Candidatus Liberibacter asiaticus (CaLas) and Xanthomonas citri pv. citri (Xcc), respectively. Endolysins from bacteriophages are a possible option for disease resistance in plant breeding. Here, we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas. LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc, according to inhibition zone analyses. The two genes, driven by a strong promoter from Cauliflower mosaic virus, 35S, were integrated into Carrizo citrange via Agrobacterium-mediated transformation. More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB, allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term. LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc, indicating that LasLYS2 had dual resistance to HLB and citrus canker. A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations, which might boost the citrus defense response, according to transcriptome analysis. We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2. Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2. Overall, the work shed light on the mechanisms of resistance of CaLas-derived endolysins, providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.The most damaging citrus diseases are Huanglongbing (HLB) and citrus canker, which are caused by  Candidatus Liberibacter asiaticus (CaLas), which belongs to the phloem-limited, unculturable, fastidious Alphaproteobacteria, and its vector in the field is the Asian citrus psyllid (Diaphorina citri) [Xanthomonas citri pv. citri (Xcc), is another severe bacterial disease of citrus in the world. Especially in recent years, it has shown an outbreak trend, mainly because many eradication programs are too costly to continue for a long time [Citrus, and almost all citrus cultivars are susceptible to citrus canker [Citrus huanglongbing (HLB), which occurs frequently in all major citrus-growing regions in the world, is undoubtedly the world’s most devastating citrus disease. HLB is caused by a citri) . HLB caua citri) . Thus faong time , 4, whics canker , 4, 5. Ts canker . Genetics canker .Endolysins produced by bacteriophages have been extensively used for pathogen control in humans and animals , 12. EndPropionibacterium phage P1.1 endolysin LysP11 in tobacco demonstrated significant antibacterial action against Erysipelothrix rhusiopathiae [Clavibacter michiganensis subsp. michiganensis phage CMP1, also conferred resistance to C. michiganensis in transgenic tomatoes [There have been a few cases of a bacteriophage-encoded endolysin being used to boost disease resistance in plants. Ectopic production of opathiae . CMP1, atomatoes . Bacteritomatoes , showingCaLas genome carries two prophages, SC1 and SC2, which were predicted to have a lytic and lysogenic cycle, respectively [CaLas prophage or its lytic genes is believed to negatively correlate with CaLas pathogenicity or infection [CaLas prophages and their lytic genes are a powerful means to control HLB by triggering bacterial ‘suicide’ to suppress CaLas pathogenicity. There are two predicted endolysin genes, LasLYS1 (CLIBASIA_04790) and LasLYS2 (CLIBASIA_04800), in the CaLas genome [LasLYS1 and LasLYS2 in Carrizo citrange (Citrus sinensis × Poncirus trifoliata) exhibited enhanced resistance to HLB. LasLYS2 has strong and dual resistance to HLB and citrus canker and can completely inhibit CaLas bacteria from colonizing transgenic plants. We also evaluated the structure of the bacterial community and plant immune responses affected by LasLYS1 and LasLYS2 expression in roots and midribs, where plants directly challenge CaLas attack, and highlighted the important characteristics associated with protein structure for engineering endolysins and promoting broad-spectrum disease resistance in citrus.The ectively . Activatnfection , 23, sugs genome , but thein vitro using the Oxford cup method. CaLas belongs to the Rhizobiaceae [A. tumefaciens and A. rhizogenes) species. The citrus canker pathogen Xcc was also tested. Because CaLas is an unculturable bacterium, it was not possible to include it in this test. LasLYS1 and LasLYS2 were expressed in Escherichia coli cells for harvesting the proteins. The bactericidal activities of different concentrations of recombinant proteins were first tested against A. tumefaciens EHA105 and Bacillus thuringiensis HD73 strains. Both LasLYS1 and LasLYS2 at 1.5 mg/ml concentration can induce visible inhibition zones against EHA105  since it was reported that CaLas tends to first colonize roots [CaLas were detected in the LasLYS1 lines L1-1, L1-2, L1-4, L1-7, L1-10, and L1-12, and the LasLYS2 lines L2-1, L2-3, L2-5, L2-6, and L2-8, compared with the WT control  of WT plants  and Rhizobiaceae while augmenting Burkholderiaceae and Rhodanobacteraceae in the microbial community of the root.Comparison of microbial relative abundance showed that, compared with WT controls, the populations of Xanthomonadaceae and Rhizobiaceae decreased, while the populations of Comamonadaceae, Pseudonocardiaceae, Rhodanobacteraceae, and Enterobacteriaceae increased in the roots of L2-6 and L1-2 lines S7. In thEndophytic bacteria community changes in transgenic plants may influence the host immune response ,28. ThusLasLYS1 and LasLYS2 expressions were strongly correlated with ‘plant hormone signal transduction’, ‘plant–pathogen interaction’, and ‘MAPK signaling pathway–plant’ pathways, according to KEGG pathway enrichment analysis  encode transcription factors; four (three RPM1s and one RPS2) encode disease resistance proteins; two (RBOHDRBOHB and ) encode respiratory burst oxidase homologs; and 17 are kinase family protein genes -mediated defense responses were obviously affected by pression . SA-medic plants , revealiEnterobacteria phage T  domains 1 and 2. These domains correspond to the sequences from 24 to 66 amino acids (including H1 α-helix and S1 and S2 β-sheet) and from 67 to 170 amino acids (including H2–H7 α-helices and S3 and S4 β-sheets), respectively  and confirmed that they also possess potential resistance to HLB and citrus canker, which provided new candidates for genetic improvement of disease resistance in citrus.Bactericidal peptides or proteins from other species have displayed great potential in citrus breeding for broad-spectrum disease resistance. Our previous study showed that expressing synthesized cecropin B genes in transgenic citrus alleviated the susceptibility to HLB and citrus canker , 33. Haoet al.  showed tet al. . Endolyset al. , 36. Esp [et al.  demonstrobiaceae . These dCaLas, which multiplies in the cytoplasm of sieve elements in the phloem once it enters the plant [Xcc, which multiplies in the intercellular space of citrus, indicating that LasLYS2 can kill Xcc without the help of a signal peptide. It is possible that LasLYS2 is transported outside the host cell to execute its bactericidal function through passive diffusion due to the characteristics of its primary and tertiary structures. In fact, it would be worthwhile to increase expression of endolysin genes without being concerned about the transport of the enzyme by adding an effective signal peptide for efficiently lysing bacteria at the site of infection [CaLas and Xcc, which live in the cytoplasm and intercellular space of host cells, respectively.The function prediction showed that LasLYS1 and LasLYS2 are lysozymes. It has been shown that the lysozyme genes, which originated from different species, such as bacteriophage, chicken, goose, and human, confer broad resistance against different pathogenic bacteria and even fungi , 36–38. nfection . Thus, oLBA4404, Ar1193, and Xcc, although it still inhibited CaLas growth in citrus. This further suggests that the H2 α-helix could be a core structure essential for the function of Lyz domain 2. Further studies are needed to confirm the role of the α-helices in endolysin activity. Lyz domain 1 in LasLYS1 contains the predicted catalytic triad Glu-8aa-Asp/Cys-6aa-Thr, responsible for the hydrolysis reaction [in vitro activity against the tested strains in LasLYS1N and LasLYS1N:LYS2. Lyz domain 1 also showed potential inhibition of Lyz domain 2 in LasLYS1 but not in LasLYS2, explaining the functional difference between LasLYS1 and LasLYS2. The different impacts of Lyz domain 1 on the activity of Lyz domain 2 in LasLYS1 and LasLYS2 may be related to the distinct amino acid sequences in LasLYS1 and LasLYS2  and LasLYSI (5.7 kDa), exhibited stronger inhibitory activity against CaLas compared with the larger proteins, LasLYS1C (11.5 kDa) and LasLYS1N:LYS2 (19.0 kDa). Furthermore, we speculate that Lyz domain 2 contains unidentified catalytic residues, as LasLYS2, LasLYS1C, and LasLYSI all demonstrated bacteriolytic activity. The catalytic triad of lysozyme typically involves Glu as a general acid responsible for the catalytic mechanism, while Asp is not essential [LasLYS2 displayed a wider bactericidal spectrum and higher resistance to HLB and citrus canker, despite having similar secondary and tertiary structures to LasLYS1. Informatics analysis revealed that LasLYS2 only possesses Lyz domain 2, while LasLYS1 has both Lyz domains 1 and 2. Bacteriostatic zone tests demonstrated that Lyz domain 2 was crucial for the activity of both LasLYS1 and LasLYS2. Lyz domain 2 consists of five α-helices (H2–H7 α-helix), whereas Lyz domain 1 only contains an H1 α-helix. This suggests that the H2–H7 α-helix plays an important role in the function of Lyz domain 2. Furthermore, LasLYSI, lacking the H2 α-helix, lost its activity against reaction . However LasLYS2 , which w LasLYS2  suggestsssential . In concLasLYS1 and LasLYS2 in citrus transgenic plants, we performed 16S amplicon sequencing of the midrib and root since the tissues are the main habitat of CaLas [CaLas [LasLYS1 and LasLYS2 transgenic plants. Members of the Burkholderiaceae family are believed to be beneficial bacteria for defending against HLB disease [Burkholderia territorii strain A63 and Burkholderia metallica strain A5 have antagonistic activities against Sinorhizobium meliloti, a relative of CaLas [Xcc [CaLas infection [Plant endophytes are activists of host health in a commensal or beneficial manner, which can inhibit pathogen infection through direct competition, secreting effectors or antimicrobial compounds, or stimulating plant immunity , 43. As s [CaLas , 26. The disease , 44, 45.of CaLas , and sigLas [Xcc . RhodanoCaLas infection [LasLYS1 and LasLYS2 augmented defense-related transcription activities in healthy transgenic plants. Inoculation of Burkholderiaceae strains can activate the expression of genes involved in the induced systemic resistance of citrus, which has the potential to promote plant health [SAMT1, NPR1, TGA1, PR1, and PR5), participating in SA-dependent systemic/induced acquired resistance, were upregulated in our transgenic plants. Therefore, increasing some beneficial bacteria, such as Burkholderiaceae and Rhodanobacteraceae, might promote an SA-mediated defense response in transgenic plants. We also demonstrated that the MAPK signaling pathway was upregulated by LasLYS1 and LasLYS2 expression in transgenic plants. The activation of the MAPK signaling pathway plays a positive role in citrus resistance to HLB [CaLas infection, expressions of MAPKK19 (an MEKK), WRKY40, and RBOHD homologs increased in rough lemon, which is HLB-tolerant, compared with that of ‘Madam Vinous’ sweet orange, which is HLB-susceptible [RBOHD induced by CaLas infection is possibly responsible for ROS accumulation in citrus phloem [Arabidopsis enhanced resistance to Pseudomonas syringae [External application of host-derived antimicrobial peptides or proteins can prime citrus innate immunity to reduce nfection ,47. The t health , 46. Seve to HLB . Furtherceptible . Expresss phloem . ROS accs phloem . Constitsyringae ,51. ThusCaLas can simultaneously provide good protection for citrus plants against HLB and citrus canker that are induced by CaLas and X. citri pv. citri, respectively. Importantly, the LasLYS2 transgene can maintain low CaLas titers in citrus plants without visible symptoms for a long time, and it is even possible to completely clear CaLas from infected plants. The study also demonstrated that LasLYS1 and LasLYS2 positively affected the host’s endophytic bacteria community and defense transcription activities, which were beneficial to plant resistance. We highlighted the observed characteristics of Lyz domain 2 of the CaLas endolysins that are associated with its structure and function, which will provide a good reference for screening existing endolysins or bactericidal proteins for genetically generating disease resistance in citrus.This study demonstrated that transgenic expression of LasLYS2 endolysin from CaLaswere taken from a citrus plantation in China’s Guangxi Province. Grafting the scions onto Jincheng orange (Citrus sinensis) seedlings in a greenhouse was done to preserve and reproduce CaLas. This research employed the Xcc strain XccYN1 [In the National Citrus Variety Improvement Center’s nursery in Chongqing, China, Carrizo oranges were grown for citrus transformation. Sweet orange scions infected with n XccYN1 .LasLYS1 (CLIBASIA_04790) and LasLYS2 (CLIBASIA_04800), the two genes were amplified from CaLas-infected Wanjincheng orange midribs by PCR, cloned into pGEM-T® Easy Vector , and confirmed by Sanger sequencing. The TMHMM server (v.2.0) and signalP5.0 tool were used to predict the transmembrane structure and signal peptide of LasLYS1 and LasLYS2, respectively. The Phyre5 tool [Based on the coding sequences of re5 tool  was usedre5 tool .LasLYS1 and LasLYS2, we synthesized the LasLYS1N, LasLYS1C, LasLYSI, and LasLYS1N:LYS2 genes by PCR. These genes, including LasLYS1 and LasLYS2, were cloned into the pCZN1 vector. The resulting constructs were transferred into the Arctic-Express (DE3) strain. Protein expression was induced by 0.4 mM isopropyl-thio-2-d-galactopyranoside for 4 h at 37°C. Recombinant proteins with a His-tag were purified using Ni2+-NTA agarose following the manufacturer’s instructions . Protein purity and concentrations were determined by SDS–PAGE and the Bradford assay [Based on the sequences of rd assay , respect600 of 0.2. Five Oxford cups were placed onto 25 ml of LB solid medium and 2 ml of bacterial resuspension to create LB plates. The Oxford cup received separate additions of 50 μl recombinant protein. The inoculation was kept at 28°C for 3 days before being photographed. Using a Vernier caliper, the inhibitory zone’s diameter was determined. The test was repeated three times.Recombinant proteins’ bactericidal properties were evaluated using the Oxford cup technique. In a shaking incubator at 28°C for 16 h, bacterial strains were grown in Luria–Bertani broth  medium. Centrifugation was used to separate the bacteria for 1 min at 10 000 rpm, after which they were diluted in LB to an ODLasLYS1, LasLYS2, LasLYS1N, LasLYS1C, LasLYSI, and LasLYS1N:LYS2 were optimized based on codon usage bias in plants, and the optimized sequences were artificially synthesized by PCR. The optimized LasLYS1 and LasLYS2 were cloned into a pLGN vector [LasLYS1 and LasLYS2 transcription was driven by a strong 35S constitutive promoter. These constructs were transformed into the A. tumefasciens EHA105 strain for producing transgenic plants. The optimized LasLYS1N, LasLYS1C, LasLYSI, and LasLYS1N:LYS2 were inserted into a pNMG vector, and their expressions were controlled by the 35S promoter  solution. The infected explants were put vertically into vermiculite in seedling-raising discs and then maintained in an artificial climate chamber with 80% relative humidity and a 16 h photoperiod at 26°C. Two months later, transgenic hairy roots were detected using GFP fluorescence detection and PCR analysis.escribed . In brieCaLas-infected Wanjingcheng orange leaf discs. Prior to grafting, PCR was used to detect the presence of CaLas in the petioles of the leaves . Seventy-two punctures per independent transgenic line were sorted based on the diseased area using the following index: 0, <0.2 mm2; 1, 0.2–0.4 mm2; 2, 0.4–0.6 mm2; 3, 0.6–0.8 mm2; 4, 0.8–1.0 mm2; 5, 1.0–1.2 mm2; 6, 1.2–1.4 mm2; 7, >1.4 mm2. The disease index (DI) for each independent transgenic was calculated with the formula:Using WT plants as a control, resistance to citrus canker in transgenic plants was evaluated by the pinprick method described in previous work . Three fDI = Σ (no. of each index × the corresponding index)/(72 × 7) × 100.The growth of XccYN1 in the leaves of transgenic plants was also evaluated daily 0–9 dpi) after infection, as described previously [ dpi afteIn vivo infiltration was also used to determine the canker resistance of transgenic plants [8 CFU/ml) were injected into 3-month-old leaves. Daily observations of the development of canker symptoms in inoculated plants were made.c plants . XccYN1 All of the preceding experiments were repeated three times.Healthy transgenic and healthy WT plants were used to analyze the endophytic bacterial community of the roots and midribs. Three biological replicates were carried out. For each biological replicate, citrus DNA was extracted from at least three plants. The V3 and V4 regions of the bacterial 16S DNA were amplified with the primers 335F/769R  to constwww.biocloud.net). ASVs with an adjusted P < .05 and bacterial taxa with P < .05 were considered to have a significant difference compared with the WT controls.The processing and assembly of raw data were performed as described . ASVs weC. sinensis genome (v.3.0) (http://citrus.hzau.edu.cn). The databases GO, KO, KOG/COG, Nt, Nr, Pfam, and Swiss-Prot were utilized to predict gene function. featureCounts [P-value <.01. On the BMKCloud platform (www.biocloud.com), principal component analysis of gene expression and KEGG pathway enrichment, GSEA, and Venn diagram and heat map construction of DEGs were conducted. Using the STRING platform (v.11) [Arabidopsis homologies.The investigation was conducted using three biological replicates. BioMarker Technologies Illumina, Inc.  was entrusted with the RNA-seq analysis of roots and midribs from healthy transgenic and WT control plants. Using HISAT 2.0.5 , all thereCounts  was usedreCounts , DEGs inm (v.11) , proteinGAPDH gene [−ΔΔCt) [The extraction of citrus RNA, synthesis of cDNA, and quantification of gene expression were carried out in accordance with previous methods . The GAPPDH gene  was used [−ΔΔCt) . SupplemWeb_Material_uhad159Click here for additional data file.


Chromosome 1p36 deletion syndrome (1p36DS) is one of the most common terminal deletion syndromes , due to a heterozygous deletion of part of the short arm of chromosome 1. The 1p36DS is characterized by typical craniofacial features, developmental delay/intellectual disability, hypotonia, epilepsy, cardiomyopathy/congenital heart defect, brain abnormalities, hearing loss, eyes/vision problem, and short stature. The aim of our study was to (1) evaluate the incidence of the 1p36DS in the French population compared to 22q11.2 deletion syndrome and trisomy 21; (2) review the postnatal phenotype related to microarray data, compared to previously publish prenatal data. Thanks to a collaboration with the ACLF (Association des Cytogénéticiens de Langue Française), we have collected data of 86 patients constituting, to the best of our knowledge, the second‐largest cohort of 1p36DS patients in the literature. We estimated an average of at least 10 cases per year in France. 1p36DS seems to be much less frequent than 22q11.2 deletion syndrome and trisomy 21. Patients presented mainly dysmorphism, microcephaly, developmental delay/intellectual disability, hypotonia, epilepsy, brain malformations, behavioral disorders, cardiomyopathy, or cardiovascular malformations and, pre and/or postnatal growth retardation. Cardiac abnormalities, brain malformations, and epilepsy were more frequent in distal deletions, whereas microcephaly was more common in proximal deletions. Mapping and genotype–phenotype correlation allowed us to identify four critical regions responsible for intellectual disability. This study highlights some phenotypic variability, according to the deletion position, and helps to refine the phenotype of 1p36DS, allowing improved management and follow‐up of patients. Through several published European, American, and Japanese cohorts , we have collected data of 86 patients postnatally diagnosed with 1p36DS. Data from the corresponding prenatal observations have been previously published  try to assess all cases of 1p36 deletion diagnosed in France and with previously reported prenatal cases, try to evaluate its incidence; (2) review the phenotype, related to CMA data and help to establish a link between genotypes and phenotypes associated with this genetic syndrome to provide more information to families.22.1A multicenter, nationwide, retrospective study was set up to collect data on patients with 1p36DS with the cooperation of the ACLF.Ninety‐one patients diagnosed postnatally between 1996 and 2018 using various diagnostic methods , or CMA) were reported by 18 French laboratories out of the 37 surveyed centers. The following data were requested by a questionnaire addressed to the referring geneticist: indication for genetic testing, clinical information, age at diagnosis, technique used for diagnosis, and mode of inheritance.GNB1 gene.Five patients were excluded from the clinical analysis: two patients for whom genetic and clinical information was lacking, one patient with a derivative chromosome one resulting from an unbalanced translocation (with 1p36 deletion associated with a 6 Mb chromosome 16 duplication), two other patients with very small deletions (41 and 166 kb) encompassing the 2.2https://genome.ucsc.edu/) genetic tools, UCSC Genome Browser (build GRCh37/hg19), with the creation of two groups according to deletion position. Distal and proximal 1p36 critical regions  were described in the literature  for most of them. A map of CMA results was generated using the University of California Santa Cruz . Statistical analyses were performed with Epi Info™ version 3.5.4 (CDC). For all tests, https://www.face2gene.com/) to evaluate facial features.Patients' photographs were analyzed using Face2gene .Age at diagnosis: The patient's age at diagnosis was known in 85 cases. The mean and median age ± SD (range) were respectively 6.12 and 2 ± 8.3 (0.005–33.7) years old (from 2 days to 33 years). Among all cases, 9 patients were newborns (age below 28 days), 34 infants (age between 1 month and 2 years), 15 young children (age between 2 and 6 years), 12 older children (age between 7 and 12 years), 8 adolescents (age between 13 and 19 years), and 7 adults (age > 20 years).Mortality: Information about survival was known in 51 cases. Among these 51 patients, 8 children were dead (15.7%), and 6 of them died before 2 years old. Deletions sizes of these patients  varied from about 3.7 to 8.3 Mb , interstitial deletions (24), and derivative chromosomes (1). In five patients, CMA allowed to bring out complex rearrangements (1p36 deletion associated with other deletion or duplication on chromosome 1). In our cohort, minimum and maximum deletions sizes varied from 532 kb to 11.8 Mb Table .Out of the 86 patients included in the study, CMA was performed on 72 patients. Among them, additional aberrations were reported in six patients and summarized in Table The cohort was divided into two groups according to the deletion position: Group A including 56 patients, and Group B including 11 patients Figure  correspo3.3Of the 54 patients for whom a parental analysis was performed: the 1p36 deletion was found de novo in 50 patients, inherited in two cases , resulted from an unbalanced translocation in one case (plus another case excluded from the study), and one 1p36 deletion was found in mosaic .3.4Clinical features in all patients, in Group A , and Group B  were summarized in Figure We also found: pre‐ and/or post‐natal growth retardation in 45 patients (45/57), behavioral disorders in 33 patients (33/44), microcephaly in 32 patients (32/57), eye/vision problems in 28 patients (28/40), hands (33/51), and feet (25/46) abnormalities, gastrointestinal anomalies in 24 patients (24/36) and also spinal defects (24/40).3.4.1Data were available in 75 patients, and developmental delay or ID was reported in 100% of patients (75/75). The severity degree of this delay was unknown in most cases. Walking (data was available in 17 patients) was acquired for the earliest at 16 months and was not acquired at the time of the consultation for three patients at 3, 5, and 10 years, respectively. A regression of acquisitions was reported in four patients , including aggressivity, self‐injury/self‐aggressivity, temper tantrums, low frustration tolerance, social interaction difficulties, and stereotypies.3.4.2Data were available in 78 patients with details in 69 patients. Facial dysmorphism was reported in 94% 73/78)  of patients presented brain malformations, including corpus callosum abnormalities for 13 patients (13/62) and ventricular dilation for 12 patients (12/62). We also observed polymicrogyria, pachygyria, cortical dysplasia, heterotopia, cerebral atrophy, delay in myelination, and white matter anomalies Tables  and S4. 3.4.4p < 0.05).(Data were available in 68 patients) was found in 46 patients (deletions were mapped in 33 patients) and associated with brain malformations in 28 patients. Interestingly brain malformations were more frequent in Group A  as well as epilepsy  (3.4.5p < 0.05). Cardiac abnormalities were found in 77.8% of newborns (7/9), 59.4% of infants (19/32), 33.3% of young children (5/15), 37.5% of older children (3/8), 14.3% of adolescents (1/7), and 20% of adults (1/5).Data were available in 77 patients. Cardiovascular malformations or cardiomyopathy were reported in 47% (36/77). Of these 36 patients, deletions were mapped in 27 of them. Structural heart defects were observed in 32% of patients with mostly ventricular septal defect  and patent ductus arteriosus . Cardiomyopathy  were noted in 21% of patients (left ventricular non‐compaction and dilated cardiomyopathy in 7/77 patients (9%) and 6/77 patients (8%), respectively). Four patients presented both structural heart defects and cardiomyopathy. Cardiac abnormalities were more frequent in Group A (25/49) than in Group B (2/11) (3.4.6(Data were available in 51 and 46 patients): short hands, bilateral talus valgus, clinodactyly, flat feet, brachydactyly, and transverse palmar crease were reported.3.4.7p < 0.05). Deletions were mapped in 24 patients. It was associated with brain abnormalities in 16 patients.Data were available in 57 patients. Microcephaly was found in 32 patients and was more frequent in Group B (8/9) than in Group A (16/35) (3.4.8Hearing loss (11), skin abnormalities (11), joint abnormalities (13), limbs abnormalities (9), overweight (9), lungs abnormalities (8), abnormalities of the external genitalia (8), hypertrichosis (7), renal defects (6), early puberty (5), hypertonia/ pyramidal syndrome (5), hypothyroidism (4), hepatic defects (3), and hematologic disorders (2). All clinical signs reported, are shown in Tables 3.5GABRD, SKI, or PRDM16 genes. Nevertheless, he exhibited facial dysmorphism, partial agenesis of the corpus callosum, hypotonia, spinal abnormalities, short fingers, hypertrichosis, feeding difficulty with gastroesophageal reflux requiring a gastrostomy, myopia, hearing loss, and sleep apnea syndrome requiring a tracheotomy. He presented psychomotor delay  and mild to severe ID. So, even if he did not present all the major features of 1p36 deletion, he had a severe neurodevelopmental phenotype.In our cohort, deletions over 10 Mb were associated with a more severe presentation. Two patients had deletions larger than 10 Mb. The first one presented dysmorphism; left ventricular non‐compaction; ventricular septal defect; microcephaly; large anterior fontanel; corpus callosum hypoplasia; delayed myelination; hypotonia, feet abnormalities; growth retardation; hearing loss, and psychomotor delay. The second carried the largest deletion: 11.8 Mb (del54). Interestingly, this patient had neither cardiomyopathy nor congenital heart defect, microcephaly, epilepsy, or growth delay but he presented intra‐uterine growth delay. It was an interstitial deletion that did not include the 3.6Using previously published data, we calculated the number of new cases of 1p36DS per year between 2012 and 2016. As shown in Table 4With the cooperation of the members of the ACLF, we reported 86 patients with 1p36DS diagnosed postnatally between 1996 and 2018. According to the deletion position, we have delineated two groups: 56 patients with distal deletions (Group A) and 11 patients with more proximal deletions (Group B). Patients in our cohort presented mainly: facial dysmorphism, microcephaly, developmental delay or ID, hypotonia, epilepsy, brain malformations, behavioral disorders, cardiomyopathy or cardiovascular malformations, pre‐ and/or postnatal growth retardation. Cardiac abnormalities, brain malformations, and epilepsy appeared to be more frequent in Group A than in Group B, whereas microcephaly seemed to be more common in Group B.4.1This large retrospective study has the inconvenience of lacking some clinical data, however, it gives information about the frequency of the deletion 1p36, in continuum with our previous del1p36 prenatal study, in the same population.4.2We estimated an average of 10 cases of 1p36DS diagnosed per year in France. However, only 18 laboratories reported cases, so we probably could not assess all the cases of 1p36DS in France; thus, the number of cases per year is probably underestimated in our cohort. However, our results give an approximation of the incidence of this syndrome, about 1 case per 18,000 live births, which seems to be slightly lower than that described in the literature  , as already described in the literature  and 11 patients with more proximal deletions (Group B), so classical distal deletion is more frequent in our cohort Figure . We obse4.4In our study, all patients who died suffered from heart defects, and among them, two died from cardiogenic shock, and heart failure. Shimada and colleagues have reported two deceased patients, one of whom died at 10 months old of heart failure . In Shimada's report, most patients had the first SRO and some had the first and second SRO, authors explained that severe neurodevelopmental prognosis may be provided by haploinsufficiencies of KCNAB2 and CHD5. Interestingly in our cohort, all patients presented ID, and some have deletions of MOR2 (KCNAB2 and CHD5) not encompassing MOR1 (GNB1). This suggests that genes in MOR2 may also contribute to the neurological phenotype observed in the 1p36DS. Petrovski et al. have shown that de novo mutations in GNB1 cause severe neurodevelopmental disability, hypotonia, and seizures. Two unique patients (del64 and del39) carried very small deletions (41 and 166 kb) encompassing only the GNB1 gene , SKI (MIM:164780), KCNAB2 (MIM:601142), RERE (MIM:605226), UBE4B (MIM:613565), SPEN (MIM:613484), and CDC42 (MIM:116952) . Finally, we report another even more proximal MOR (MOR4) (21–22.7 Mb region) including the CDC42 gene. Heterozygous variants of CDC42 have been reported in ID  , 16.1–16.2(hg19)) recently published as causing neurodevelopmental disorder  encodes for ligand‐gated chloride channels for GABA (gamma‐aminobutyric acid), the major inhibitory neurotransmitter in the mammalian brain  gene has been associated with epilepsy in 1p36DS  than in Group A, and in the literature, 5/6 patients with proximal deletions presented microcephaly, and the last one has a head circumference at the 5th centile. Therefore, microcephaly seems to be a frequent feature in proximal 1p36 deletions. It has been suggested that another gene: RERE (Arginine‐glutamic acid dipeptide repeats) may be responsible for microcephaly in 1p36DS. Indeed, Kim et al. observed a reduced brain size in Rere mutant mice  disruption and chromatin disorganization leading to the deregulation of several genes or candidate genes for microcephaly as CDC42 (Takenouchi et al., SKI, or RERE (Figures MTOR gain of function mutations have been described in a neurodevelopmental disorder associated with macrocephaly (Rodríguez‐García et al., MTOR is included in MOR3 and deleted in 10 patients, among these 10 patients, 8 presented microcephaly. Also, we can hypothesize that MTOR deletion may also be responsible for microcephaly (Figure Microcephaly was observed in 32/57 patients in our cohort and 60% (Heilstedt, Ballif, Howard, Lewis, et al.,  Figures . Moreove4.9In our post‐natal cohort, we observed more facial abnormalities, with 94% of patients reported with dysmorphism, than in the ACLF prenatal cohort (Guterman et al., 4.10PRKCZ, SKI, and PRDM16 (Zaveri et al., PRDM16 gene (Figure PRDM16 have been described in patients with cardiomyopathy and left ventricular non‐compaction (Arndt et al., PRDM16 did not have any type of cardiac abnormalities suggesting a variable penetrance. Arndt and colleagues suggested a possible interaction between PRDM16 and SKI. They coinjected subthreshold doses of PRDM16 and SKI morpholinos in zebrafish and observed a reduced cardiac output (Arndt et al., PRDM16 but no SKI did not present cardiomyopathy. Some other genes may be responsible for cardiomyopathy as PDPN, and CASZ1 (Jordan et al., RERE gene may be responsible for clinical signs recorded in 1p36 deletion, including heart defects notably ventricular septal defect (Figure ECE1 gene may also contribute to cardiac features. ECE1 has been implicated in Hirschsprung disease, autonomic dysfunction, and cardiac defects. Hofstra et al. described a patient with Hirschsprung disease, ductus arteriosus, small subaortic ventricular septal defect, and small atrial septal defect and dysmorphism, carrier of a missense mutation in ECE1 and they suggested that this variant may cause or contribute to the patient's phenotype (Hofstra et al., Guterman found 40% of cardiac malformations in the prenatal cohort (Guterman et al., t Figure . The ECE5Thanks to a collaboration with the ACLF we have collected data of 86 patients constituting the second‐largest cohort of 1p36DS patients in the literature to our knowledge. This study enables a continuum with prenatal published data. We estimated an average of, at least, 10 cases per year in France. 1p36DS seems to be much less frequent than 22q11.2 deletion syndrome and trisomy 21. We observed that 1p36DS may be a life‐threatening condition and can lead to early death in the most severe cases. Mapping and genotype–phenotype correlation allowed us to identify four critical regions responsible for ID and some phenotypic variability, particularly according to the deletion position. This study helps to refine the phenotype of 1p36DS, which is diagnosed more and more early, especially prenatally with the generalization of the CMA and more recently with genome analysis. A better knowledge of this syndrome allows improved management of patients, particularly in the detection of malformations, complications, and in the follow‐up of patients. Moreover, even in the face of characteristics specifically suggesting 1p36DS, a CMA would provide more information to families.Clémence Jacquin, Emilie Landais, Céline Poirsier, Alexandra Afenjar, Ahmad Akhavi, Nathalie Bednarek, Caroline Bénech, Adeline Bonnard, D. Bosquet, Lydie Burglen, Patrick Callier, Sandra Chantot‐Bastaraud, Christine Coubes, Bruno Delobel, Margaux Descharmes, Jean‐Michel Dupont, Vincent Gatinois, Nicoles Gruchy, Sarah Guterman, Abdelkader Heddar, Lucas Herissant, Delphine Heron, Bertrand Isidor, Pauline Jaeger, Guillaume Jouret, Boris Keren, Paul Kuentz, Cedric Le Caignec, Jonathan Levy, Nathalie Lopez, Zoe Manssens, Dominique Martin‐Coignard, Cyril Mignot, Chantal Missirian, Céline Pebrel‐Richard, Lucile Pinson, Jacques Puechberty, Sylvia Redon, Damien Sanlaville, Marta Spodenkiewicz, Anne‐Claude Tabet, Alain Verloes, Gaelle Vieville, Catherine Yardin, François Vialard, Martine Doco‐Fenzy clinically examined patients, provided clinical and genetics data, and/or performed genetic analysis. Clémence Jacquin designed the study and wrote the manuscript. Emilie Landais performed part of the data analysis. All authors read and approved the final version of the manuscript. Emilie Landais and Martine Doco‐Fenzy supervised the study, and critically reviewed the article.The authors declare no conflict of interest.Appendix S1: Supporting Information.Click here for additional data file.Table S4: Summary of clinical features of all the patientsClick here for additional data file.


Mespilus germanica) extract (ME) supplementation on growth performance, antioxidant, immune status, and stress responses in rainbow trout (Oncorhynchus mykiss) were studied. Six hundred fish  were distributed randomly into five experimental groups (assayed in triplicates). The experimental diets were formulated as follows: 0 , 0.5% (T2), 1% (T3), 1.5% (T4), and 2% (T4). After 60 days feeding trial, the fish were confined, and the density increased (60 kg/m3) for further 14 days. Results showed significant increases in final weight (FW), weight gain (WG), specific growth rate, and feed intake in the T4 compared to the control (P < 0.05). The feed conversion ratio (FCR) in T4 significantly decreased compared to the control (P < 0.05). Also, the treated groups showed significant improvements in catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), lysozyme (LYZ), total immunoglobulin , respiratory burst activity (RBA), total protein, and phagocytosis (PHA) (P < 0.05). Moreover, compared with the control group, supplementation could significantly decrease glucose (GLU) and cortisol (CORT), alanine transaminase (ALT), lactate dehydrogenase (LDH), aspartate transaminase (AST), and alkaline phosphatase (ALP) (P < 0.05). After the challenge, FW and WG in all treated challenge groups were significantly improved compared to the control group (P < 0.05). FCR showed a significant decrease in all treated challenged groups compared to the control group (P < 0.05). However, malondialdehyde, CAT, GPx, SOD, LYZ, complement activity (C3 and C4), total Ig, RBA, peroxidase, and PHA in challenged treated groups were significantly increased compared to the control group (P < 0.05). All treated challenged groups showed lower ALT, LDH, AST, ALP, GLU, and CORT levels than the control group (P < 0.05). The experiment herein successfully demonstrated that dietary ME stimulated fish growth, antioxidant status, and immune responses in crowding conditions and can be recommended as beneficial feed additives for rainbow trout.High stocking density is a stress factor that potentially affects physiological and immune responses. In this study, the effects of medlar ( Stressors generally challenge the various physiological functions due to recovering the homeostatic status of fish. Besides, elevating the innate immune mechanism, health condition, and growth rate of the host is an ideal approach to disease and stress management strategies in modern aquaculture , 2.Nutritional treatment has been recommended as a useful method to mitigate fish stress . ImmunosMespilus germanica L.) belongs to the Rosaceae family and mainly grows in the temperate regions of southwest Asia and southeastern Europe [One of these medical herbs is medlar  extract (ME) on growth performance, antioxidant, and immune responses of rainbow trout exposed to crowding stress. To the best of our knowledge, little information is available about the effect of medlar extract on rainbow trout immunity.One of the freshwater aquaculture species commercially farmed under intensive or semi-intensive systems is rainbow trout in Iran, s mykiss . High sts mykiss . The preFresh leaves of medlar were purchased from a local market in Rasht, Iran. Leaves were washed using sterile distilled water and oven-dried at 50°C for 48 hr. 80% ethanol was added to 200 g dried medlar and then incubated in a shaker incubator for 24 hr. The extract was filtered through a Whatman paper (no. 1) . The alcμl aliquot of the dissolved extract was mixed with 50 μl Folin–Ciocalteu 10% reagent. Then, 30 µl Na2CO3 20% was added and incubated for 1 hr at 37°C; the absorbance was measured at 735 nm in the dark and compared to the calibration curve of gallic acid [Total phenol content was determined based on the Folin–Ciocalteu method. Briefly, a 120 lic acid . The resμl extract was added to 1,250 μl of distilled water, and then 75 μl of 5% sodium nitrate solution was added. After 5 min, 150 μl of 10% aluminum chloride solution was added and kept at room temperature for 5 min. Finally, 500 μl of sodium hydroxide solution and 775 μl of distilled water were added to the mixture. The mixture was homogenized, and the adsorption rate was read at 510 nm. A standard quercetin curve was used, and the results were expressed in mg of quercetin equivalent (QE)/g.Total flavonoid content was measured using aluminum chloride . In thisμM) was added to the 100 µl of seaweed extract. The sample was then incubated for 30 min at room temperature. The absorbance was measured at 520 nm. The radical scavenging activity of DPPH was calculated using the following formula [About 0.2 ml of 0.1 mM 1, 1-diphenyl-2-picrylhydrazyl (DPPH) ethanol (150  formula :(1)%inhiβ-carotene was dissolved in 1 ml of chloroform. β-Carotene methanolic solution (28 μl) was homogenized with linoleic acid (28 μl) and Tween 40 (200 mg). After evaporation of chloroform using rotary at 40°C, 50 ml of distilled water was added. The same procedure was repeated using butylate hydroxy anisole as the antioxidant standard. Readings were performed every 15 min at 470 nm. Where Ac and As are the absorbances of the control and samples, respectively. The potency of essential oil oxidation was calculated based on the following formula:The inhibition of linoleic acid peroxidation was determined according to Miller . About 2There were four diets in this experiment, containing 0 , 0.5% (T2), 1% (T3), 1.5% (T4), and 2% (T5) ME. Such concentrations were chosen based on a previous study . The ing3). The experiment lasted for 60 days and five test groups in triplicates. Fish were fed based on ad libitum, three times daily [3 for further 14 days [Rainbow trout were cultured in spring water on a local farm, and the water quality parameters, including temperature, dissolved oxygen, pH, and unionized ammonia nitrogen, were monitored daily at 14–15°C, and 7.5–8.2, 7.1–7.3, and 0.002 mg/l, respectively . Water tes daily . After t 14 days . After t 14 days , 26.Growth performance and survival parameters were calculated using the following equations :(3)WeighAfter the experimental time, the fish were fasted for 24 hr and anesthetized (75 mg/l eugenol) for 1 min . Blood wμM/l) content was determined with the thiobarbituric acid reaction by a commercial kit . Superoxide dismutase (SOD) was defined as the quantity of enzyme (U/ml) that inhibits the reduction of oxidase-cytochrome C . Catalase  was utilized to decrease H2O2 absorbance according to Góth's [Malondialdehyde , alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activity were detected by biochemistry analyzer, using commercial kits  .Micrococcus luteus in 0.2 mg/ml in a 0.05 M sodium phosphate buffer (pH = 6.2) as the substrate according to the method of Ellis [Lysozyme  activity was measured through turbidometric assay and lysis of of Ellis . The comof Ellis .µg/ml) activity in serum or leukocytes was measured according to Cordero et al. [µl of serum was diluted with 45 µl of Hank's Balanced Salt (without Ca2+ or Mg2+) in flat-bottomed 96-well plates. About 100 ml of the solution, including 40 ml distilled water, one pill of 3, 30, 5, 50- tetramethylbenzidine , and 10 µl H2O2 were added. The color-change reaction happened in the 30–60 s. Then, 50 µl of 2 M sulfuric acid (2 M H2SO4) was added. Finally, the optical density was read at 450 nm in a plate reader. Standard samples without serum were used as blanks.The peroxidase  was incubated in 2% sucrose (pH = 3–4) at 30°C for 2 hr, then boiled for 30 min. The yeast was centrifuged (800 g for 10 min) and washed twice with 0.85% NaCl (concentration of 2 × 108 cells/ml). Approximately 40 µl of blood heparin and 20 µl suspension were collected in the microtube and were incubated in a shaker incubator at 30°C for 30 min. Finally, dried smears were stained with Wright–Giemsa. One hundred phagocytoses were analyzed per slide under the optical microscope by using the below formula:Leukocyte PHA (%) activity was determined with yeast as an indicator according to the Zhou et al.  method. Cortisol  concentration in serum samples was quantified using an ELISA kit . Serum glucose  content was measured by a commercial kit  .P < 0.05 as the significance level.Before performing the analysis of variance (ANOVA), the normality of the data was checked using the Kolmogorov–Smirnov test, and Levene's test was used to assay the variances of data. Statistical analysis of data performed using SPSS software version no. 20.00 , and the results represent the mean ± SE. Differences in studied parameters among the experimental groups were processed by one-way ANOVA followed by Tukey's multiple comparison test considering β-carotene/linoleic acid (74.11 ± 4.91%) of ME are shown in Bioactive compounds such as total polyphenol content (189.22 ± 6.22 g mg GAE/g), total flavonoids (57.55 ± 5.31 mg QE/g), and antioxidant capacities evaluated by DPPH (56.77 ± 3.66%), and P < 0.05). However, no statistical variations were recorded in FI and SR in fish-fed supplemented diets and the control group (P > 0.05).P < 0.05). Determination of WG showed a significant increase in all experimental treatments over the control treatment (P < 0.05), whereas FCR analysis showed a significant decrease in experimental treatments over the control treatment (P < 0.05). 1.5% and 2% ME significantly increased FI. No significant differences were observed for the SGR and SR between the fish-fed dietary ME and the control group (P > 0.05).After the challenge, 1%, 1.5%, and 2%  ME significantly improved FW compared to the control group (P > 0.05). The value of SOD and CAT increased in T3 and T4 compared to the control group (P < 0.05). Other treatments displayed no significant difference compared to the control (P > 0.05). In addition, GPx analyses indicated a significant increase in all ME treatments compared to the control group (P < 0.05).P > 0.05); however, significant decreases were observed in other treatments compared to the control group (P < 0.05). Also, the SOD, CAT, and GPx activities in fish fed T3, T4, and T5 were significantly increased than that in the control group (P < 0.05).After the challenge, the MDA value didn't influence in T2 and T3 groups (P < 0.05), however, the T2 displayed no significant difference (P > 0.05). AST contents in fish fed 1.5% (T4) ME were significantly lower than those in fish fed the control diet (P < 0.05). Notably, there were no significant differences between the other groups (P > 0.05). Moreover, the value of ALP decreased in fish groups fed the supplemented diets compared to the control group (P < 0.05). ME administration significantly decreased LDH levels in T3, T4, and T5 compared to the control group; the lowest level of LDH was recorded in T3 (P < 0.05).Enzyme parameters of rainbow trout fed different levels of ME are presented in P < 0.05). Moreover, all experimental groups showed lower AST levels than the control group (P < 0.05), though no statistical variations were recorded among them (P > 0.05). Compared to the control group, ALP and LDH contents were significantly lower in all treated groups. The lowest level of them was found in the T4 (P < 0.05).After the challenge, the value of ALT decreased in fish groups fed the supplemented diets, compared to the control group (P < 0.05); other treatments (T2 and T5) had shown no significant difference compared to the control treatment (P > 0.05). No statistical variations were observed in C3 and C4 values between the fish-fed experimental groups and the control treatment (P > 0.05). Significantly higher total Ig level was found in all experimental treatments compared with the control groups (P < 0.05); however, there were no significant differences between them (P > 0.05). Moreover, results exhibited a significantly higher amount of RBA in fish-fed ME diets than in the control group (P < 0.05). Fish treated with T3, T4, and T5 have significantly higher PRO activity than T1 (P < 0.05). Besides, T2 had shown no significant differences than T1 (P > 0.05). The highest level of PHA was recorded in the T4 group (P < 0.05); other treatments showed no significant difference than that of the control (P > 0.05).The effect of dietary ME on the immune parameters of rainbow trout is presented in P < 0.05). The other treatments displayed no significant differences. Additionally, the serum total Ig, RBA, PRO, and PHA levels were significantly higher in the T3, T4, and T5 compared to the control group (P < 0.05), T2 had shown no significant difference compared to the control treatment (P > 0.05).After the challenge, results exhibited the most significant amount of LYZ and C4 in the fish-fed T4 diet than in the control group (P < 0.05); other treatments had shown no significant difference. CORT assessment showed a significant decrease in T4 and T5 compared to the T1 (P < 0.05); meanwhile, no significant differences were observed among other dietary groups (P > 0.05).According to P < 0.05). Moreover, the value of CORT decreased in fish groups fed different doses of ME diets compared to the control group (P < 0.05).After the challenge, the GLU both in T4 and T5 were recorded as significantly lower than the control group (Stressors such as overcrowding depend on the duration and intensity and can cause acute or chronic stress responses . DevelopDicentrarchus labrax) [Sparus aurata) [Oreochromis niloticus) [The findings of this study showed that before the challenge, supplementing the diet with 1.5% ME had growth-promoting effects  on rainbow trout, whereas at high stocking density, 1%, 1.5%, and 2% ME increased fish growth performance compared to the control group. Although the exact mechanism for this effect is not clear, ME may have beneficial effects to reduce stress detrimental effects. Daily FI and growth rate reduction at short-term crowding stress were observed on several species, such as European seabass ( labrax)  and gilt aurata) . High st aurata) , 40. Add aurata) , 42. For aurata) . Similarloticus)  and rainloticus)  treated 2O2 in a concentration-dependent manner though indirectly raises the capability to mitigate oxidative stress and resist stress [Scophthalmus maximus) held at a high density. Similarly, Xie et al. [Cyprinus carpio) fed rhubarb  had higher hepatic CAT and SOD activities after 1 and 7 days of crowding stress. In another study, 1, 8-cineole (cineole) as an herb essential oil increased serum CAT, SOD, and decreased MDA. Meanwhile, after the stress, MDA decreased and CAT and SOD increased in the treated rainbow trout [Malvae sylvestris), oregano (Origanum vulgare), and Persian shallot (Allium hirtifolium boiss) [Coriandrum sativum), common mallow (M. sylvestris) fed oak acorn (Quercus brantii) [Momordica charantia) [Thymus vulgaris) [Under high stocking density, fish can disturb by the production of ROS in excess of body antioxidant capacity . Fish mut stress . Liu et t stress  reportede et al.  demonstrow trout . The enhm boiss) , coriandbrantii) , bitter arantia) , Nile tiulgaris) .Ctenopharyngodon idella) [Glycyrrhiza glabra) supplementations increased ALP activity in 10–30 g/kg licorice groups before and after stress [Liver metabolic enzymes are raised in cases of acute injury or dysfunction following disease, stressful situations, and toxicant exposure . ALT and idella) . In addi idella) . In contr stress . FurtherEucalyptus sp. EOs supplementation increased its activity in rainbow trout.An evaluation of serum immune parameters may demonstrate the potential of herbal additives as promising feed supplements. LYZ is an important natural antimicrobial protein of the innate immune system . It exerC. idella), which reflects a reduction tendency in nonspecific immune response [Complement proteins, such as C3 and C4, participate in different immune functions, including inflammatory responses, opsonization of pathogens, and clearance of homeostatic cells . The redresponse . Followiresponse .Salmo salar) subjected to crowding stress [A. hirtifolium) can regulate the immune system response by affecting the total Ig in Zebrafish (Danio rerio).Total Ig is a vital immune constituent and contributes to producing specific antibody responses , which ag stress . Meanwhig stress . Ghafarig stress  reportedApostichopus japonicus) [Withania sominefera root [RBA is an indication of oxidative potential in phagocytes through stimulation by ROS or foreign agents . The preponicus)  Also, RBera root .2O2) [2O2 is scavenged by CAT to form water and by PRO to oxygen which participates in the immune defense [β-glucan (BG) or/and vitamin C in red sea bream (Pagrus major) diets [PRO is an important microbicidal agent that eliminates hydrogen peroxide (H2O2) . H2O2 is defense . Resultsr) diets . Despiter) diets . Also, nr) diets .Echinacea purpurea extract [Boesenbergia rotunda) powder [Litopenaeus vannamei) [Labeo rohita) [Phagocytes like neutrophils, macrophages, and dendritic cells are recognized to avoid pathogen attacks and eliminate microorganisms in fish immune systems . PHA is  extract  and Thai) powder  on Nile annamei) ; and dri rohita) .GLU is used to estimate the stress response, and it is also the main energy source during starvation , 82. It Lippia alba  EO in silver catfish (Rhamdia quelen) [Myrcia sylvatica EO in gilthead seabream reduced CORT levels in high stocking density [Hormonal variations are the first level of the stress reaction . Overcro quelen) , as well density .The overall data of the current study showed that dietary 2% ME had greater synergistic interactions in stress-related responses of rainbow trout reared in high-density conditions. Therefore, the application of ME could be of interest as feed supplements to enhance the growth and health status and prevent stress-related immunosuppression of rainbow trout under intensive culture. However, further research is recommended to confirm the results obtained.


Lycium ruthenicum were subjected to NaCl treatments, and the growth characteristics, antioxidant enzyme activities, proline accumulation, and metabolic enzyme content were analyzed. The results revealed that NaCl concentrations between 50 to 150 mM have a positive effect on the growth of L. ruthenicum explants. Increasing NaCl concentrations elevated the activities of superoxide dismutase (SOD) and catalase (CAT), while hydrogen peroxide (H2O2) content was inhibited, suggesting that the elevated antioxidants play a central protective role in superoxide anion (O2•−) and H2O2 scavenging processes in response to NaCl treatments. Also, high proline levels also protect antioxidant enzyme machinery, thus protecting the plants from oxidative damage and enhancing osmotic adjustment. Increasing levels of pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR), and ornithine-δ-aminotransferase (δ-OAT) were observed, resulting in elevated level of proline. In addition, the expression levels of LrP5CS1, -2, -3, LrOAT-1, and -2 were promoted in NaCl treatments. According to the combined analysis of metabolic enzyme activities and their relative expression, it is confirmed that the glutamate (Glu) pathway is activated in L. ruthenicum faced with different levels of NaCl concentrations. However, Glu supplied by δ-OAT is fed back into the main pathway for proline metabolism.Salinity influences the level of antioxidants and proline content, which are both involved in the regulation of stress responses in plants. To examine the interplay between the antioxidant system and proline metabolism in plant stress acclimation, explants of  It has been shown that the key abiotic factor limiting plant growth is soil salinity, which negatively affects a number of biochemical and physiological processes. In order to preserve vital cellular functions such as photosynthesis, antioxidant synthesis, mineral homeostasis, osmolyte synthesis, and respiration from salt stress, plants modify their metabolic mechanisms ,2. These2•−) with itself to form hydrogen peroxide (H2O2) in chloroplasts and mitochondria, while catalase (CAT) and peroxidase (POD) can eliminate H2O2 and scavenge superoxide anion (O2•−) [2O2. The accumulation of H2O2, which results from the canalization reaction of the SOD enzyme, is prevented by the ascorbate–glutathione cycle [2O2 formed during the β-oxidation of fatty acids to water and molecular oxygen [Salt stress in plants leads to oxidative stress and an increase in the production of reactive oxygen species (ROS), which are regulated by the antioxidant system ,5. To prn (O2•−) ,7. SOD, ne cycle . The hydne cycle . CAT is r oxygen . POD andr oxygen ,11.2O2, buffering cytosolic pH and redox adjustment status of cells, stabilizing protein structure, and finally acting as a transmitter for stress tolerance [Proline is one of the numerous osmolytes, and among them is the most frequently accumulated in stressed plants . It is nolerance ,14,15. Aolerance . Also, eolerance .Arabidopsis thaliana, the Orn pathway has been shown to be more important for arginine catabolism and not for proline generation [In plants, the biosynthesis of proline consists of two sequential pathways: the glutamate (Glu) and ornithine (Orn) pathways ,19. Firsneration ,21. Howeneration ,23.Lycium ruthenicum Murr. (Solanaceae), commonly known as wolfberry, is a member of xerohalophytes that has developed strong adaptability to saline conditions and is widely distributed in Asia, Europe, and Africa’s arid and semiarid regions [L. ruthenicum have predominantly concentrated on ecological adaptation, anthocyanin content, economic, and medicinal benefits [L. ruthenicum showed a drastic increase in proline and antioxidant contents [L. ruthenicum. It has been vividly reported that proline accumulation in plants is a combined process of catabolism, biosynthesis, and transport [L. ruthenicum, investigate their expression profiles and the activities of enzymes involved in the steps of proline metabolism, and further confirm the proline metabolism pathway under NaCl treatments in L. ruthenicum. regions . It is a regions ,26,27. T regions ,29,30,31benefits ,33. Alsocontents ,34; howeransport ; therefop ≤ 0.05) trends were observed in shoot length, root length and root fresh weight of explants treated with 100 mM NaCl, which were 1.03, 1.04, and 0.34 times greater than the control, respectively. At high concentration (300 mM NaCl), the size of explants decreased compared with the control. NaCl treatments also resulted in changes in biomass, for instance, fresh weight of leaves and stems was significantly increased when treated with 150 mM NaCl, while the fresh weight of roots was higher when treated with 100 mM NaCl compared to control explants. The highest decrease in biomass occurred in explants treated with 300 mM NaCl. These changes are used as indicators of growth inhibition by NaCl treatment. These findings were confirmed in our study, in which 50 and 100 mM promoted root length; 100 mM promoted shoot length, root length, and root fresh weight; while 150 induced leaf and stem fresh weight.To explore phenotypic traits and biomass response to NaCl treatments, shoot height, root length, leaf fresh weight, stem fresh weight, and root fresh weight were measured. We found differences in biomass and morphological traits, although no visible damage was observed. Compared to the control, explants treated with different NaCl concentrations showed phenotypic changes in shoot height and root length and also resulted in changes in fresh biomass . SignifiL. ruthenicum were amphistomatic, with stomata on both the adaxial and abaxial surfaces. Our results revealed that the size of stomata was significantly smaller on both leaf surfaces in the control and high NaCl treatment than in moderate (50 mM) NaCl. The stomata density decreased with increasing NaCl, and the adaxial leaf surface had highest number of stomata than abaxial surface. The size of guard cells was higher in 250 mM NaCl treatments on the adaxial surface but higher on the abaxial surface in control. With increasing NaCl concentration, the size of leaf epidermal cells increased by 46.7% and 58.6% on both adaxial and abaxial surfaces, respectively, compared with the control . The H2O2 content showed a decreasing trend as a function of NaCl treatments, with the highest content in leaf and stem under control, whereas 50 mM NaCl treatment provided the highest concentration in the root compared with other NaCl treatments . Among the different NaCl concentrations, 250 and 300 mM exhibited the highest P5CS activity in leaf and stem, with 3.74- and 3.04-fold increases, respectively, compared with the control. The results in p ≤ 0.05) at 200, 250, and 300 mM NaCl for leaf, stem, and root, respectively, by 2.10-, 5.97-, and 2.56-fold compared to the control treatment  increase δ-OAT activity in L. ruthenicum explants, with the highest increase under 250 mM treatment. The root initially increased, then decreased at 150 mM treatment and increased again to optimum at 300 mM treatment with a 9.17-fold increase compared with control  values ≥ 5 were selected for further analysis under NaCl treatments , Lycium chinense (AIS36041.1), and Capsicum chinense (PHU13933.1) in the GenBank. The LrP5CR candidate protein also showed very high identity with P5CRs from other plants and had the highest identity of 95% with P5CR in Solanum stenotomum (XP_049384604.1). Both LrOAT1 and -2 candidate proteins also had a high identity of 95% with OAT in C. chinense (PHU09018.1) in the GenBank. The putative LrPDH1 and -2 proteins were 89% and 83% identical to Solanum verrucosum (XP_049346113.1) and Capsicum baccatum (PHT57387.1), respectively. The deduced protein sequences were further characterized by their basic features such as the number of amino acid residues, molecular weight, prediction of subcellular localization, and isoelectric point (pI)  .P5CS genes are expressed at different times in different tissues depending on NaCl concentration. The relative expression level of LrP5CS1 was significantly expressed in almost all tissues under 50 and 250 mM NaCl treatments. The relative expression of P5CS1 was much higher in all tissues under 250 mM treatment, especially with increasing treatment time; the relative expression under 50 mM treatment was also higher in leaf and stem after 6 h, and in stem and root after 12 h compared with control. LrP5CS2 is also strongly regulated in almost all tissues under both NaCl concentrations, although there are differences in expression patterns at different concentrations and treatment durations. Relative expression peaked in root and stem under 50 mM NaCl at 12 h compared with control but expressed higher in leaf at 6 h. Expression in the root peaked after 12 h at 50 mM treatment but decreased significantly at 250 mM treatment; an increasing trend in expression was observed in the leaf and stem. LrP5CS3 showed a higher expression pattern at both NaCl concentrations, except in the leaf under 250 mM treatment, which was not expressed throughout the treatment periods. In general, the expression of P5CS3 under 50 mM is higher than under 250 mM, except for 250 mM treatment under 6 h leaf ; the expression of leaf was significantly higher under 250 mM treatment than under control and 50 mM treatments. In 250 mM treatment, expression in the stem peaked after 6 h and was 11.6-fold higher than the control  increased in the leaf, stem, and root under 50 mM treatment and peaked at 12 h compared with the control. Expression of LrProDH1 peaked only in the root, whereas LrProDH2 peaked in the leaf and root at 6 h under 250 mM treatment. Also, under 50 mM at 12h, the expression levels are much higher compared to 250 mM NaCl treatment  are the main antioxidant enzymes that scavenge excess ROS in plants [2•− to H2O2 whereas CAT rapidly decomposes H2O2, producing H2O and O2 [2O2 level. Li et al. [2O2 to seedlings of glycophyte Triticum aestivum under salt stress increased SOD and CAT activities. Thus, our results suggest that the coordination of CAT activity with SOD activity plays a central protective role in the O2•− and H2O2 scavenging process in L. ruthenicum explants for better survival under stress. POD is reported to be involved in scavenging of H2O2, but its role is unclear in L. ruthenicum since NaCl treatments inhibited its activity.When exposed to salt stress, plants always produce a large amount of reactive oxygen species (ROS), which then cause oxidative stress. In this study, Hn plants . SOD is O and O2 ,54. In ti et al.  reportedL. ruthenicum to high NaCl concentrations. In xero-halophyte species A. canescens, proline accumulation was highly elevated by high salinity (200 and 400 mM NaCl) but was insignificantly affected by moderate concentration (100 mM) of NaCl. Proline can also reduce oxidative damage through stabilization of antioxidant enzymes in stressed plants [Pancratium maritimum; however, their activities were promoted significantly in the presence of exogenous proline [O. europaea leaves under stress [L. ruthenicum may protect antioxidant enzyme machinery and thus protect the plant from oxidative damage under stress conditions.Proline content in all tissues was elevated significantly under 250 mM NaCl treatment, with no evidence of explant injury, suggesting that proline may also be involved in physiological response of  proline . Also, er stress . This inAtriplex canescens, it was found that proline accumulation was significantly correlated to Ψs under high salinity conditions (200 and 400 mM NaCl) [L. ruthenicum.The increase in the accumulation of soluble protein and proline indicate their contribution to osmotic adjustment under NaCl treatments. In addition, the contribution of proline to Ψs showed significant increase under high concentration of NaCl treatments and accounted for 1.5% under 250 mM NaCl. Also, in halophytic mM NaCl) ,44. This+ that can be further used as an electron acceptor in the oxidative pentose phosphate pathway [L. ruthenicum, P5CS activity and expression of LrP5CS1, -2 and -3 genes were promoted under high NaCl treatments. Other scholars have reported that in hybrid glycophyte Medicago sativa, increased in P5CS activity and P5CS expression after NaCl treatment revealed an involvement of P5CS in proline biosynthesis [K. virginica, it is reported that overexpression of KvP5CS1 resulted in proline hyperaccumulation [VaP5CS and LrP5CS in some glycophyte species such as Vigna aconitifolia and Lilium regale revealed proline accumulation and consequently enhanced stress tolerance [L. ruthenicum under high NaCl concentration. In the cytosol, the P5C intermediate is reduced to proline by P5CR. Similar to L. ruthenicum, it was confirmed by Armengaud et al. [L. ruthenicum, analysis of P5CR activity and LrP5CR gene revealed that NaCl treatments elevated its activity and relative expression. Correlation between increasing P5CR activity and proline accumulation was observed in halophytic Mesembryanthemum crystallinum, and a moderately salt-tolerant Morus alba in NaCl stress [L. ruthenicum faced with different levels of NaCl treatments, as LrP5CS1, -2, -3 and LrP5CR genes were greatly expressed.Since proline accumulation is the result of a combination of biosynthesis and catabolism, it was necessary to analyze the activities of proline enzymes, the genes associated with metabolism, and their relative expression patterns involved in these processes to understand the mechanism of proline metabolism. Proline biosynthesis is a reductive pathway and uses NADPH to reduce glutamate to P5C and P5C to proline and generates NADP pathway . Plants  pathway . Prolineynthesis . Also, imulation , and theolerance ,61,62,63d et al.  and Verbd et al.  that P5Cl stress ,67. OverL. ruthenicum, two isoforms of δ-OAT were discovered, δ-OAT activity and the relative expression levels LrOAT1 and -2 were promoted when faced with NaCl concentrations. Funck, Stadelhofer, and Koch [L. ruthenicum. However, there is no clear evidence of mitochondrial P5C moving to the cytoplasm or chloroplast for proline synthesis; hence, Glu supplied by δ-OAT is fed into the standard pathway for proline synthesis. More direct studies are required to confirm proline biosynthesis via δ-OAT in L. ruthenicum.Studies have revealed that the Glu pathway is essential for proline biosynthesis during stress regulation in most species rather than the Orn pathway . δ-OAT hand Koch  showed land Koch . Our resL. ruthenicum; a similar number of genes has also been described in the genomes of Nicotiana tabacum cv. [LrProDH1 and -2) and ProDH activity analysis revealed an elevated response in NaCl treatments in this study. However, it has been suggested that one ProDH gene serves general metabolic functions, whereas the other is involved in stress-induced adaptation mechanisms [LrP5CDH, which is the second enzyme of proline catabolism.The function of proline is dose-dependent, and a high concentration could cause a toxic effect in plants ,68. Prolacum cv. . The relchanisms ,69; thusL. ruthenicum used for this study were conserved in Tissue Culture Laboratory of College of Forestry, Gansu Agricultural University, China. About 2–3 cm young shoots were used as explants, and the roots of the explants were induced using ½ Murashige and Skoog nutrient medium (MS medium) supplemented with 0.2 mg/mL indol-3-butyric acid (IBA) and 1mg/mL naphthalene acetic acid (NAA). All cultured explants were incubated in growth chamber at 25/22 °C (day/night) with a relative humidity of 65% and a photoperiod of 16/8 h (light/dark) for three weeks. After rooting, explants of uniform growth (10 cm) were transplanted into rectangular shape plastic vessels with modified ½ Hoagland nutrient solution [Explants of solution  to develL. ruthenicum explants, whereas 6 and 12 hours (h) were used for analysis of gene expression in response to NaCl treatment. For gene expression, the explants were exposed to 0, 50, and 250 mM NaCl and placed in a growth chamber at 25 °C and 65% relative humidity using the same experimental design. Under the same light condition, the treatments were applied from 8 a.m. to 8 p.m., and samples were collected at 2 p.m. for 6 h and then at 8 p.m. for 12 h. The leaves, stems, and roots were separated after thorough cleaning with double distilled water and quickly frozen in liquid nitrogen and stored at −80 °C for further analysis. Stress responses involve slower adjustments in enzymatic activity that may not be fully evident in shorter time periods, but transcript levels of genes have been reported to exhibit rapid and transient induction of their expression upon stress exposure, which is important for activation of defense mechanisms and initiation of adaptive processes . Therefo2), which is defined as an ellipse with its major axis equal to guard cell length and its minor axis equal to the width of the entire stoma; guard cell size (μm2), which is length and width of the guard cell; and leaf epidermal cell area (μm2), which was calculated through the number of pixels inside the borderline. The epidermal surface of leaves from 14 d explants under NaCl treatments  was examined with a scanning electron microscope , and 10 mm pieces were cut out of the leaves. The pieces were mounted on SEM stubs with microscopic slide; the slide was cooled with ice close to 0 °C prior to insertion into the SEM in order to slow down desiccation. Four images on both abaxial and adaxial sides of the leaves were taken. ImageJ version 1.47  was used for determination of the following parameters: stomata density, which was manually counted from all the pictures; stomata size . Fresh leaf tissue of about 0.5 g was collected in a freezing point mode at room temperature, transferred to a microcentrifuge tube, and finely ground using TGrinder . The sample supernatant was collected after centrifugation at 12, 000× g for 15 min at room temperature [The leaf Ψs was analyzed using Fiskeperature . The vanperature ,73. Then2O2 (no. H2O2-1-Y) content were determined using assay kits from Comin Biotechnology Co., Ltd., Suzhou, China (www.cominbio.com). All absorbance values were read using Perkin Elmer Singapore Pte. Ltd., Enspire® Multimode plate reader.The proline accumulation (no. PRO-1-Y) and soluble protein (no. BCAP-2-W), antioxidant enzyme activity (CAT (no. CAT-1-Y), SOD (no. SOD-1-Y) and POD (no. POD-1-Y)), metabolic enzyme activity ((P5CS (no. P5CS-1-Y), P5CR (no. P5CR-2-W), δ-OAT (no. OAT-1-Y), ProDH (no. ProDH-1-Y)), and HProline and soluble protein content, CAT, SOD, and POD activities were performed according to the Comin Biotechnology Co., Ltd., Suzhou, China, methods described by Wang, Gao, Sun, Lu, Li, Li, Wang, and Liu . All the2O2 was evaluated using a test kit based on the titanium sulfate principle [2O2 and titanium sulfate form a yellow titanium peroxide complex that exhibits characteristic absorbance at 415 nm. Briefly, 0.1 g of fresh sample was homogenized in 1 mL of extraction solution, and the homogenate was centrifuged at 8000× g for 10 min at 4 °C. Absorbance values were recorded as Am415 nm for the measuring tube and Ac415 nm as the control tube. Change in A (ΔA) was calculated as A measuring tube—A control tube. H2O2 content = 1.34 (ΔA − 0.0006)/W, where W represents the sample fresh weight. Hrinciple  with somrinciple . H2O2 an2∙6H2O, KCl, EDTA, DTT, and PVP) and centrifuged at 8000× g for 10 min at 4 °C. The supernatant was collected for enzymatic reaction. The enzymatic reaction consisted of 100 µL supernatant and 100 µL Tris-HCl buffer ; then, the reaction was placed under 37 °C for 10 min and centrifuged at 8000× g for 10 min at 25 °C. The supernatant was collected for chromogenic reaction, which contained 20 µL of supernatant and 200 µL chromogenic mixture  (test sample). The reaction was then placed under room temperature for 30 min, and the absorbance was recorded at 660 nm. A blank control, standard control and positive control were also measured, which contained 20 µL of sterilized water, 20 µL of standard phosphorus application solution, and 20 µL of enzymatic control. The control tube for the enzymatic reaction was not incubated for 10 min. This method has the characteristics of traceability, sensitivity, and rapidity; therefore, the test tubes used in the determination are strictly required to be phosphorus-free. The P5CS activity (U/mg protein) was calculated as standard control × (assay sample − positive control) ÷ (standard control − blank control) × total volume of enzymatic reaction ÷ (sample protein content × volume of extract used) ÷ reaction time.P5CS activity was assayed based on the glutamyl phosphate reductase activity as described previously by Campanile et al.  with somg for 10 min at 4 °C. The supernatant was collected for the final reaction. The final reaction mixture contained 10 µL of supernatant and 190 µL of working solution ). The control reaction mixture contained 10 µL supernatant and 190 µL control solution . The reactions were incubated at 37 °C in dark for 30 min, and the absorbance was recorded at 450 nm. Change in absorbance (ΔA) was calculated as final reaction − control reaction, and the P5CR activity was calculated as (△A + 0.0285) ÷ 1.3384 × total volume of enzymatic reaction ÷ (sample protein content × volume of extract used) × 1000 ÷ reaction time.The extraction and analysis of P5CR activity was based on the method of Rena et al. . Briefly2HPO4∙H2O-KH2PO4 buffer solution containing EDTA, mercaptoethanol, and glycerol) and then centrifuged at 10,000× g for 10 min at 4 °C, and the supernatant was collected for final reaction. The final reaction mixture contained 20 µL supernatant, 60 µL ornithine, 60 µL α-ketoglutaric acid, and 60 µL NADH, and the change in absorbance was calculated at 340 nm from initial absorbance and final absorbance at 37 °C for 10 min reaction. The activity of δ-OAT was calculated according to the sample protein content. δ-OAT activity analysis was performed according to Charest et al.  with som2HPO4-NaH2PO4 buffer solution with EDTA). After centrifugation at 1500× g for 15 min at 4 °C, 10 µL of TritonX-100 was added to the supernatant and placed in an ice bath for 30 min, then centrifuged at 16,000× g for 20 min at 4 °C. The supernatant was collected for final reaction, which contained 35 µL supernatant, 15 µL PMS, and 150 µL mixed solution (2.4 mL Na2CO3-NaHCO3 buffer solution: 0.3 mL L-proline: 0.3 mL DCPIP), and the decrease in absorbance was monitored at 600 nm in an interval of 10 min. One enzyme activity unit was defined as the alternation of 0.01 absorbance in 1 mL of reaction system per minute by 1 mg of protein, and the result was expressed as U/mg protein.ProDH activity was measured using methyl isothiocyanate. Briefly, 0.1 g of fresh sample was homogenized in 1 mL of extract solution  was used to analyze the basic characteristics of the encoded proteins. Protein subcellular localization prediction was performed using the online DeepLoc-1.0  tool.The sequence information of proline metabolic enzymes P5CS, P5CR, δ-OAT, ProDH, and P5CDH was obtained from Sequence Read Achieve (SRA) database of NCBI (SRR7700825)  and our Evo M-MLV RT Kit, no. AG11705  was used to reverse-transcribe the total RNA into cDNA and for removal of any possible genomic DNA mixed in the cDNA before RT-qPCR analysis, following the manufacturer’s instruction.Total RNA was extracted from leaf, stem, and root using TransZol Up Plus RNA Kit (no.M31018)  conferring to the manufacturer’s instruction. The RNA quantity and quality were determined using TGen Spectrophotometer  based on the A260 nm/A280 nm and A260 nm/A230 nm ratios. L. ruthenicum Elongation factor1-α (LrEF1-α) gene (JX427553) was used as a housekeeping gene . Synthetic cDNA was diluted in a 4-fold series  and the method described by Chang et al. [® qPCR SYBR Green Master Mix kit , three biological replicates and triplicate quantitative analysis were performed according to the manufacturer’s protocol. The RT-qPCR assay was carried out using the QuantStudio™ 5 Real-Time PCR Instrument (ABI) . The 2−ΔΔCt method [The RT-qPCR analysis was conducted following the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines  and geneg et al.  was usedt method  was usedp ≤ 0.05. ANOVA was used to analyze the differences between treatments in each tissue and time. All graphical presentations were performed using Graphpad Prism version 8.Data of the three biological replicates tooling six explants per treatment and 3 analytical replicate were performed using a general linear model (SPSS version 23); the means and standard deviation (SD) of replicates were calculated. Data were compared between treatments and control by analysis of variance (ANOVA) in SPSS version 23 to separate significant differences, followed by Duncan’s multiple range tests with least significant difference (LSD) at L. ruthenicum can resist high NaCl concentration, and moderate NaCl concentration is useful for growth promotion and biomass production. Increasing NaCl concentration increased the contents of SOD and CAT, but inhibited the activity of POD, which enhanced the antioxidant system of L. ruthenicum to scavenge ROS. Our results revealed that NaCl treatment leads to an increase in proline level in L. ruthenicum, which suggested proline protection of antioxidant enzyme machinery and thus protected the plants from oxidative damage under NaCl treatment. The increase in P5CS, P5CR, and δ-OAT activities in response to NaCl treatments promoted the level of proline in L. ruthenicum. Proline metabolism enzymes related genes LrP5CS1, -2, -3, LrP5CR, LrOAT-1, -2, LrProDH-1, -2, and LrP5CDH were identified. RT-qPCR analysis confirmed that their expression profiles were promoted in response to NaCl treatments, indicating their roles in proline metabolism. Overall, the results of this study confirmed that the Glu pathway is involved in proline accumulation in L. ruthenicum under stress conditions.In this study, it was shown that