Loss of HLH-30 markedly impaired survival in starved larval worms and recovery upon refeeding bacteria. Remarkably, provision of simple nutrients in a completely defined medium (C. elegans maintenance medium [CeMM]), specifically glucose and linoleic acid, restored lysosomal acidification, TOR activation, and survival with refeeding despite the absence of HLH-30. Worms deficient in lysosomal lipase 2 (lipl-2), a lysosomal enzyme that is transcriptionally up-regulated in starvation in an HLH-30–dependent manner, also demonstrated increased mortality with starvation–refeeding that was partially rescued with glucose, suggesting a critical role for LIPL-2 in lipid metabolism under starvation. CeMM induced transcription of vacuolar proton pump subunits in hlh-30 mutant worms, and knockdown of vacuolar H+-ATPase 12 (vha-12) and its upstream regulator, nuclear hormone receptor 31 (nhr-31), abolished the rescue with CeMM. Loss of Ras-related GTP binding protein C homolog 1 RAGC-1, the ortholog for mammalian RagC/D GTPases, conferred starvation–refeeding lethality, and RAGC-1 overexpression was sufficient to rescue starved hlh-30 mutant worms, demonstrating a critical need for TOR activation with refeeding. These results show that HLH-30 activation is critical for sustaining survival during starvation–refeeding stress via regulating TOR. Glucose and linoleic acid bypass the requirement for HLH-30 in coupling lysosome nutrient sensing to survival.Lysosomes are ubiquitous acidified organelles that degrade intracellular and extracellular material trafficked via multiple pathways. Lysosomes also sense cellular nutrient levels to regulate target of rapamycin (TOR) kinase, a signaling enzyme that drives growth and suppresses activity of the MiT/TFE family of transcription factors that control biogenesis of lysosomes. In this study, we subjected worms lacking basic helix–loop–helix transcription factor 30 ( Lysosomes play a central role in coupling the nutrient state of the cell to growth and survival decisions. This study uncovers a critical role for HLH-30, the nematode ortholog of the mammalian MiT/TFE family of master regulators of lysosome biogenesis, in survival under starvation and refeeding conditions. Refeeding simple nutrients bypasses the requirement for HLH-30 to permit survival. Lysosomes are membrane-bound subcellular organelles that contain degradative enzymes and are ubiquitously present in all eukaryotic cell types. These specialized organelles compartmentalize an acidified milieu for enzymatic degradation of complex macromolecules and organelles that are trafficked via autophagy for recycling intracellular substrates or taken up from the extracellular space via endocytosis, macropinocytosis, and phagocytosis. Transcription Factor EB (TFEB) and related members of the MiT/TFE  family of basic helix–loop–helix leucine zipper transcriptional activators TFE3 and MiTF) were discovered as master regulators of autophagy and lysosome biogenesis programs by analyzing lysosomal gene promoters , 2. TFEB and MiTFLysosomes play a critical role in sensing the cellular nutrient state and coupling the responses to activation of target of rapamycin (TOR), a nutrient-activated serine–threonine kinase that promotes anabolism and growth and suppresses catabolism . LysosomRecent studies have identified a feedback loop whereby MiT/TFE family members transcriptionally up-regulate mTOR complex components during the starvation phase to permit mTOR reactivation upon refeeding  to channC. elegans offers numerous advantages as a model system to evaluate the effects of starvation and refeeding on survival of the whole organism, given the various adaptations it has evolved to sustain survival in the face of nutrient deprivation , N = 3/group). However, hlh-30(lf) worms starved for 33 hours displayed a marked energy deficit compared to starved wild-type worms (ATP levels in nmol/mg DNA: 37 ± 2 in hlh-30(lf) versus 171 ± 4 in the wild type, P < 0.001 by t test, N = 3/group), despite both being alive at this time point (hlh-30(lf) worms displayed higher levels of amino-acid metabolism gene transcripts and a different set of lysosomal gene transcripts compared with their fed counterparts  worms. Wild-type HLH-30 or a mutant protein deficient in nuclear translocation  or hlh-30(mNLS)(oe) (overexpression abbreviated as “oe”). Fluorescence microscopy revealed that starvation caused HLH-30::RFP to translocate to the nucleus, whereas HLH-30(mNLS)::RFP remained in the cytoplasm (hlh-30(lf) worms (To investigate the role of  protein ) was fusytoplasm . Biochemytoplasm  without ytoplasm . This inf) worms , S6C Fighlh-30(lf) worms with bacteria may not be sufficient for survival because critical metabolites required for recovery are not generated from breakdown of complex substrates as a result of defective lysosomes in this mutant. To test the hypothesis that a mixture of simple nutrients can bypass defective lysosomes and rescue the lethality of hlh-30(lf) worms, we employed C. elegans maintenance medium (CeMM), a fully defined chemical medium that contains various simple nutrients and is sufficient to sustain growth and development [hlh-30(lf) worms starved for 33 hours, followed by 15 hours of CeMM treatment and subsequent E. coli OP50 refeeding for 48 hours). Rescue was time dependent such that 3–6 hours of incubation caused partial recovery, whereas 9–15 hours of incubation caused complete recovery (hlh-30(lf) worms starved for as long as 4 days but was no longer effective after 6 days of starvation (hlh-30(lf) mutants and prolonged periods of starvation in wild-type animals may result in similar deficits.Dysfunction of autophagy or the LYNUS complex results in reduced levels of simple metabolites, such as amino acids and sugars, and lethality in mouse pups under conditions of starvation , 30. Intelopment . When strecovery . Incubatarvation . This obarvation , suggestTo determine which component of CeMM is necessary for rescue, we systematically removed each major class of macronutrients. Removal of heme, amino acids, or nucleic acids did not significantly impair recovery, indicating these factors are not necessary, whereas removal of lipids or carbohydrates abolished rescue, indicating these factors are necessary . To detehlh-30(lf) worms, we performed global metabolomic profiling in starved and CeMM-refed worms. As shown in hlh-30(lf) worms (that ultimately survive) from starved hlh-30(lf) worms  worm (E. coli OP50 on NGM dishes was sufficient to rescue starved hlh-30(lf) worms  worm . Rather,lf) worm . Moreovef) worms . Accordif) worms . By contf) worms .hlh-30(lf) worms compared to their starved counterparts (hlh-30(lf) worms compared to starved hlh-30(lf) worms  in the subset of metabolites with the highest importance to group separation in the random forest analysis , was sigterparts . While lterparts , the abuf) worms , indicathlh-30(lf) mutants demonstrated reduced glucose, maltose, and maltotriose levels compared to the starved wild type (hlh-30(lf) mutants compared with the starved wild type (hlh-30(lf) mutants  mutants compared to the wild type (hlh-30(lf) mutants, they continued to be markedly reduced compared with CeMM-refed wild type (E. coli OP50-refed hlh-30(lf) mutant worms after starvation compared to CeMM-refed animals (E. coli OP50-refed versus near complete in CeMM-refed hlh-30(lf) mutant worms  worms that permits survival. Indeed, CeMM refeeding led to the restoration of ATP levels, indicating that CeMM corrected the marked energetic insufficiency observed in starved hlh-30(lf) worms (ATP levels in nmol/mg DNA: 108 ± 11 in CeMM-refed hlh-30(lf) versus 72 ± 15 in CeMM-refed wild type, P > 0.05, N = 3/group). Also, while levels of diacylglycerols, endocannabinoids, and phospholipids robustly rebounded in CeMM-refed versus starved wild-type worms, comparable increases were not observed in starved hlh-30(lf) mutants upon CeMM feeding (hlh-30(lf) mutants, consistent with impaired autophagic protein breakdown during starvation with hlh-30 deficiency (hlh-30(lf) mutants after refeeding (hlh-30(lf) mutants, it did not restore a metabolic phenotype identical to the wild type.In wild-type worms, CeMM feeding resulted in increased levels of glucose and its metabolite, glucose-6-phosphate, as well as of maltose and maltotriose , indicatild type , consistild type , along wild type , which wild type  and was ild type  and otheild type . Similarild type ; S11 Fig animals . This di feeding . Levels ficiency . Interesefeeding . These rhlh-30–deficient worms. TFEB and HLH-30 play important roles in lysosomal lipolysis during starvation stress [lipl-2  in an hlh-30–dependent manner  mutation is a deletion of multiple exons likely to cause a strong loss of function (lipl-2(lf)); these worms displayed impaired survival during starvation compared to wild-type worms  worms ) could rescue hlh-30(lf) worms. lipl-2(oe) did not rescue the lethality of E. coli OP50-refed hlh-30(lf) worms after 33 hours of starvation  worms but does not confer survival. However, lipl-2(oe) was sufficient to confer a modest rescue of starved hlh-30(lf) worms when CeMM minimal was supplemented with glucose , which was significantly greater than the rescue observed when CeMM minimal was supplemented with lipids  animals. hlh-30(oe);lipl-2(lf) animals displayed a small but significant rescue compared to lipl-2(lf) animals, but they were still impaired compared to the wild type  worms . Taken tarvation . This isild type . OverexpC. elegans [lipl-4 transgenic worms [hlh-30(lf) worms was similar to their fed counterparts and wild-type controls (hlh-30(lf) worms, there appeared to be fewer, and the number appeared to be restored by CeMM refeeding (C. elegans ortholog of human BECN1 (bec-1), a gene critical for autophagosome formation [bec-1 mRNA levels by more than 90% did not attenuate (and even enhanced) the beneficial effect of CeMM supplementation in starved hlh-30(lf) worms (Prior studies indicate that 20-carbon polyunsaturated fatty acids (PUFAs) accumulate in starved  elegans , and recic worms , suggestcontrols . Althougefeeding . To deteormation , 37. Redf) worms .hlh-30(lf) worms, we compared gene expression changes in CeMM-refed worms at 0, 3, and 15 hours using RNAseq profiling and identified significantly regulated KEGG pathways (hlh-30(lf) worms only up-regulated a subset of these pathways focused upon lipid metabolism, indicating their potentially critical role in the observed rescue (hlh-30(lf) mutants that were distinct from their wild-type counterparts (hlh-30(lf) worms with 15 hours of CeMM exposure (and not with 3 hours of exposure) corresponding to the observed rescue (see group 3 from vha-subunit genes in hlh-30(lf) and wild-type worms that were fed, starved, or refed CeMM or E. coli OP50  worms compared to fed worms, but not in starved wild-type worms compared to fed worms  worms compared to the E. coli OP50-refed wild type  displayed significantly higher transcript levels in hlh-30(lf) mutants after CeMM refeeding compared to E. coli OP50 refeeding (vha genes (E. coli OP50-refed hlh-30(lf) mutants (hlh-30(lf) mutants compared with the similarly treated wild type (hlh-30(lf) mutant worms.To elucidate how CeMM rescues starvation lethality in pathways . Refeedipathways , indicatd rescue . Unsuperterparts . We focuoli OP50 . Two dised worms ; 2) vha-ild type . These pefeeding . Furtherha genes . In cont mutants . Four ofild type . Taken thlh-30(lf) worms, which could account for the observed rescue upon subsequent refeeding with bacterial food, a complex source of nutrients. To examine this premise, we performed staining with LysoTracker Red, an acidophilic dye, and evaluated expression of LMP-1, a lysosome membrane protein (hlh-30(lf) worms refed with E. coli OP50 compared to similarly treated wild-type worms (hlh-30(lf) worms compared to starved wild-type worms (E. coli OP50) restored LMP-1 abundance to wild-type levels in the hlh-30(lf) worm  worm , indicatvha genes, we used the specific V-ATPase inhibitors concanamycin A or bafilomycin A1 (Baf-A1) [hlh-30(lf) worms refed with CeMM  worms  worms refed with CeMM , which encodes a C. elegans ortholog of mammalian hepatocyte nuclear factor 4α (HNF-4α) (with linoleic acid as an endogenous ligand [vha gene transcription in worms [nhr-31 transcripts reduced CeMM-induced rescue of starved hlh-30(lf) mutants  mutants supplemented with either glucose or linoleic acid. A significant increase in survival was not observed, pointing to a critical role for both the nutrients (glucose and linoleic acid) in entraining nhr-31 signaling to confer rescue  , 40. Botith CeMM . In addif) worms . Reducinith CeMM , indicats ligand ), has bein worms . RNAi ta mutants , indicatr rescue .hlh-30(lf) mutants  mutants (hlh-30(lf) mutants.To investigate the role of TOR activation, we attempted CeMM rescue in the presence of rapamycin, a known inhibitor of TOR activity . Treatme mutants , pointinlet-363  worms compared to the starved wild type. These findings suggest hlh-30 is necessary to promote transcription of these genes during starvation. Importantly, refeeding with CeMM but not E. coli OP50 restored the expression of let-363 and ragc-1 to wild-type levels in hlh-30(lf) mutants  worms, whereas CeMM exposure increased ragc-1 transcript levels in hlh-30(lf) worms. We analyzed a strong loss-of-function ragc-1(tm1974) mutation caused by a deletion spanning multiple exons (ragc-1(lf). ragc-1(lf) animals displayed striking sensitivity to starvation-induced lethality (E. coli OP50 or CeMM did not restore viability (ragc-1 in hlh-30(lf) mutants was sufficient to confer partial rescue on starved animals fed E. coli OP50 le exons , termed ethality . Refeediiability . Overexpoli OP50 , S23 Fighlh-30 to drive vha gene transcription and couple lysosome nutrient sensing to TOR activation, which permits survival upon refeeding following starvation. The following lines of evidence support this claim. First, hlh-30 deficiency markedly sensitizes worms to starvation-induced death, which cannot be rescued upon refeeding. Second, HLH-30 activation (nuclear translocation) is essential to permit survival with starvation and refeeding stress. Third, hlh-30–dependent induction of lipl-2, a lysosomal lipase, during starvation is critical to permit survival with starvation and refeeding stress. Fourth, exogenous delivery of glucose and linoleic acid restores energy stores, up-regulates vha gene transcription, restores lysosome acidification, and drives TOR reactivation to permit survival upon refeeding with complex nutrients in hlh-30–deficient and lipl-2–deficient worms. Fifth, knockdown of nhr-31 (an upstream master regulator of vha gene transcription), knockdown of vha-12 (a proton pump subunit), and pharmacologic inhibition of the proton pump or TOR activity inhibit the rescue observed with simple nutrients in starved and refed hlh-30–deficient worms. Sixth, the ragc-1–deficient mutant displays starvation–refeeding sensitivity and cannot be rescued with simple nutrients after starvation–refeeding stress. Seventh, forced activation of TOR with RAGC-1 overexpression is sufficient to rescue starved hlh-30–deficient worms upon refeeding. A model that emerges  [hlh-30(lf) worms cannot generate sufficient linoleic acid from de novo synthesis under starvation conditions and require its supplementation exogenously, presumably because these worms lack the hlh-30–dependent transcriptional up-regulation of the fatty acid desaturase fat genes involved in de novo biosynthesis of unsaturated fatty acids, as described previously [E. coli OP50 restored lipl-2 expression in hlh-30(lf) worms but was also not sufficient to confer survival. Our findings also implicate a critical role for HLH-30 translocation to the nucleus because a mutant HLH-30 protein lacking the nuclear localization signal did not translocate to the nucleus and was ineffective in sustaining survival. This indicates a role of hlh-30–induced transcription of its target genes, such as lipl-2 and ragc-1, in the observed response and is less likely to be due to other factors such as a potentially toxic role of cytosolic HLH-30 protein with a mutated nuclear localization signal.The novel, to our knowledge, observation that underlies our discovery is that a brief duration of starvation is sufficient to impair critical survival processes and prevent recovery despite refeeding in the escribed  to genervatives) , namely vatives) . Howevereviously , or are vha gene) transcripts and restores acidified lysosomes in the hlh-30–deficient worms. Furthermore, our studies implicate an important role for NHR-31, a nuclear hormone receptor, in transducing CeMM-mediated restoration of viability in starved hlh-30–deficient worms. NHR-31 is expressed in the worm intestine and excretory cell throughout development and identified as a master regulator of vha gene transcription and resistance to osmotic stress [nhr-31 signaling to restore lysosome pH. This, along with CeMM-mediated restoration of lysosome abundance, facilitates LYNUS [E. coli OP50-refed hlh-30–deficient worms. Indeed, refed hlh-30–deficient worms demonstrate growth on E. coli OP50 only after prior incubation in CeMM because TOR activity is required for accessing nutrients to drive worm growth and development [ragc-1 transcript levels are significantly reduced by starvation–refeeding stress in the hlh-30(lf) mutant and are only rescued by CeMM treatment (but not by E. coli OP50). Accordingly, the increased sensitivity of the ragc-1 mutant to starvation–refeeding stress demonstrates that TOR activation is essential as a final step for survival with refeeding following starvation that cannot be rescued by CeMM. Importantly, forced up-regulation of the TOR machinery by RAGC-1 overexpression was sufficient to rescue the obligate mortality in the starved hlh-30–deficient worms upon refeeding, attesting to a central role for lysosome nutrient sensing in survival in worms as observed in mammals [C. elegans growth and development and warrant careful experimental dissection of its multifaceted effects in future studies. Interestingly, amino acids that have been demonstrated to activate TOR signaling by both rag-dependent [rag-independent mechanisms [hlh-30–sufficient state, a premise that should be further explored for potential therapeutic benefit in humans facing prolonged periods of fasting.A major finding of our study is that CeMM up-regulates proton pump  by activation of ongation . This prongation , and ablongation , pointinongation , and knont worms . Our datdaf-16, which is essential to survive starvation because daf-16–deficient worms fail to undergo L1 arrest and die without exogenous nutrients [daf-16 regulates expression of multiple genes involved in biosynthesis of unsaturated lipids, namely fat-2, fat-6, fat-7, and the fatty acid elongase elo-2 [daf-16 mutant worms [hlh-30–independent fashion to overcome the autophagy deficiency under starvation conditions and confer rescue upon refeeding. Our data demonstrating that concomitant knockdown of bec-1 gene expression does not prevent CeMM-mediated rescue rule out the role of canonical autophagy in this process. Interestingly, oleic acid, a PUFA, was sufficient to induce noncanonical bec-1–independent autophagy in C. elegans embryos and mammalian cells [Another mechanistic possibility whereby linoleic acid and PUFAs may confer survival signaling is via regulation of lipid stores, such as observed with deficiency of utrients . Indeed,se elo-2 , 55, andnt worms  in an hlan cells . WhetherThe discovery of lysosomal metabolites with signaling roles and the ability to replenish them exogenously may have profound therapeutic implications in human disease with both genetic and acquired dysfunction of the lysosomal machinery. Indeed, lysosome storage diseases (LSDs) are inborn errors of metabolism clinically characterized by a “failure to thrive,” with generalized metabolic abnormalities beyond those expected from deficiency of individual lysosomal enzymes . MultiplC. elegans strains were maintained at 20 °C on NGM dishes seeded with the E. coli strain OP50. The wild-type strain was Bristol N2. The mutant strains hlh-30(tm1978), lipl-1(tm1954), lipl-2(tm4324), lipl-3(tm4498), lipl-4(tm4417), and ragc-1(tm1974) strains ; see E. coli OP50 and Comomonas were purchased from the C. elegans Genetic Center, University of Minnesota. To inactivate E. coli OP50, the bacteria were grown overnight on NGM dishes and exposed to UV light in a UV-Crosslinker  at 3,000 joules. A culture was subsequently inoculated in Luria broth (LB) and tested for growth at 37 °C on a shaker for 18 hours. The absence of bacterial growth confirmed inactivation.All E. coli OP50, Comamonas, or UV-killed E. coli OP50 or placed in S-medium containing E. coli OP50 in suspension. The number of live worms, defined as worms exhibiting spontaneous movement, was scored by visual inspection immediately and after 48 hours. The metric “Alive after Starvation” was assessed as percentage of worms alive immediately upon placement on the food source, and the metric “Alive after Refeeding” was assessed as number of worms alive 48 hours later divided by the number of worms alive immediately upon placement on the food source. Fed worms were L1 stage larvae collected 18 hours after placement of a synchronized population of eggs on seeded NGM Petri dishes. Movies depicting spontaneous movement in these worms were obtained with a Leica M80 microscope  with a 1.25× objective and an IC80 HD camera.Gravid adults were bleached with alkaline hypochlorite to kill all contaminants and all worms not protected by the eggshell, then washed and placed in M9 , 64. TheE. coli OP50 for one hour. Pharyngeal pumping was scored as described [L1 worms were starved 33 hours in M9, then placed on NGM dishes seeded with escribed .hlh-30(oe) transgenic strains was generated by PCR amplification of a 1,497 base pair (bp) fragment containing the coding sequence for W02C12.3a from pDONR201  and a 2,000 bp fragment containing W02C12.3a promoter (Dharmacon) and subcloning these into the pBlueScript SK+ vector pDG219 to create hlh-30p::hlh-30::rfp. hlh-30(oe) carries the amEx272 extrachromosomal array. The plasmid used to generate hlh-30(mNLS) transgenic strains was created by site-directed mutagenesis of the putative nuclear localization sequence found in hlh-30p::hlh-30::rfp, based on homology with the mammalian TFEB protein [hlh-30mNLS(oe) carries the amEx291 extrachromosomal array. The co-marker pRF4 (rol-6(su1006)) was coinjected along with the hlh-30(oe) or hlh-30mNLS(oe) constructs. Please see the list of strains in The plasmid used to generate  protein . Specifiragc-1(oe) was made through microinjection of ragc-1p::ragc-1::rfp and the co-marker sur-5::GFP into the gonad arm of hlh-30(lf). ragc-1 was PCR amplified from genomic DNA to produce a 2,016 bp amplimer that extends 560 bp upstream of the translational start and 219 bp downstream of the translational stop codon. Constructs were subcloned into pBlueScript SK+, and the sequence was verified. Fed L4 worms were collected in 15-mL Falcon tubes, washed eight times with M9 to remove debris and bacteria, and starved in M9 at a density of one worm per μL. The 15-mL tubes were shaken horizontally on a Labnet Orbit 1000 at 100 revolutions per minute at 20 °C. An aliquot was taken every 24 hours, the worms were placed on NGM dishes, and the number of live worms compared to total worms in the aliquot was scored to assess “Alive after Starvation (%).” ragc-1(oe) transgenic animals were identified by expression of GFP prior to the starvation–refeeding assay. Worms were starved for 36 hours, then refed by placement on NGM dishes seeded with OP50. The number of live worms was scored upon refeeding 72 hours later and scored as Alive After Refeeding . This extrachromosomal array was named amEx324. Please also see the list of strains in The lipl-2(oe) was made through microinjection of lipl-2p::lipl-2::rfp and the co-marker pRF4 (rol-6(su1006)) into the gonad arm of hlh-30(lf). lipl-2 was generated through PCR amplification of fosmid DNA. The construct extends 1,468 bp upstream of the ATG translational start site and 1,631 bp downstream to include the unspliced lipl-2 genomic sequence without the stop codon. This construct was subcloned into pDG219, a modified pBlueScript SK+ plasmid containing RFP and 3′ UTR of unc-54. Please also see the list of strains in The hlh-30(oe) transgenic strain and the co-marker pRF4 (rol-6(su1006)) were injected into the gonad arm of lipl-2(tm4324) to generate hlh-30(oe);lipl-2(lf). Please also see the list of strains in The plasmids used to generate nhr-31p::3XFLAG::nhr-31 were subcloned into pBlueScript SK+ to produce a 3,414 bp amplimer that extends 1,000 bp upstream of the translational start for C26B2.3a.1, contains a DYKDHD-DYKDHD-DYKDDDD (3×FLAG) motif, and is connected to the coding sequence for C26B2.3a.1 CDS and its 593 bp 3′ UTR via a linker. The sequence was verified, followed by microinjection of nhr-31p::3XFLAG::nhr-31 and the co-marker sur-5::GFP into the gonad arm of hlh-30(lf) to generate nhr-31(oe);hlh-30(lf) worms. Please also see the list of strains in gBlock fragments  containing the proximal and distal halves of C. elegans lines expressing extrachromosomal arrays as above were generated with coinjection markers as indicated in All 2× CeMM medium was prepared as described, using D-glucose as the energy source . The vitFree fatty acids were conjugated to fatty-acid–free bovine serum albumin  as previously described . A 20% BE. coli OP50 in an equimolar ratio, spread on the NGM dish and allowed to dry, and placed in a 20 °C incubator for one hour. Bacterial concentration was estimated using the method described [9 per dish. To measure intact bacteria in the intestine, we seeded NGM plates with a strain of E. coli OP50 expressing GFP obtained from the Caenorhabditis Genome Center (CGC), as previously described [Fluoresbrite YG Carboxylate (0.5-μm diameter) microspheres  were mixed with escribed . The finescribed . Worms wE. coli OP50-seeded NGM dishes, and scored as Alive After Refeeding 48 hours later.Worms were starved 33 hours, then placed in CeMM containing 25 μM Baf-A1 , 400 nM concanamycin A (Sigma C9705), or respective controls 0.34% DMSO or 1.2% DMSO for 15 hours, followed by transfer to OP50. Worms were grown to L4 stage, washed seven times in M9, adjusted to a density of one worm per μL, then placed in M9 containing 200 μM rapamycin. After 36 hours at 20 °C, worms were washed in M9, then transferred to CeMM containing 200 μM rapamycin for 15 hours. Then, worms were washed in M9 and placed on NGM dishes containing OP50 and 200 μM rapamycin for 72 hours, where the number alive after refeeding was scored.Rapamycin  was dissolved in 100% DMSO at 50 mg/mL and added to plate agar, M9, and CeMM at 200 μM. Controls contained an equivalent DMSO concentration. Eggs were placed on NGM dishes containing rapamycin and E. coli OP50 that was grown overnight with LysoTracker. 18 hours after bleaching, worms were washed twice in fresh M9 (without LysoTracker) and imaged. For collecting starved worms, worms were bleached and placed in M9 containing LysoTracker and starved for 33 hours, followed by two washes with fresh M9 (without LysoTracker) and imaged. For collecting refed worms, worms were starved for 33 hours in M9 with LysoTracker as described, then refed with CeMM containing LysoTracker for 15 hours, washed twice with fresh M9 (without LysoTracker), and imaged. To allow for operator viewing, all fluorescent images were configured in ImageJ to have a maximum brightness and contrast of 50. Worms were outlined and measured for area and integrated density. The corrected total worm fluorescence (CTWF) was calculated for each worm by “CTWF = Integrated density − (Area × mean fluorescence of control),” where the control was taken from worms not exposed to LysoTracker.For all experiments, LysoTracker Red DND-99  was used at a final concentration of 1 μM, and worms were incubated at 20 °C. For collecting fed worms, gravid adults were bleached, and eggs were washed and resuspended in 1 mL M9 containing LysoTracker. A glass Pasteur pipet was used to transfer eggs from M9 to an NGM dish containing dried C. elegans protein extracts, we washed worm pellets  three times with M9 and homogenized in 200 μL of ice-cold buffer containing 50 mM Tris HCl (pH 7.4), 2.5 mM EDTA, 25 mM NaCl, 0.2% NP 40, 10 mM EGTA, 20 mM NaFl, 25 mM Na4O7P2, 2 mM Na3VO4 with protease and phosphatase inhibitor . Mechanical action was used to break apart worm tissue. Samples were then centrifuged 200 × g for 20 minutes at 4 °C, and supernatant was transferred to a fresh 1.7-mL Eppendorf tube waiting on ice. Protein was measured with the Bradford assay performed on a Bio-Rad Smart Spec Plus Spectrophotometer . 10 μg of protein was loaded on SDS-PAGE gels, and immunoblotting was performed as previously described [To generate escribed . AntibodL1 stage worms  were harvested and washed three times in M9 buffer, then followed by three freeze/thaw cycles and boiled for 15 minutes. The samples were spun at 11,000g for 10 minutes at 4 °C. The supernatant was collected and measured for ATP activity by the ATP determination kit  and further normalized to the amount of dsDNA with Quant iT PicoGreen dsDNA reagent  as described .hlh-30(lf) adults were bleached, and eggs were placed on NGM dishes containing 5 mM IPTG and 25 μg/ml carbenicillin. An HT-115(DE3) bacterial colony containing L4440 or bec-1 plasmid [bec-1 plasmid. Worms were scored alive after refeeding 48 hours later. For RNAi to nhr-31, we employed dsRNA soaking. An nhr-31 RNA construct was generated through PCR amplification of 1,569 bp from GE Dharmacon C. elegans clone C26B2.3 ORF. To check for potential cross interference, defined by greater than 80% identity over 200 bp, a nucleotide Blast search was performed using WormBase. There were no matches greater than 200 bp except for exon sequences found in nhr-31. The amplimer was subcloned into the multiple cloning site of L4440 at the HindIII and NotI restriction site, between the T7 RNA polymerase binding sites, which was verified with sequencing. The clone for expressing RNAi targeting vha-12 was obtained from the Ahringer RNAi library. To generate dsRNA from these clones, a PCR produced a linear template of dsDNA that included the subcloned nhr-31 fragment or the vha-12 fragment and the flanking T7 RNA polymerase binding sites. The product of the DNA polymerase reaction was purified using the Monarch PCR and DNA Cleanup Kit . Subsequently, in vitro transcription was performed with 500 ng of purified dsDNA using the HiScribe T7 RNA Polymerase Kit . The product of the RNA polymerase reaction was purified using the RNeasy MinElute Kit . The starvation and refeeding assay for L1 stage larvae was performed as described above in the presence of 1 μg of L4440, nhr-31, or vha-12 dsRNA.To deliver dsRNA to worms by feeding, gravid wild-type and  plasmid  was inocLysoTracker Red DND-26  was diluted in M9 and dispensed on NGM dishes, M9, or CeMM to yield a final concentration of 1 μM. Worms were cultured on these dishes or liquid solutions in the dark and transferred to NGM dishes or fresh liquid without dye for 30 min. The animals were paralyzed with 25 mM levamisole  in M9, mounted on 3% agarose pads on microscope slides, and imaged with an Olympus FV1200 Confocal Microscope .OP50 conditions were prepared and subjected to transmission electron microscopy analyses as described [Samples containing L1 worms in fed, starved, CeMM, and refed escribed . After tg for 15 minutes at 4 °C. The aqueous phase and an equal volume of 70% ethanol were added to a fresh 1.7-mL tube. Nucleic acids were isolated with the RNeasy Mini Kit  following manufacturer’s instructions. cDNA was generated with the iScript Reverse Transcription Supermix for RT-qPCR . cDNA was mixed with Syber Green Master Mix  as recommended by the manufacturer and analyzed on an Applied Biosystems 7500 Real Time PCR System. Cycle threshold (Ct) values were normalized to the control gene ama-1 or act-1. See Worms were collected in Eppendorf tubes and washed three times in M9. We added 400 μL of TRIzol  to the worm pellet , and the tube was frozen immediately in liquid nitrogen and stored at −80 °C. Following three cycles of freeze–thaw, 200 μL of TRIzol was added with incubation at room temperature for five minutes. Thereafter, 140 μL of chloroform  was added, and the samples were shaken by hand to mix for 60 seconds and incubated at room temperature for two minutes, followed by centrifugation at 12,000 × hlh-30(lf) worms  at L1 stage under fed, starved, and CeMM-refed conditions (at 3 and 15 hours), necessitating the multiplexing of multiple libraries into two lanes for sequencing. Total reads per individual sample averaged 39.3 million, with 99% alignment to the C. elegans genome (average across samples) to the Ensembl release 76 assembly with STAR version 2.0.4b. Sequencing performance was assessed for total number of aligned reads, total number of uniquely aligned reads, genes and transcripts detected, ribosomal fraction, known junction saturation, and read distribution over known gene models with RSeQC version 2.3. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.5. All gene-level counts were then imported into the R/Bioconductor package EdgeR, and TMM normalization size factors were calculated to adjust for samples for differences in library size. Genes not expressed in any samples  were excluded from further analysis. The TMM size factors and the matrix of counts were then imported into R/Bioconductor package Limma, and weighted likelihoods based on the observed mean-variance relationship of every gene were then calculated for all samples with the Voom function. Overall similarity and difference of the samples was assessed with a Spearman correlation matrix and multidimensional scaling plots  was used to obtain single-ended 50-nucleotide reads. Two samples were prepared for wild-type and lots see . Read mohlh-30(lf) C. elegans samples  were starved for 33 hours in M9 media devoid of nutrients or starved 33 hours, then refed with a fully defined liquid diet (CeMM) for 15 hours; then worms were pelleted, snap frozen, and submitted for global metabolic profiling through Metabolon Inc. . Small biochemicals in the samples were extracted, separated by ultra-high-performance liquid chromatography, and identified in a semiquantitative manner by accurate mass tandem mass spectrometry as previously described [y-axis for metabolite data reflects scaled intensity for each metabolite. ANOVA contrasts were used to identify biochemicals that differed significantly between experimental groups. An estimate of the false discovery rate  was calculated to take into account the multiple comparisons that normally occur in metabolomic-based studies and is reported alongside the P-value. Instrument variability was determined by calculating the median relative standard deviation (RSD) for the internal standards that were added to each sample prior to injection into the mass spectrometers. Overall process variability was determined by calculating the median RSD for all endogenous metabolites  present in 100% of the Client Matrix samples, which are technical replicates of pooled samples. Median RDS for instrument variability and total process variability were 2% and 8%, respectively. Hierarchical cluster analysis was performed with Array Studio . Supervised statistical analysis was performed with random forest analysis, which classifies samples based on a consensus of decision trees built from the biochemical profiles [Wild-type and escribed . A totalprofiles . For ranprofiles .C. elegans  in methanol–water (4:1) was prepared. The C. elegans samples were homogenized in internal standard solution (2 nL/worm) using an Omni Bead Ruptor  and centrifuged to obtain clear homogenate. For analysis of LPC(18:3), clear homogenate was directly injected into the LC-MS/MS system. For analysis of glucose, glucose-6-phosphate, maltose, maltotriose, citrate, α-ketoglutarate, malate, and fumarate, the clear homogenate (0.5 mL) was dried, the residue was partitioned between water (0.2 mL) and chloroform (0.75 mL), and the aqueous phase was injected into the LC-MS/MS system. For analysis of α-FFA(18:3) and γ-FFA(18:3), clear homogenate (0.2 mL) was dried and derivatized with N-(4-aminomethylphenyl)pyridium, and the derivatives were dissolved in methanol for LC-MS/MS analysis. Measurement of LPC(18:3), α-FFA(18:3), and γ-FFA(18:3) was performed on a Shimadzu 10A HPLC system  coupled to a Thermo Fisher Scientific TSQ Quantum Ultra triple quadrupole mass spectrometer, and data processing was conducted with Xcalibur (Thermo Fisher Scientific). The analysis of glucose, glucose-6-phosphate, maltose, maltotriose, citrate, α-ketoglutarate, malate, and fumarate was performed on a Shimadzu 20AD HPLC system coupled to an AB Sciex 4000QTRAP mass spectrometer , and data processing was conducted with Analyst 1.5.2. Quality control (QC) samples were prepared by pooling the aliquots of the study samples and were used to monitor the instrument stability. The QC samples were injected six times in the beginning to stabilize the instrument and were injected between every five study samples. The relative quantification was provided, and the data were reported as the peak area ratios of the analytes to the corresponding internal standards. All metabolites in QC samples show CV < 15%, except for maltose and maltriose, which were undetectable.Liquid chromatography–tandem mass spectrometry (LC-MS/MS) targeted metabolomics was performed for detecting linoleoyl-GPC, linoleoyl-lyso phosphatidyl choline (LPC)(18:3), α-FFA(18:3), γ-FFA(18:3), glucose, glucose-6-phosphate, maltose, maltotriose, citrate, α-ketoglutarate, malate, and fumarate. An internal standard solution for t test for two independent groups and one-way ANOVA or two-way ANOVA for comparing results across multiple groups for one or two variables, respectively, with GraphPad Prism software. Bonferroni’s post hoc test was employed after ANOVA for testing all pairwise comparisons. A 2-tailed value of P < 0.05 was considered statistically significant.All results are expressed as mean ± standard error of the mean (SEM). Each “replicate” indicates a biological replicate performed at a different time along with the controls (which were tested at the same time as that replicate). Statistical differences were assessed with the unpaired Student S1 Fighlh-30(lf) worms were analyzed after variable periods of starvation for Alive after Starvation (A) and Alive after Refeeding (B) as described in the legend for N = 5 biological replicates of approximately 50 worms/time point; values indicate mean ± SEM. *P < 0.05 by post hoc test following two-way ANOVA. The data are similar to hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L1, first larval stage; SEM, standard error of the mean.Wild-type and (TIF)Click here for additional data file.S2 Fighlh-30(lf) worms were analyzed after refeeding for 48 hours following variable periods of starvation (Alive after Refeeding), as described in the legend for E. coli OP50 on NGM dishes (+ OP50), UV-killed E. coli OP50 on NGM dishes , live Comamonas bacteria on NGM dishes , or live E. coli OP50 in S-basal liquid medium . N = 50 worms/time point from one biological replicate. Raw data are located in hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L1, first larval stage; NGM, nematode growth medium; S-medium, liquid medium containing concentrated OP50.Wild-type and (TIF)Click here for additional data file.S3 Figphlh-8::gfp, which expresses GFP in the precursor M cell, were imaged by fluorescent microscopy. See hlh-30 (hlh-30(+)) and hlh-30(lf). If the hlh-30(lf) mutation abrogated the L1 arrest, then development would progress, resulting in M cell division and multiple green cells. Fifty worms per genotype were examined. (B) Pharyngeal pumping rate was scored using a dissecting microscope. hlh-30(lf) and wild-type L1 worms were scored after 33 hours of starvation and 1 hour of refeeding on NGM dishes with live E. coli OP50. Values are the average of 50 worms, and bars are standard error. The values were not significantly different . Raw data are located in hlh-30(lf) and wild-type L1 worms were starved for 33 hours in M9 medium and cultured for 1 hour on NGM dishes with live E. coli OP50 admixed with fluorescent microspheres (in a ratio of 1:1). Representative confocal images without (left) and with merged bright-field (DIC) images (right) show green fluorescence (white arrows) that indicates ingestion of the microspheres. 100 worms were examined in each group. Scale bar is 20 μm. (D) hlh-30(lf) and wild-type L1 worms were starved for 33 hours followed by 48 hours of refeeding on NGM dishes seeded with live GFP-expressing E. coli OP50. Representative confocal images with merged bright-field (DIC) and GFP fluorescence demonstrate lack of green bacteria inside the worm intestines. GFP-expressing bacteria were only visualized within and proximal to the pharynx (see arrows). 100 worms were examined in each group. Scale bar is 20 μm. DIC, differential interference contrast; GFP, green fluorescent protein; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L1, first larval stage; NGM, nematode growth medium; NS, not significant; phlh-8::gfp, transcriptional fusion of basic helix–loop–helix promoter and GFP integrated into the genome.(A) Transgenic worms with the integrated array (TIF)Click here for additional data file.S4 Figama-1 determined by qPCR for autophagy and lysosomal machinery genes (as named) in L1 stage wild-type and hlh-30(lf) animals in the fed state (fed), after starvation for 33 hours (starved), and after starvation for 33 hours followed by refeeding on E. coli OP50 for 15 hours (OP50). N = 3 biological replicates/group. Bars indicate mean ± SEM. *P < 0.05 by post hoc test after one-way ANOVA. Raw data are located in ama-1, amanitin-binding subunit of RNA polymerase II; au, arbitrary unit; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L1, first larval stage; qPCR, quantitative PCR; SEM, standard error of the mean.(A–N) mRNA abundance in au with values normalized to the control gene (TIF)Click here for additional data file.S5 Fighlh-30(lf) L1 worms that were fed or starved for 33 hours and subjected to RNAseq analysis. N = 2 biological replicates/group. (B) Unsupervised hierarchical clustering of significantly altered transcripts from A. Lists of genes identified under groups labeled A–E are presented in hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; KEGG, Kyoto Encyclopedia of Genes and Genomes; L1, first larval stage; RNAseq, RNA sequencing.(A) Venn diagram depicting significantly regulated Click here for additional data file.S6 Fighlh-30 mRNA abundance determined by qPCR was analyzed in the wild type; hlh-30(lf); hlh-30(lf);amEx272, a strain that overexpresses HLH-30::RFP (hlh-30(oe)); and hlh-30(lf);amEx291, a strain that overexpresses HLH-30::RFP with a mutation of the nuclear localization signal: hlh-30(mNLS)(oe). All worms were analyzed at the L1 stage in the fed state. N = 3 biological replicates/group. Bars indicate mean ± SEM. *P < 0.05 by post hoc test after one-way ANOVA. (B) hlh-30(oe) and hlh-30(mNLS)(oe) animals were analyzed at the L1 stage in the fed state or after 33 hours of starvation. Representative images display DIC (left) and fluorescence to reveal HLH-30::RFP (center), and 2.5× magnified insets  to reveal starvation-induced nuclear localization of HLH-30::RFP (arrows). Scale bar is 20 μm. (C) Wild-type, hlh-30(lf), hlh-30(oe), and hlh-30(mNLS)(oe) worms were analyzed after 33 hours of starvation for “Alive after Starvation” (C) as described in the N = 3 biological replicates/group of approximately 50 worms. Bars indicate mean ± SEM. *P < 0.05 by post hoc test after one-way ANOVA. Data for hlh-30(oe) and hlh-30(mNLS)(oe) are the analysis of one transgenic strain depicted in A. Eleven other independently derived hlh-30(oe) strains and one other independently derived hlh-30(mNLS)(oe) strain displayed similar results in these assays. Raw data are located in hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(mNLS)(oe), overexpressed HLH-30 with a mutant nuclear localization signal; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; hlh-30(oe), overexpressed HLH-30; qPCR, quantitative PCR; RPF, red fluorescent protein; SEM, standard error of the mean.(A) (TIF)Click here for additional data file.S7 Fighlh-30(lf) worms were starved for the indicated duration of time (on the y-axis), refed with E. coli OP50 or CeMM, and analyzed for “Alive after Refeeding” as described in the legend for N = 3 biological replicates with approximately 50 worms/time. Data are shown as mean ± SEM. *P < 0.05 versus hlh-30(lf) CeMM by post hoc test after two-way ANOVA. Raw data are located in C. elegans maintenance medium; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; SEM, standard error of the mean.(TIF)Click here for additional data file.S8 Fighlh-30(lf) L1 stage worms subjected to 33 hours of starvation and analyzed immediately (St.) or analyzed after 15 hours in complete CeMM. N = 6 biological replicates/group with approximately 150,000 animals per replicate. (B) Random forest analysis of metabolites that accurately segregated hlh-30(lf) worms into starved or refed groups. See C. elegans maintenance medium; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L1, first larval stage.(A) Hierarchical cluster analysis of metabolites measured in wild-type and (TIF)Click here for additional data file.S9 Fighlh-30(lf) worms were analyzed after 33 hours of starvation and 15 hours of exposure to complete or modified formulations of CeMM for “Alive after Refeeding” as described in the legend for N = 3 biological replicates/group for panels A and B, and N = 1 replicate for panel C with approximately 50 worms/condition. *P < 0.05 by post hoc test after two-way ANOVA for A and B. Raw data are located in C. elegans maintenance medium; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; SEM, standard error of the mean.(A–C) Wild-type and (TIF)Click here for additional data file.S10 Fighlh-30(lf) and wild-type worms subjected to 33 hours of starvation in M9 medium (labeled as St.) followed by 15 hours of incubation in CeMM, depicted in heat maps and graphs. Data on some metabolites presented in N = 6 biological replicates/group. *P < 0.05 by post hoc test after two-way ANOVA. y-axis values are scaled intensity for each metabolite. See C. elegans maintenance medium; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; SEM, standard error of the mean.Depiction of significantly altered lipid metabolism pathways, along with significantly regulated candidate metabolites in (TIF)Click here for additional data file.S11 Fighlh-30(lf) and wild-type worms subjected to 33 hours of starvation in M9 medium (labeled as St.) followed by 15 hours of incubation in CeMM, depicted in heat maps and graphs. Data on some metabolites presented in N = 6 biological replicates/group. *P < 0.05 by post hoc test after two-way ANOVA. y-axis values are scaled intensity for each metabolite. See C. elegans maintenance medium; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; SEM, standard error of the mean; TCA, tricarboxylic acid cycle.Depiction of various metabolic pathways that glucose is channeled into, along with significantly regulated metabolites in (TIF)Click here for additional data file.S12 Fighlh-30(lf) worms were subjected to metabolomics analyses after 33 hours of starvation and 15 hours of exposure to CeMM or E. coli OP50 to determine levels of α-ketoglutarate (A), fumarate (B), malate (C), citrate (D), glucose-6-phosphate (E), glucose (F), α-FFA (G), γ-FFA (H), LPC (18:3)-1 (I), and LPC (18:3)-2 (J). Data are presented as mean ± SEM. N = 4 biological replicates/group. *P < 0.05 by t test. Raw data are located in C. elegans maintenance medium; FFA, linolenate; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; LPC, lyso phosphatidyl choline; SEM, standard error of the mean; TCA, tricarboxylic acid cycle; α-FFA, α-linolenate; γ-FFA, γ-linolenate.(A–J) (TIF)Click here for additional data file.S13 Fighlh-30(lf) and wild-type worms subjected to 33 hours of starvation in M9 medium (labeled as St.) followed by 15 hours of incubation in CeMM, depicted in heat maps and graphs. Bars indicate mean ± SEM. N = 6 biological replicates/group. *P < 0.05 by post hoc test after two-way ANOVA. y-axis values are scaled intensity for each metabolite. See C. elegans maintenance medium; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; SEM, standard error of the mean.Depiction of significantly altered endocannabinoid and protein metabolism pathways, along with significantly regulated candidate metabolites in (TIF)Click here for additional data file.S14 Fighlh-30(lf) and wild-type worms subjected to 33 hours of starvation in M9 medium (labeled as St.) followed by 15 hours of incubation in CeMM, depicted in heat maps and graphs. N = 6 biological replicates/group. *P < 0.05 by post hoc test after two-way ANOVA. Bars indicate mean ± SEM. y-axis values are scaled intensity for each metabolite. See C. elegans maintenance medium; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; SEM, standard error of the mean.Depiction of significantly altered nucleic acid breakdown and salvage pathways, along with significantly regulated candidate metabolites in (TIF)Click here for additional data file.S15 Fighlh-30(lf) and wild-type worms subjected to 33 hours of starvation or with 15 hours of refeeding with E. coli OP50. Fed worms are included as controls. Values were normalized to the control gene ama-1. Bars indicate mean ± SEM. N = 3 biological replicates/group. *P < 0.05 by post hoc test after two-way ANOVA.  Wild-type, lipl-1(lf), lipl-3(lf), and lipl-4(lf) worms were analyzed after variable periods of starvation for Alive after Starvation (E) and Alive after Refeeding (F) as described in the N = 3 biological replicates of approximately 25 worms/time point. *P < 0.05 versus wild type by post hoc test after two-way ANOVA. Raw data are located in ama-1, amanitin-binding subunit of RNA polymerase II; au, arbitrary unit; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; lipl-1(lf), loss-of-function mutation in lysosomal lipase 1; SEM, standard error of the mean.(A–D) mRNA abundance determined by qPCR in au for indicated genes in (TIF)Click here for additional data file.S16 Figlipl-2(lf), and hlh-30(oe);lipl-2(lf) worms were analyzed after variable periods of starvation for Alive after Refeeding as described in the legend for N = 3 biological replicates of approximately 50 worms/time point. *P < 0.05 for hlh-30(oe);lipl-2(lf) versus wild type and #P < 0.05 for hlh-30(oe);lipl-2(lf) versus lipl-2(lf) by post hoc test after two-way ANOVA. Raw data are located in hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; hlh-30(oe), overexpressed HLH-30; lipl-2(lf), loss-of-function mutation in lysosomal lipase 2; SEM, standard error of the mean.Wild-type, (TIF)Click here for additional data file.S17 Fighlh-30(lf) worms analyzed in the fed state, after 33 hours of starvation, and after 15 hours of exposure to complete CeMM. Representative images are from one experiment; we performed two biological replicates/group. Black arrowheads point to the intestinal brush border, large white arrows point to mitochondria, and thin white arrows point to autophagic structures (autophagosomes and autolysosomes). Representative of N = 2 independent trials per group. Scale bars = 500 nm. CeMM, C. elegans maintenance medium; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L1, first larval stage.Transmission electron microscopic analyses of L1 stage wild-type and (TIF)Click here for additional data file.S18 Fighlh-30(lf) worms were analyzed after 33 hours of starvation and 0 or 15 hours of exposure to complete CeMM for “Alive after Refeeding” as described in the legend for bec-1 mRNA or L4440 as control and analyzed at the L4 stage. Bars indicate mean ± SEM. N = 3 biological replicates/group. *P < 0.05 by post hoc test after two-way ANOVA versus hlh-30(lf) with 0 hours of CeMM treatment (labeled OP50) or as indicated by horizontal line. (B) bec-1 mRNA abundance in au with values normalized to the control gene ama-1 determined by qPCR for fed wild-type and hlh-30(lf) worms. Bars indicate mean ± SEM. P = NS indicates that no statistically significant differences were noted by t test. (C) bec-1 mRNA abundance was analyzed in wild-type and hlh-30(lf) L4 stage worms 72 hours after starvation. The control RNAi (L4440) value was set equal to 1.0 for both genotypes, and bec-1 values were normalized to the expression of ama-1. Bars indicate mean ± SEM. N = 3 biological replicates/group. *P < 0.05 by post hoc test after one-way ANOVA. Raw data are located in ama-1, amanitin-binding subunit of RNA polymerase II; au, arbitrary unit; bec-1, C. elegans ortholog of human BECN1; CeMM, C. elegans maintenance medium; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L1, first larval stage; L4, fourth larval stage; NS, not significant; qPCR, quantitative PCR; RNAi, RNA interference; SEM, standard error of the mean.(A) Wild-type and (TIF)Click here for additional data file.S19 Figama-1 determined by qPCR for the indicated genes. hlh-30(lf) L1 worms were starved for 33 hours and then transferred to NGM dishes with E. coli OP50 for 15 hours or fed CeMM minimal supplemented with glucose and linoleic acid , loss-of-function tm1978 mutation hlh-30; L1, first larval stage; NGM, nematode growth medium; qPCR, quantitative PCR; SEM, standard error of the mean; vha, vacuolar H+-ATPase.(A–L) mRNA abundance in au with values normalized to the control gene (TIF)Click here for additional data file.S20 Figama-1 determined by qPCR for the indicated genes. Wild-type and hlh-30(lf) L1 worms were starved for 33 hours and transferred to NGM dishes with E. coli OP50 for 15 hours or fed CeMM for 15 hours. Bars indicate mean ± SEM. N = 3 biological replicates/group. *P < 0.05 by post hoc test after two-way ANOVA. Raw data are located in ama-1, amanitin-binding subunit of RNA polymerase II; au, arbitrary unit; CeMM, C. elegans maintenance medium; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L1, first larval stage; NGM, nematode growth medium; qPCR, quantitative PCR; SEM, standard error of the mean.(A–S) mRNA abundance in au with values normalized to the control gene (TIF)Click here for additional data file.S21 Fignhr-31 overexpressing hlh-30(lf) worms (nhr-31(oe);hlh-30(lf)) have approximately 17-fold higher levels of nhr-31 transcripts compared to the wild type. Animals were subjected to 33 hours of starvation followed by refeeding with CeMM minimal , loss-of-function tm1978 mutation hlh-30; nhr-31, nuclear hormone receptor 31; nhr-31(oe), overexpressed nhr-31; SEM, standard error of the mean.imal see  suppleme(TIF)Click here for additional data file.S22 Figama-1 for genes (as named) in L1 stage wild-type and hlh-30(lf) animals in the fed state (fed), after starvation for 33 hours (starved), and after starvation for 33 hours followed by refeeding on E. coli OP50 (OP50) or CeMM (CeMM) for 15 hours. Bars indicate mean ± SEM. N = 6 biological replicates/group. *P < 0.05 by post hoc test after one-way ANOVA. Raw data are located in ama-1, amanitin-binding subunit of RNA polymerase II; au, arbitrary unit; CeMM, C. elegans maintenance medium; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L1, first larval stage; qPCR, quantitative PCR; SEM, standard error of the mean; TOR, target of rapamycin.(A–H) mRNA abundance determined by qPCR in au with values normalized to the control gene (TIF)Click here for additional data file.S23 Figragc-1 mRNA abundance in au with values normalized to the control gene ama-1 was analyzed in fed L4 stage worms in the following groups: wild-type, hlh-30(lf), and hlh-30(lf);ragc-1(oe) (hlh-30(lf);amEx324 worms that overexpress ragc-1 from an extrachromosomal array utilizing its endogenous promoter). Bars indicate mean ± SEM. N = 3–6 biological replicates/group. *P < 0.05 by post hoc test after one-way ANOVA. ama-1, amanitin-binding subunit of RNA polymerase II; au, arbitrary unit; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L4, fourth larval stage; ragc-1, the ortholog for mammalian RagC/D GTPases; ragc-1(oe), overexpressed ragc-1; SEM, standard error of the mean.(TIF)Click here for additional data file.S24 Fighlh-30(lf) L1 worms subjected to 33 hours of starvation, 33 hours of starvation followed by 3 or 15 hours of CeMM exposure , loss-of-function tm1978 mutation hlh-30; L1, first larval stage; RNAseq, RNA sequencing. Spearman correlation matrix (A) and multidimensional scaling plot (B) depicting various RNA samples from wild-type and re as in , or stud(TIF)Click here for additional data file.S1 MovieL1, first larval stage.(MP4)Click here for additional data file.S2 MovieL1, first larval stage.(MP4)Click here for additional data file.S3 Moviehlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L1, first larval stage.(MP4)Click here for additional data file.S4 Moviehlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L1, first larval stage.(MP4)Click here for additional data file.S5 MovieC. elegans maintenance medium; L1, first larval stage.CeMM, (MP4)Click here for additional data file.S6 MovieC. elegans maintenance medium; hlh-30, basic helix–loop–helix transcription factor 30; hlh-30(lf), loss-of-function tm1978 mutation hlh-30; L1, first larval stage.All movies were obtained at identical magnification. CeMM, (MP4)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 TableqPCR, quantitative PCR.(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.


Correction to: Translational Psychiatry10.1038/s41398-018-0331-3 Published online 15 Feb 2019.Author forgot to attach a supplementary doc file which includes the supplementary methods and supplementary figure legends.


The data provide heart rates, left ventricular contractile functions, coronary flow, phosphocreatine concentrations, and adenosine 5’-triphosphate (ATP) concentrations throughout hypoxia in mdx mouse hearts. This data article also provides left ventricular contractile functions after low flow ischemia with and without glucose, glycogen levels before ischemia or hypoxia, glucose uptake rates during low flow ischemia and insulin stimulation, and insulin-stimulated phospho-Akt protein levels, a protein in insulin signaling, in mdx mouse hearts.The data contain body weights, plasma free fatty acids concentrations and cardiac uncoupling protein-3 protein levels for wild-type and  Mdx mouse hearts have decreased left ventricular contractile functions and faster ATP loss rates during hypoxia compared with wild-types. Mdx mouse hearts also have higher left ventricular end-diastolic pressures and lower glucose uptake rates during low flow ischemia than wild-types. Insulin-stimulated phospho-Akt protein levels are similar in all mouse hearts while mdx mouse hearts have lower glucose uptake rates in response to insulin than wild-types.2n = 61) and age-matched mdx (n = 62) mice at 8 months. All the procedures were approved by the Animal Ethics Review Committees, University of Oxford, and by the Home Office, United Kingdom. Plasma free fatty acids concentrations were measured using NEFA kit . Western blot quantified protein levels with anti-uncoupling protein-3, anti-phospho-Akt  and anti-GAPDH (Abcam). Isolated whole Langendorff-perfused mouse hearts were equilibrated with fatty acids free Krebs-Henseleit buffer gassed with 95% O2 – 5% CO2 (PaO2 = ~ 90 mmHg), and subjected to hypoxia with buffer gassed with 95% N2 – 5% CO2 (PaO2 = ~ 45 mmHg) and reoxygenation. Left ventricular pressures and coronary flow were recorded. Protocols were shown in 3H] glucose during low flow ischemia (0.5 ml min−1 gww−1) and in response to insulin t-test.Experiments were carried out using male wild-type (C57BL10) (


E3 ubiquitin ligases are the most expanded components of the ubiquitin proteasome system (UPS). They mediate the recognition of substrates and later transfer the ubiquitin (Ub) of the system. Really Interesting New Gene (RING) finger proteins characterized by the RING domain, which contains 40–60 residues, are thought to be E3 ubiquitin ligase. RING-finger proteins play significant roles in plant growth, stress resistance, and signal transduction. In this study, we mainly describe the structural characteristics, classifications, and subcellular localizations of RING-finger proteins, as well the physiological processes of RING-finger proteins in plant growth and development. We also summarize the functions of plant RING-finger proteins in plant stress resistance. Finally, further research on plant RING-finger proteins is suggested, thereby establishing a strong foundation for the future study of plant RING-finger proteins. Ub targets cellular proteins via three distinct enzymes: the Ub-activating enzyme E1, the Ub-conjugating enzyme, enzyme E2, and Ub-ligase enzyme E3 in UPS (the ubiquitin proteasome system) ,5,6. Hows-X2-Cys  9]. It . It 2-Cyonserved . These c-Cys-X4-4-Cys-X2-CArabidopsis thaliana proteome. These members are mainly divided into seven subtypes according to structural differences: RING-H2(241), RING-HC(186), RING-v(25), RING-C2(10), RING-D(10), RING-S/T(4), and RING-G(1). RNIG-H2 and RING-HC are two canonical subclasses of RING-finger domains, accounting for 50% and 39% of RING-finger domains, respectively [Arabidopsis thaliana due to the improved annotation of the A. thaliana genome. These are also divided into seven subtypes: RING-H2(258), RING-HC(191), RING-v(26), RING-C2(16), RING-D(7), RING- S/T(3), and RING-G(1) [in vitro, even the RING-finger proteins with substituted zinc binding acid residues or with slightly altered spacing [Oryza sativa) proteome and are divided into four types: RNIG-H2 (281), RING-HC (119), RING-v (23), and RING-C2 (2) [Brassica oleracea proteome. These domains can be further classified into eight RING types: RING-H2 (355), RING-HCa (215), RING-HCa (47),RING-v (49), RING-C2 (86), RING-D (11), RING-S/T (4), and RING-G (1) [Malus domestica) proteome which were further divided into NINE RING types: RING-H2 (367), RING-HC (208), RING-v (35), RING-S/T (11),RING-C2(10), RING-D (1), RING-G (2), RING-mHC (44), and RING-mH2 (10) [Solanum lycopersicum genome, which are further divided into 7 RING types: RING-H2 (248), RING-HCa (142), RING-HCb (21), RING-v (40), RING-C2 (20), RING-S/T (2), and RING-G (1) [Ostreococcus tauri, only 65 RING domains were identified from 65 predicted proteins and further divided into 8 RING types: RING-H2 (25), RING-HC (28), RING-v (7), RING-C2 (1), C3HCHC2 (1),C2HC5 (1), C3GC3S (1), and C2SHC4 (1) [With more extensive research, many RING-finger proteins have been identified in plants. There are 477 RING domains detected from 469 predicted proteins in the whole ING-G(1) . Accordi spacing ,14. A toG-C2 (2) . Eighty , RING-C2, RING-D(G-C2 (2) . At the , RING-HC6, RING-v, RING-HC1, RING-v RING-HC 8, RING-vRING-HCa 5, RING-HSHC4 (1) .Arabidopsis RFI2 is located in the nucleus [Most of the plant RING-finger proteins are found in the nucleus, cytoplasm, and cell membrane. For example,  nucleus , and ric nucleus . Meanwhi nucleus   is a negative regulator of photomorphogenesis. It directly targets the bzip transcription factor hy 5 (HY5), a positive regulator of photomorphogenesis, for degradation via the proteasome pathway in the dark [Photomorphogenesis is critical to plant flowering. the dark ,38. COP1the dark .rfi-2 promotes the expression of CONSTANS (CO), a central activator of photoperiodic flowering, and FLOWERING LOCUS T (FT) under long-day conditions, leading to an early flower phenotype. Moreover, under red and far-red light, the phenotype exhibits hypocotyl elongation [Arabidopsis, a RING-finger protein with a high expression of osmotically responsive gene 1 (HOS1) (an E3 ubiquitin ligase), under intermittent cold stress treatment, leads to the degradation of CO via the ubiquitination pathway. A decrease of the CO protein leads to a delay in flowering. It was also found that HOS1 controls the transcriptional activity of phytochrome interacting factor 4 (PIF4) to participate in phyB-mediated signal light morphogenesis [The photoperiod phenomenon is an important factor for affecting flower formation, which is the core process of plant growth and development. Red and far-red insensitive 2 (RFI2) is a RING-finger protein that participates in the photoperiod flowering pathway. The ongation . Howeverogenesis . Anotherogenesis .Arabidopsis thaliana, Medicago sativa RING-H2 zinc finger protein (MsRH2-1) in M. sativa and Capsicum annuum ring zinc finger protein 1 (CaRZFP1) in pepper are involved in plant root development. Overexpression of the SINAT5 gene in Arabidopsis showed fewer lateral roots, while the lateral roots of the sinat5 mutant plants increased. These lateral root phenotypes correlate with SINAT5, which can degrade the NAC domain containing protein 1 (NAC1) via the ubiquitin pathway [MsRH2-1 gene is closely related to the development of lateral roots and nodules, with the highest level of transcription in roots and nodules of M. sativa. The MsRH2-1 overexpression line shows inhibited development of lateral roots and suggests that MsRH2-1 may function as an E3 ligase and perform a function of the E3 ligase for substrate-specific degradation via the ubiquitin-proteasome system involved in auxin signaling [CaRZFP1 gene in tobacco revealed a phenotype with a larger primary root and more lateral roots in transgenic lines. CaRZFP1 was mainly related to the up-regulation of Root-hair-specific Cell Wall Proline-rich Protein (PRP) root hairs and lateral roots in overexpressing lines [Lotus japonicus is a positive regulator of symbiotic nodulation, possibly through interaction with LHK1 (Lotus Histidine Kinase 1), which is essential for nodule formation [HUB1 gene in Arabidopsis exhibited slower growth of primary roots. HUB1 as a RING E3 ligase regulates the growth rate of plant roots [RING-finger proteins are also involved in the growth and development of roots. The RING-finger drosophila protein sina (SINAT5), histone monoubiquitination1 (HUB1) in  pathway . The MsRignaling . Overexpng lines . An lhk1ormation . HoweverMsRH2-1 gene in alfalfa can degenerate leaves and inhibit leaf vein formation. However, overexpression of this gene in Arabidopsis leads to the discovery of rosettes [CaRZFP1 gene in tobacco leads to fast growth, size, an increased number of leaves, and heavier fresh vegetation [BRH1) gene in Arabidopsis thaliana is overexpressed, the rosette leaves of the transgenic lines were extremely curled, suggesting that it may be involved in the Brassinosteroid (BR) signaling pathway to regulate the shape of the leaves [Nicotiana benthamiana zinc finger (NbZFP1) hampered fruit development. Compared to the WT (wild type), overexpression of NbZFP1 displayed a short internode length and a sturdy stem phenotype [The RING-finger protein is also involved in leaf and height development. Overexpression of the rosettes . Overexpgetation . When the leaves . Howevere leaves . Silencihenotype .RING-finger proteins are also involved in fruit development. Flying saucer1 (FLY1) and big brother (BB) are used as E3 ligases for floral organs of the seed pectins and control the degree of methyl esterification. However, the regulation mechanisms are not clear for these two proteins ,41. It iStress is an important limiting factor affecting plant growth and crop production. Over the long process of evolution, plants have produced many different biotic/abiotic stress responses and regulation methods. In recent years, many studies have shown that RING finger proteins are involved in responding to biotic and abiotic stress in plants .XERICO in Arabidopsis, and its homologous genes ZmXerico1/2 in Zea mays, are overexpressed in Arabidopsis and improve resistance to drought. However, XERICO raises the biosynthesis of ABA by degrading the ASK1-interacting F-box protein (AtTLP9) in the proteasome system, while ZmXerico1/2 makes the ABA more stable via the ubiquitin of ABA 8′-hydroxylases to improve drought resistance [osdsg1 mutant line and Delayed Seed Germination 1 (OsDSG1)-RNAi plants, the expression level of ABA signaling-related genes was significantly increased and lead to greater resistance to drought than the wild-type. It was estimated that OsDSG1 degrades aba insensitive 3 (OsAIB3) via the 26S proteasome system and negatively regulates plant drought resistance by participating in the ABA-pathway [Capsicum annuum ADIP1 interacting ring finger protein 1 (CaAIRF1) degrades Capsicum annuum Type 2C Protein Phosphatase (CaADP1) through the ubiquitination pathway by changing the sensitivity to ABA to improve drought resistance [Capsicum annuum drought sensitive RING finger protein 1(CaDSR1) exhibited E3 ligase activity and promoted CaDILZ1 expression through the 26S proteasome pathway to alter ABA content in the modulation of drought tolerance [Capsicum annuum ABA sensitive RING finger E3 ligase 1 (CaASRF1) positively modulates ABA signaling via modulation of CaAIBZ1’s stability to drought stress [Arabidopsis ABA-insensitive RING protein 1 (AtAIRP1), AtAIRP4, Nicotiana tabacum RING-H2 Finger Gene 1 (NtRHF1), OsBIRF1, TaRZF70, and AdZFP1 [Arabidopsis thaliana, acts as a negative regulator in drought-responsive gene expression by targeting dehydration-responsive element binding protein 2 (ADREB2A) to facilitate 26S proteasome proteolysis [Arabidopsis thaliana [Oryza sativa RING domain-containing protein OsRDCP1, as a RING E3 ligase, may be involved in the transportation or degradation of a negative transcription factor or factors that inhibit(s) the expression of water stress-induced genes [Oryza sativa drought-induced SINA protein OsDIS1, via the 26S proteasome-dependent pathway, degrades Oryza sativa NIMA-related kinase 6 (OsNek6), plaing a negative role in drought stress tolerance. The orthologue protein in wheat (Triticum aestivum L.) TaDIS1 may perform a negatively function in drought stress by regulating the stress response-related genes [Drought is a major abiotic stress factor affecting plant survival. It is necessary to analyze drought resistance genes and analyze their drought resistance mechanisms. Recently, it has been reported that several RING-containing proteins function as E3 ligases in response to the Abscisic Acid (ABA) dependent defense mechanism against drought stress. A study found that sistance ,64. In t-pathway . The Capsistance . The Capolerance . Capsicut stress . Many RId AdZFP1 ,68,69,76teolysis . NUCLEARthaliana . RING methaliana . The Oryed genes . The Oryed genes ,67.Arabidopsis thaliana and its homologous protein ZmRFP1 are found to participate in the regulation of drought’s stress response. However, SDIR1 also acts as an E3 ligase to ubiquitinate the modification of SDAIR-interacting protein 1 (SDIR1P1) to regulate the expression of transcription factor ABA-INSENSITIVE5 (ABI5), a key ABA-pathway gene, thereby participating in the salt response process of Arabidopsis thaliana [Oryza sativa salt, ABA, and drought stress-induced RING finger protein 1 (OsSADR1) act as E3 ligases and function negatively in drought and salt stress [Arabidopsis and Oryza sativa salt-induced RING finger protein 1 (OsSIRP1), as E3 ligases, participate in the response of salt stress. STRF1 mainly regulates the expression of membrane transport-related proteins. OsSIRP1 is a negative regulator of salt tolerance, and its target protein needs to be further studied [O. sativa chymotrypsin protease inhibitor 2 (OsCPI2), but anther rice RING H2-type E3 ligase, OsSIRH2-14 (previously named OsRFPH2-14), plays a positive role in salinity tolerance by regulating salt-related proteins, including an HKT-type Na+ transporter  [M. esculenta RZF (MeRZF) and SpRing were found to respond to salt stress in cassava and wild tomato, respectively. However, the mechanism by which they play a role remains to be further studied [Some RING-finger proteins are also involved in salt stress. The salt and drought-induced ring finger 1 (SDIR1) in thaliana . Oryza st stress . CaRFP1 t stress . Salt to studied ,60. The HKT2; 1) ,59. The  studied ,77.Arabidopsis thaliana Al tolerance RING finger 1 (AtATRF1), acting as an E3 ligase, mediates the aluminum tolerance of Arabidopsis thaliana. Studies have shown that plants overexpressing AtATRF1 enhance tolerance to Al, while Al can induce the expression of AtATRF1. AtATRF1 is located in the nucleus and may interact and ubiquitinate Ataxia telangiectasia-mutated and RAD3-like Protein (AtATR), a transcriptional regulator, which plays an important role in plant growth and development [Aluminum (Al) toxicity is a major limiting factor in the production of acid soil crops. In recent studies, a series of E3 ubiquitin ligases have been discovered to regulate plant Al tolerance or resistance. elopment . Soybeanelopment .Arabidopsis thaliana acts as a negative regulator of low-temperature response gene transcription and can function as an E3 ubiquitin ligase. Under low temperature conditions, HOS1 can degrade an inducer of cbf expression 1 (ICE1) through the 26S protease pathway, while overexpression of HOS1 makes plants sensitive to low temperatures [Oryza sativa cold-inducible (OsCOIN) could respond to low-temperature stress, relying on the ABA pathway [OsLti6b and OsP5CS, could be induced in the overexpression line of OsCOIN, making it useful for enhancing tolerance to low temperatures [BrRZFP gene in Brassica rapa can be induced by low temperatures; the ABA, drought, and salt stress resistance of tobacco plants heterologously expressing this gene are also enhanced [Oryza sativa heat and cold induced 1(OsHCI1) and Oryza sativa heat-induced RING finger protein 1(OsHIRP1) both act as E3 ligases to positively regulate heat stress responses [OsRZFP34 gene and HEAT TOLERANCE AT SEEDLING STAGE (OsHTAS) gene can participate in the ABA pathway, change the stomatal switch state in leaves, and improve high-temperature tolerance. However, only OsHTAS was proven to be an E3 ligase [Low-temperature or high-temperature stress affect the normal life metabolism of plants, thereby affecting their growth and development. Recently, there have been many reports about RING-finger proteins in response to low-temperature stress. The RING-finger protein HOS1 in eratures ,78. Oryz pathway . Furthereratures . The expenhanced . Howeveresponses ,29. The 3 ligase ,57.Arabidopsis thaliana acts as an E3 ligase ubiquitinating transcription factor Myb domain protein 30 (MYB30) and degrades transgenic MYB30, thereby reducing the expression of disease-resistant genes and reducing the plant’s immune response [Arabidopsis toxicos en levadura 9 (ATL9) is also involved as an E3 ligase in regulating plant resistance to viable nutrient pathogens. When a pathogen infects a plant, it can induce the expression of ATL9 and ATL9 to resist the inhibitory protein of the pathogen by ubiquitination hydrolysis, thereby promoting an immune response [CaRFP1 gene was overexpressed in Arabidopsis thaliana, the transgenic plants became more sensitive to tomato bacterial spot disease. This may be due to the fact that the protein acts as an E3 ligase, degrading the expression levels of disease-related genes such as (synthesis of pathogenesis-related) PR-2 and PR-5 [Vitis pseudoreticulata by degrading the transcription factor VpWRKY11 through the ubiquitin proteasomal system, which enhances the ability of East China Grapes to resist pathogens [Vitis pseudoreticulata VpRH2, as an E3 ligase, improves resistance to powdery mildew by interacting with VpGRP2A [OsBIRF1 gene and overexpression of the NbZFP1 gene in tobacco could enhance resistance to the tobacco mosaic virus, and these two RING-finger proteins may enhance disease resistance by regulating the expression of PR genes [Biotic stress is general term for various biological factors that are unfavorable to survival and development, including pests, fungi, bacteria, and viruses. Recent studies have shown that RING-finger proteins are involved in biotic stress responses in many species. The RING-finger protein MYB30-interacting E3 ligase 1 (MIEL1) in response . Arabidoresponse . When thand PR-5 . The Eryathogens . Another VpGRP2A . HeterolPR genes .The growth and development of plants and their ability to adapt to a variety of stresses are mainly realized by changing their protein expression and metabolic pathways. It is of great significance to study the expression and function of these proteins in order to improve plant growth and tolerance to stress ,80,81. P


Caenorhabditis elegans through a cross-talk between the DELTA/NOTCH and hypoxia signaling pathways. These mechanisms may permit the animals to adapt their development to changes in the environment.Atmospheric oxygen levels modulate the RAS/MAPK signaling pathway in the nematode  Caenorhabditis elegans as a model to investigate how variations in oxygen concentrations affect cell fate specification during development. Here, we show that several processes controlled by the conserved RTK/RAS/MAPK pathway are sensitive to changes in the atmospheric oxygen concentration. In the vulval precursor cells (VPCs), the hypoxia-inducible factor HIF-1 activates the expression of the nuclear hormone receptor NHR-57 to counteract RAS/MAPK–induced differentiation. Furthermore, cross-talk between the NOTCH and hypoxia-response pathways modulates the capability of the VPCs to respond to RAS/MAPK signaling. Lateral NOTCH signaling positively regulates the prolyl hydroxylase EGL-9, which promotes HIF-1 degradation in uncommitted VPCs and permits RAS/MAPK–induced differentiation. By inducing DELTA family NOTCH ligands, RAS/MAPK signaling creates a positive feedback loop that represses HIF-1 and NHR-57 expression in the proximal VPCs and keeps them capable of differentiating. This regulatory network formed by the NOTCH, hypoxia, and RAS/MAPK pathways may allow the animals to adapt developmental processes to variations in oxygen concentration.Animals need to adjust many cellular functions to oxygen availability to adapt to changing environmental conditions. We have used the nematode  Caenorhabditis elegans, RAS/MAPK signaling is involved in several developmental processes, such as the specification of the excretory duct cell precursor, the differentiation and maturation of meiotic germ cells or the development of the hermaphrodite vulva  ras gain-of-function allele , and pfd-3, a prefoldin orthologous to the human VHL-binding protein 1. Both genes have independently been identified in a screen for suppressors of the egl-9 prolyl hydroxylase egg-laying–defective phenotype, suggesting that F44F1.3 and pfd-3 interact with the core hypoxia-response pathway  mutants (C. elegans wild isolates (zh121gf) and CB4856 Hawaii (zh122gf) let-60 ras locus (let-60(zh121gf) and CB4856 let-60(zh122gf) animals raised under different oxygen concentrations. Under normoxia (21% O2), CB4856 animals carrying the G13E mutation exhibited a higher VI than N2 animals carrying the same mutation, as had been observed earlier by introgressing the canonical G13E allele let-60(n1046gf) into the CB4856 background  irrespective of the genetic background.The two polymorphic genes  mutants . We, thuisolates  originatas locus . We thenckground  (Milloz ckground . VI was round (2 . However2) on different RAS/MAPK pathway mutants in the N2 Bristol background. We included the Wnt pathway–mutant bar-1(ga80) and the NOTCH alleles lin-12(n137gf) and lin-12(n137n720lf) in this analysis. Even though hypoxia had no effect on the VI in the N2 Bristol wild-type strain , hypoxia reduced the VI in animals carrying rf mutations in let-23 egfr, let-60 ras, or lin-45 raf or in animals expressing an activated MAPK (MPK-1) from a transgene (mpk-1(gaIs37gf)) (2.) By contrast, the increased VI in lin-1(lf) mutants, which encodes an Ets family transcription factor that represses VI downstream of MPK-1 ) .  allele causes an accelerated exit of meiotic germ cells from the pachytene stage at the restrictive temperature and results in the stacking of smaller oocytes in the proximal gonad arms (let-60(n1046gf) mutants frequently contains two duct cells expressing the lin-48::gfp marker (let-60(gf) phenotypes in both tissues . For this purpose, we calculated the change in VI (∆VI) between the indicated double mutants and let-60(n1046gf) single mutant control siblings obtained from each cross (let-60(n1046gf) mutants was reduced when combined with the egl-9(sa307lf) (vhl-1(ok161lf) (hif-1(ia04) allele  that likely represents a null allele (let-60(n1046gf); hif-1(zh111lf) double mutants exhibited an even stronger increase in VI (let-60(n1046gf); egl-9(sa307lf) hif-1(ia04lf) triple mutants exhibited the same increase in VI as let-60(n1046gf); hif-1(ia04lf) double mutants, indicating that hif-1 counteracts RAS/MAPK signaling downstream of egl-9 prolyl hydroxylase (egl-9(sa307lf) also reduced the VI of mutants in other RAS/MAPK pathway components, such as the guanine nucleotide exchange factor sos-1 or the raf homolog lin-45   or vhl-1ok161lf)  alleles,) allele . Becausel allele . let-60(se in VI  and S1B.roxylase . egl-9(sg lin-45  (Sternbeg lin-45 .hif-1(lf) mutations under normoxia was somewhat surprising. It, thus, appears that low residual levels of HIF-1 present under normoxia are sufficient to oppose RAS/MAPK signaling. We, therefore, investigated the effect of wild-type and hydroxylase deficient egl-9::gfp transgenes on VI (egl-9::gfp transgene efficiently rescued the reduced VI of let-60(n1046gf); egl-9(sa307lf) and further increased the VI of let-60(n1046gf) single mutants  background caused a further increase in the VI. Thus, low levels of HIF-1 that persist under normoxia are sufficient to repress VI.Because HIF-1 is efficiently degraded under normoxia, the increased VI caused by es on VI . A wild- mutants . By cont pathway . Moreoveegl-9(sa307lf),hif-1(ia04lf), or hif-1(zh111lf) single mutants raised at normoxia or hypoxia exhibited normal vulval development . Because VI is very robust even under varying environmental conditions (egl-9(sa307) and no increase in hif-1(ia04lf) or hif-1(zh111lf) animals relative to the wild-type controls  Thus, we did not detect substantial changes in MPK-1 activity within the dynamic range of the ERK-nKTR biosensor. We conclude that the hypoxia-response pathway attenuates the response of the VPCs to MAPK signaling, rather than directly inhibiting RAS/MAPK pathway activity.Even though mutations in the hypoxia pathway mutants significantly affected VI in sensitized genetic backgrounds, nditions , small anditions   or vhl-1(ok161) mutants ; let-60(n1046gf) RNAi-resistant mutants expressing rde-1(wt) from the Pn.p cell–specific lin-31 and the intestine-specific elt-2 promoters  mutation, as reported previously , lin-12(n137n720lf), and lin-12 (n137gf) mutants and quantifying the EGL-9::GFP signal intensities. EGL-9::GFP expression was strongly reduced in let-60(n2021rf); egl-9(sa307lf) mutants (let-60(n2021rf) mutants carrying the egl-9::gfp transgene in the egl-9(sa307lf) background). Furthermore, EGL-9::GFP expression was absent in the VPCs of lin-12(n137n720lf) mutants, whereas lin-12(n137gf) mutants showed strong and uniform expression in all VPCs (lin-12(n137gf) mutants, the RAS/MAPK is not activated in lin-12(n137gf) mutants. Furthermore, we identified eight predicted CSL (LAG-1)-binding sites (G/ATGGGAA) within a 6-kb egl-9 genomic region  double mutants. The egl-9(sa307lf) allele neither affected the VI of lin-12(n137n720lf) nor of lin-12(n137gf) mutants egl-9 locC. elegans (let-60(n1046gf); egl-9(sa307lf) and let-60(n1046gf); hif-1(zh111lf) double mutants grown under normoxia (Tables S1 and S2). We used these two double-mutant backgrounds to distinguish between RAS/MAPK pathway modifiers that are partially or fully HIF-1 dependent. We hypothesized that the knockdown of a gene that inhibits VI and depends on HIF-1 for its expression should increase the VI of let-60(n1046gf); egl-9(sa307lf) mutants in which HIF-1 is stabilized. However, knockdown of the same gene in let-60(n1046gf); hif-1(zh111lf) mutants should not increase the VI because this gene would not be expressed in the absence of HIF-1.HIF-1 up-regulates the expression of numerous target genes under hypoxia and induces multiple cellular responses . Previou elegans . We usedlet-60(n1046gf); egl-9(sa307lf) and let-60(n1046gf); hif-1(zh111lf) mutants that had an effect in a primary screen using let-60n1046gf); egl-9(sa307lf) mutants only. The empty vector negative controls are shown at the bottom and for each RNAi experiment, the matching control is indicated with the superscript numbers. Note that some genes were not analyzed in the let-60(n1046gf); hif-1(zh111lf) strain because there was no effect in let-60(n1046gf); egl-9(sa307lf) mutants. Furthermore, the knock-down of CC8.2 caused lethality and could not be analyzed. P-values were derived by bootstrapping 1,000 samples.RNAi screen to identify HIF-1 targets inhibiting vulval development. Knock-down of selected HIF-1 target genes in Table S1 let-60(n1046gf); egl-9(sa307lf) animals and did not change the VI as estimated from the number of Muv animals on the plates.HIF-1 targets without an effect on vulval development. List of HIF-1–regulated genes that were knocked down in Table S2 nhr-57, as a negative regulator of RAS/MAPK-induced VPC differentiation (nhr-57 encodes a nuclear hormone receptor similar to the Drosophila estrogen-related receptor ERR2 and the vertebrate glucocorticoid hormone receptor NR3C1 (nhr-57(tm4533lf) deletion allele to confirm that NHR-57 acts as an inhibitor of VPC differentiation. The VI of let-60(n1046gf); hif-1(zh111lf); nhr-57(tm4533lf) triple mutants was not significantly different from let-60(n1046gf); hif-1(zh111lf); nhr-57(+) control siblings (nhr-57(tm4533lf) allele increased the VI of let-60(n1046gf); egl-9(sa307lf) double and of let-60(n1046gf) single mutants compared with the corresponding nhr-57(+) control siblings.Based on these criteria, we identified a single HIF-1 target gene, or NR3C1 . We nextsiblings . Howevernhr-57::gfp reporter transgene. The reporter includes the complete nhr-57 promoter/enhancer region and contains the predicted HIF-1–binding sites  mutants with the signal being most prominent in the descendants of uninduced VPCs ; hif-1(lf) double and let-60(n1046gf); egl-9(sa307lf) hif-1(ia04lf) triple mutants was significantly decreased under hypoxia  mutants that uniformly express hif-1::gfp ; hif-1(lf) mutants, the VI of let-60(n1046gf); nhr-57(tm4533lf) double mutants did not change under hypoxia  (this study). Strong hypoxia (<1% O2) not only reduces vulval differentiation to a baseline level but also eliminates the difference between the two strains. Thus, differences in their hypoxia response may account for some of the differences in VI between the Bristol N2 and Hawaii CB4856 strains. Most wild C. elegans isolates, including CB4856, are adapted to lower oxygen levels and aggregate when grown under normoxia  or egl-9(lf) mutants, the hypoxia-response pathway and NHR-57 must act downstream of or in parallel with the RAS/MAPK pathway. NHR-57 may interact with the ETS family transcription factor LIN-1, which is an MAPK substrate that represses VPC differentiation when the RAS/MAPK pathway is inactive  by the LIN-3 EGF signal secreted from the AC. EGFR/RAS/MAPK signaling then up-regulates the expression of multiple DELTA family ligands in P6.p to activate lateral NOTCH signaling in the neighboring VPCs P5.p and P7.p and focus the inductive signal on P6.p, the future 1° VPC  allele that were maintained at 20°C and shifted to 25°C for mutant analysis. C. elegans Bristol refers to the wild-type N2 strain and Hawaii to CB4856 (LGII:let-23(sy1); LG III: unc-32(e189) lin-12(n137n720), dpy-19(e1259) lin-12(n137), hT2[bli-4(e937) let-?(q782) qIs48] . LG IV: let-60(n1046), let-60(zh121) (this study), let-60(zh122) (this study), let-60(ga89), let-60(n2021), lin-1(n304), lin-3(e1417), lin-45(sy96). LG V: egl-9(sa307), hif-1(ia04), hif-1(zh111) (this study), nhr-57(tm4533) (Mitani lab). LG X: vhl-1(ok161), rde-1(ne219), bar-1(ga80). Transgenes: gaIs37 ] ::tag, unc-119(+)] ; pRF4], zhEx418, saIs14[lin-48p::gfp], opIs206 ] (this study), arTi85 (ocedures  except No CB4856 . The trao CB4856 . The folmek(gf)] , iaIs38[-119(+)] , iaEx101-119(+)] , duIs?[e-119(+)] , zhEx6054 3′UTR] .www.wormbase.org.Detailed information on the alleles used can be found under A list of all strains used can be found in Table S3.List of strains used.Table S3 rpn-6.1 as a positive control. VI was scored in the F1 generation. Animals were anesthetized in in a drop of 5 mM tetramisole and analyzed using differential interference contrast and fluorescence microscopy.RNAi was performed as described in . Larvae Synchronized population of L1 larvae were obtained by hypochloride treatment of gravid adults and letting the embryos hatch in S-basal buffer without OP50 bacteria for 16–20 h. Arrested L1 larvae were then fed with OP50 bacteria until they reached the stage to be analyzed. The developmental stages were further distinguished using Nomarski image stacks taken along with the fluorescent images by scoring VPC divisions and the dorsal turning of the distal tip cell (DTC) and by measuring the gonad length as described . DevelopVI was scored by examining worms at the L4 stage under Nomarski optics as described in . The numComplete dataset of VI counts.Table S4 arTi85), which is based on the MPK-1 activity-dependent nuclear export of the biosensor (MPK-1 activity in the VPCs was measured in mid-L2 larvae (Pn.p stage) using the recently established ERK-nKTR biosensor , 500 nM of dpy-10(cn64) repair template AF-ZF-827, and 50 ng/μl of pDD162. Candidate worms were selected from plates harboring a large number of animals showing a roller phenotype progeny and analyzed by PCR. The zh111 deletion spans 2,464 bp and removes three to five exons .CRISPR/CAS9 was performed as described in let-60 genomic locus was partially amplified using the primers AAGGAGCAAATCGAACAGAC and ATCCATTTTATTAGGCACGCAC and cloned into p-GEM-T easy. The silently mutated PAM and the G13E mutation were obtained using the primers TGAGTGCTGATTTACCAACTCCTCCATCTCCAACTACCACAAGCTTGTACTCCGTC and AAGCTTGTGGTAGTTGGAGATGGAGGAGTTGGTAAATCAGCACTCACCATTCAACTCATC. N2 Bristol or CB4856 Hawaii animals were injected with 50 ng/μl sgRNA plasmid, 50 ng/μl pDD162, 50 ng/μl repair template, and 5 ng/μl pCFJ104. The progeny was screened for the multivulva phenotype, and candidate animals were verified by PCR and sequencing.The sgRNA sequence AATGACGGAGTACAAGCTTG was inserted into plasmid #46169 (PU6::unc-119_sgRNA) via site-directed mutagenesis. The nhr-57 transcriptional start site was amplified together with the coding genomic region (without the stop codon) using the primers ctaacaacttggaaatgaaatCACCAACACCTTCTACACAGCTGC and gttcttctcctttactcatTTGTCCATCAA-TGATTTTATAGATTTTGTCG. The gfp sequence was amplified from pPD95.75 using the primers ATGAGTAAAGGAGAAGAAC and gagatctggttcaaatagCTATTTGTATAGTTCATCCATGC. 459 bp of the nhr-57 3′ UTR were amplified using the primers GCTATTTGAACCAGATCTCTTC and cagtacggccgactagtagGAATAAATCATCCCAAAGCCGTTTTTG. A vector backbone containing a CB-unc-119(+) rescue as well as the AmpR gene was amplified using the primers ATTTCATTTCCAAGTTGTTAGCG and CTACTAGTCGGCCGTACTGAGGTGTTGTCGCTT-TTATTGGG. The four fragments were combined into a 10.170-kb plasmid (pSMa32) using Gibson Assembly. N2 animals were injected (because of mutation in the CB-unc-119(+) gene) with 5 ng/μl pSMa32, 2.5 ng/μl pCFJ104 and 100 ng/μl pBS. Transformants were isolated based on the presence of the co-injection marker.A 1,206-bp fragment upstream of the