectively; and reduced enzymatic activity of SUS. These results suggest that critical factors affecting vegetative regrowth upon defoliation are de novo carbohydrate synthesis by newly emerged leaves and proper carbohydrate management in leaves and stubble. Physiological and genetic analyses have demonstrated that the reduced sensitivity to and inhibited biosynthesis of cytokinin enhance regrowth vigor. Proper regulation of these metabolic and hormonal pathways identified in this study can lead to the development of new grass varieties with enhanced regrowth vigor following defoliation.

Glucosylceramides impact cellulose deposition and cellulose synthase complex motility in Arabidopsis.

Cellulose is an abundant component of plant cell wall matrices, and this para-crystalline polysaccharide is synthesized at the plasma membrane by motile Cellulose Synthase Complexes (CSCs). However, the factors that control CSC activity and motility are not fully resolved. In a targeted chemical screen, we identified the alkylated nojirimycin analog N-Dodecyl Deoxynojirimycin (ND-DNJ) as a small molecule that severely impacts Arabidopsis seedling growth. Previous work suggests that ND-DNJ-related compounds inhibit the biosynthesis of glucosylceramides (GlcCers), a class of glycosphingolipid associated with plant membranes. Our work uncovered major changes in the sphingolipidome of plants treated with ND-DNJ, including reductions in GlcCer abundance and altered acyl chain length distributions. Crystalline cellulose content was also reduced in ND-DNJ-treated plants as well as plants treated with the known GlcCer biosynthesis inhibitor N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenyl ethyl]-decanamide (PDMP) or plants containing a genetic disruption in GLUCOSYLCERAMIDE SYNTHASE (GCS), the enzyme responsible for sphingolipid glucosylation that results in GlcCer synthesis. Live-cell imaging revealed that CSC speed distributions were reduced upon treatment with ND-DNJ or PDMP, further suggesting an important relationship between glycosylated sphingolipid composition and CSC motility across the plasma membrane. These results indicate that multiple interventions compromising GlcCer biosynthesis disrupt cellulose deposition and CSC motility, suggesting that GlcCers regulate cellulose biosynthesis in plants.

Genetic Mutations in FKS1 Gene Associated with Acquired Echinocandin Resistance in Candida parapsilosis Complex.

Candida parapsilosis complex has recently received special attention due to naturally occurring FKS1 polymorphism associated with high minimal inhibitory concentrations for echinocandin and the increase of clonal outbreaks of strains resistant to commonly used antifungals such as fluconazole. Despite the previous fact, little is known about the genetic mechanism associated with echinocandin resistance. Therefore, the present study was designed to investigate the mechanism of acquired echinocandin resistance in C. parapsilosis complex strains. A total of 15 clinical C. parapsilosis complex isolates were sub-cultured for 30 days at a low concentration of micafungin at ½ the lowest MIC value of the tested isolates (0.12 µg/ml). After culturing, all the isolates were checked phenotypically for antifungal resistance and genotypically for echinocandin resistance by checking FKS1 gene hot spot one (HS1) and HS2 mutations. In vitro induction of echinocandin resistance confirmed the rapid development of resistance at low concentration micafungin, with no difference among C. parapsilosis, C. metapsilosis, and C. orthopsilosis in the resistance development. For the first time we identified different FKS1 HS1 and or HS2 mutations responsible for echinocandin resistance such as R658S and L1376F in C. parapsilosis, S656X, R658X, R658T, W1370X, X1371I, V1371X, and R1373X (corresponding to their location in C. parapsilosis) in C. metapsilosis, and L648F and R1366H in C. orthopsilosis. Our results are of significant concern, since the rapid development of resistance may occur clinically after short-term exposure to antifungals as recently described in other fungal species with the potential of untreatable infections.

NnSUS1 encodes a sucrose synthase involved in sugar accumulation in lotus seed cotyledons.

Fresh lotus seeds are gaining favor with consumers for their crunchy texture and natural sweetness. However, the intricacies of sugar accumulation in lotus seeds remain elusive, which greatly hinders the quality improvement of fresh lotus seeds. This study endeavors to elucidate this mechanism by identifying and characterizing the sucrose synthase (SUS) gene family in lotus. Comprising five distinct members, namely NnSUS1 to NnSUS5, each gene within this family features a C-terminal glycosyl transferase1 (GT1) domain. Among them, NnSUS1 is the predominately expressed gene, showing high transcript abundance in the floral organs and cotyledons. NnSUS1 was continuously up-regulated from 6 to 18 days after pollination (DAP) in lotus cotyledons. Furthermore, NnSUS1 demonstrates co-expression relationships with numerous genes involved in starch and sucrose metabolism. To investigate the function of NnSUS1, a transient overexpression system was established in lotus cotyledons, which confirmed the gene's contribution to sugar accumulation. Specifically, transient overexpression of NnSUS1 in seed cotyledons leads to a significant increase in the levels of total soluble sugar, including sucrose and fructose. These findings provide valuable theoretical insights for improving sugar content in lotus seeds through molecular breeding methods.