Leukaemia occurs when specific mutations promote aberrant transcriptional and proliferation programs, which drive uncontrolled cell division and inhibit the cell’s capacity to differentiate. In this review, we summarize the most frequent genetic lesions found in myeloid leukaemia of Down syndrome, a rare paediatric leukaemia specific to individuals with trisomy 21. The evolution of this disease follows a well-defined sequence of events and represents a unique model to understand how the ordered acquisition of mutations drives malignancy.CTCF or EZH2—and in signalling mediators of the JAK/STAT and RAS pathways. Most of them are also found in non-DS myeloid malignancies, albeit at extremely different frequencies. Intriguingly, mutations in proteins involved in the three-dimensional organization of the genome are found in nearly 50% of cases. How the resulting mutant proteins cooperate with trisomy 21 and mutant GATA1 to promote ML-DS is not fully understood. In this review, we summarize and discuss current knowledge about the sequential acquisition of genomic alterations in ML-DS.Children with Down syndrome (DS) are particularly prone to haematopoietic disorders. Paediatric myeloid malignancies in DS occur at an unusually high frequency and generally follow a well-defined stepwise clinical evolution. First, the acquisition of mutations in the GATA1 transcription factor gives rise to a transient myeloproliferative disorder (TMD) in DS newborns. While this condition spontaneously resolves in most cases, some clones can acquire additional mutations, which trigger myeloid leukaemia of Down syndrome (ML-DS). These secondary mutations are predominantly found in chromatin and epigenetic regulators—such as cohesin,  Trisomy 21 is the most common chromosomal disorder in humans and is the genetic basis of Down syndrome (DS) ,4,5, indML-DS is characterized by a distinctive multi-step evolution in which it is always preceded by a pre-leukaemic condition known as transient abnormal myelopoiesis (TAM) or transient myeloproliferative disorder (TMD) ,8 Figur. All TMDGATA1 mutations [CTCF, the two main drivers of three-dimensional (3D) genome folding. This high frequency is unexpected, especially for CTCF which is rarely mutated in AML or other myeloid malignancies. Moreover, chromatin modifiers such as EZH2 are more frequently mutated in ML-DS than in AML. Signalling pathway mutations are mostly found in receptors and members of the Janus kinase-signal transduction and activator of the transcription (JAK-STAT) signalling cascade, as well as in the RAS pathway. These mutations may interfere or cooperate with altered signalling pathways in DS. In general, however, the interplay between the three genetic elements comprising this disease—trisomy 21, GATA1 mutations and secondary mutations—is poorly understood. Here, we provide an overview of the current knowledge on how these elements may promote ML-DS.The molecular mechanisms involved in the progression from TMD to ML-DS are not fully understood. Large-scale sequencing studies have identified the most frequent mutations acquired in ML-DS in addition to utations ,10,21. SERG, ETS2 and RUNX1, are located on chromosome 21. Overexpression of Erg and Ets2 induce megakaryocytic expansion and contribute to a myeloproliferative phenotype in mice [Dyrk1a, induces a marked megakaryocytic expansion [ERG and RUNX1 [RUNX1, ETS2 and ERG in human trisomy 21-induced pluripotent stem cells (iPSCs) suppresses the altered differentiation phenotypes [Trisomy 21 foetal livers have an abnormal haematopoietic development. Specifically, there is an increased frequency of haematopoietic stem cells (HSCs), which also show increased clonogenicity and megakaryocytic–erythroid output. This results in an expansion of megakaryocyte–erythroid progenitors (MEPs) and a decrease in granulocyte–macrophage progenitors (GMP). In addition, B-cell differentiation is impaired ,25,26,27 in mice ,29,30. Ixpansion . Howeverenotypes .RUNX1 and FLI1) were found to be hypermethylated in DS samples [Trisomy 21 can also interfere with gene expression by altering DNA methylation patterns. DNA methylation profiling at different stages of ML-DS development revealed hypomethylation in early stages and hypermethylation in advanced stages . Interes samples .Gata1 mutation) and a third event (MplW515L) [Different mouse models of partial trisomy 21 have been used to understand DS haematopoietic development. In mice, the orthologous regions of human chromosome 21 are located in chromosomes 10, 16 and 17. The most commonly used DS mouse model (Ts65Dn) carries an extra copy of 104 genes of mouse chromosome 16 . These mplW515L) . While tplW515L) ,31.GATA1, GATA2 and GATA3 are expressed in haematopoietic cells, while GATA4, GATA5 and GATA6 are expressed in various tissues including the heart, intestine and lung [GATA1 is located on chromosome X [Gata1 in mice leads to impaired erythropoiesis. In addition, mice with selective loss of Gata1 expression in the megakaryocytic lineage have a markedly lower frequency of platelets and impaired megakaryopoiesis [The GATA family consists of six transcription factors (GATA1 to GATA6) that bind to the same DNA consensus sequence through a highly conserved zinc finger domain ,37. Accoand lung . In humamosome X  and is mmosome X ,41, eosimosome X , mast cemosome X , megakarmosome X ,44 and Smosome X ,46. The mosome X . GATA1 hopoiesis .GATA1 resulting in the introduction of a premature stop codon which promotes the exclusive translation of GATA1s [All TMD and ML-DS cases carry somatic mutations in f GATA1s . Mechanif GATA1s ,50,51,52GATA1 mutations lead to aberrant and accelerated production of poorly differentiated haematopoietic cells [hi megakaryocyte precursors accumulate during late megakaryopoiesis, causing a reduction in apoptosis and an increase in the number of cells in S-phase [Cellular models of TMD and GATA1s show that trisomy 21 and ic cells . During ic cells . Specifi S-phase . Moreove S-phase , favouriCTCF is also recurrently mutated in non-DS AMKL (10–21%) [EZH2 and SUZ12 or the chromatin modifier KANSL1. Analysis of the clonal origin of mutations shows that cohesin, CTCF and EZH2 mutations may have essential roles in the early stages of ML-DS progression [RUNX1, TP53, WT1, CREBBP and MYC (~8%). The spliceosome component SRSF2 is mutated in ~8.5% of cases. Among signalling-related genes, the JAK-STAT pathway shows the higher frequency of mutations (48%), followed by members of the RAS family (14%) [The progression from TMD to ML-DS requires at least one additional mutation in the GATA1s clones. The advent of large-scale sequencing studies over the last decade has enabled the identification of recurrent mutations in ML-DS patients ,10,21. T(10–21%) ,55. In agression . DeletioCTCF in less than 1%. JAK1 and JAK3 are rarely mutated in AML, whereas NRAS is more often found mutated in AML than in ML-DS. In addition, the most frequent mutations in AML are almost absent in ML-DS. This is the case of FLT3, DNMT3A and NPM1 [IDH1/2 or CEBPα. Similarly, spliceosome and epigenetic proteins such as SF3B1 and TET2, which are found among the most frequently mutated proteins in myelodysplastic syndromes (MDS), are only mutated in less than 3% of cases in ML-DS.Most of these mutations are also found in AML and other myeloid malignancies, but the frequencies at which they are found can be extremely different . Cohesinand NPM1 ,57, and GATA1 mutations occur in 39.2% and ~10.0% of cases, respectively, even though none exhibit physical phenotypes consistent with DS. Paired-sample analyses reveal that 90% of patients harbouring GATA1 mutations also carry an extra copy of chromosome 21 [While ML-DS and non-DS AMKL share common morphological and immunophenotypic features ,59, theyosome 21 .Transcriptional and chromatin regulators are extremely frequently mutated in myeloid malignancies ,64, inclSTAG2, SMC1A, SMC3, RAD21 and NIPBL, are frequently found in several cancer types such as bladder cancer, Ewing sarcoma and glioblastoma [STAG2, representing ~42% of all cohesin mutations [Mutations in members of the cohesin complex, including blastoma . Cohesinblastoma ,66,67,68blastoma ,70,71. Mblastoma ,57,68. Cblastoma , mutatioutations ,21. Thesutations ,21. The utations , negativSmc3 or Stag2 conditional haploinsufficiency [STAG2 knock-out [In order to understand the function of the cohesin complex in normal and malignant haematopoiesis, several groups have studied the impact of partially or totally deleted cohesin in haematopoietic cells ,76,77,78ficiency ,74, as wficiency , all disficiency . In lineficiency . On the nock-out .Apart from its role in sister chromatid cohesion, cohesin has a critical function in the 3D organization of the genome. The formation of TADs is thought to result from the combined action of cohesin and the DNA-binding factor CTCF. Current models posit that cohesin extrudes chromatin until it encounters two convergently oriented CTCF molecules, resulting in chromatin loops anchored at CTCF sites ,80,81. AEbf1 gene, a key lymphoid lineage determining transcription factor. In conditional mouse models of Stag2 deficiency, the altered 3D structure surrounding Ebf1 impairs its expression, leading to a blockade of B-cell differentiation [RUNX1 mutations in MDS. STAG2 and RUNX1 colocalise at a subset of enhancer elements, and the combined absence of the two factors alters chromatin contacts in a cooperative manner [Etv6 from its binding sites, which impairs normal differentiation [The deregulation of specific subsets of enhancer-dependent genes may explain the altered balance of HSPC subsets and impaired lineage commitment of cohesin-deficient mouse models. Inflammatory signals are key regulators of the balance between self-renewal and differentiation of HSPCs ,90,91. Tntiation . It has e manner . An intentiation .Different therapeutic strategies have been proposed to target cohesin-mutant cells (reviewed in ). RecentCTCF mutations are recurrently observed in both ML-DS (11.3–20.0%) and non-DS AMKL (10–21%). These can be frameshift, nonsense, missense or splice-site mutations, and they mostly occur in the region containing the zinc finger domains [CTCF mutations are due to large deletions [GATA1, suggesting that they are early events in the clonal expansion during the TMD to ML-DS transition [CTCF mutations are much less frequent in AML (2%) [ domains ,21,55, w domains ,98. A toAML (2%)  and in MAML (2%) . In contAML (2%) ,101 and AML (2%) ,103.As part of its key role in the formation of TADs ,106,107,Ctcf-hemizygous mice features altered CTCF binding at poorly conserved sites. Consequently, several hundred genes are deregulated, many of which are involved in cancer signalling pathways such as PI3K-AKT [Ctcf allele in mice results in more aggressive tumours, with high rates of local invasion and metastatic dissemination [CTCF expression is induced in cancer cell lines, it promotes a decrease in proliferation and in clonogenic capacity [Ctcf haploinsufficiency promotes cellular proliferation, colony formation and cell cycle progression [CTCF are found in many cancers, including uterine (~25%), stomach (~7%), bladder (~6%) and breast-invasive carcinoma (~4%) [PI3K-AKT . The losmination . Additiocapacity . The oppgression . It is tma (~4%) ,121,122.CTCF knock-out trisomic GATA1s progenitors from foetal liver did not drive leukemic transformation upon transplantation in mice [CTCF mutations may not promote the same leukaemogenic effects, and that CTCF mutation may require additional events on top of GATA1 mutations. Further studies set in a DS genetic background are needed to elucidate the role of CTCF in this disease and to identify actionable targets.As in the case of cohesin mutations, the unusually high frequency found in ML-DS suggests that the partial absence of CTCF function may cooperate with the dosage imbalance of chromosome 21 genes, with specific megakaryocytic transcriptional regulators, or with both. Unlike cohesin knock-out, however,  in mice , suggestEZH2 is over-expressed [EZH2 gene in ML-DS [EZH2 mutations in MDS and AML are associated with bad prognosis and acquired chemoresistance [Ezh2 or Suz12 loss-of-function mouse models show increased HSC repopulation capacity, suggesting that normal levels of PRC2 restrict HSC activity [HOX genes de-repression, which can be reversed by a combination of bortezomib and cytarabine [EZH2 mutations can cooperate with other mutations to promote malignancy, such as with RUNX1 [NRAS [RUNX1 gene resides on chromosome 21 and NRAS mutations are frequently found in the disease. Similar to the case of CTCF, EZH2 knock-out foetal liver progenitors with trisomy 21 and GATA1s expression did not drive leukemic transformation upon transplantation in mice [EZH2 mutations act in cooperation with additional mutations in ML-DS.EZH2 and SUZ12 are subunits of the PRC2 complex that catalyses the deposition of di- and tri-methylation on H3K27, which is then recognized by PRC1, which mono-ubiquitinates H3K119. Altogether, the activity of PRC1 and PRC2 promotes chromatin compaction and transcriptional silencing . Both gexpressed ,125 or cxpressed . Howeverxpressed ,127,128.in ML-DS . Howeverin ML-DS  , all of which are ubiquitously expressed, except for JAK3, which is restricted to a few tissues, including the haematopoietic system [The JAK-STAT signalling pathway is critical for haematopoietic development and immune function as it is involved in the rapid transduction of multiple cytokines, hormones and growth factor signals from the cell membrane to the nucleus. Specifically, the JAK-STAT pathway mediates the transmission of type I and type II cytokines, including interleukins, interferons, erythropoietin (EPO), thrombopoietin (TPO), GM-CSF, prolactin and the growth hormone . The binc system ,139. Memc system . Once inc system . Unphospc system .JAKs are mostly found in haematological malignancies. In myeloproliferative neoplasms, the JAK2V617F mutation accounts for 98% of cases of polycythaemia vera and around 50% of essential thrombocythaemia and myelofibrosis [JAK1, JAK2 and JAK3 have been found in lymphoblastic leukaemia [STAT3, STAT5 and STAT6 also carry somatic mutations in B- and T-cell leukaemias [STAT3 overexpression has been noted in solid tumours as in uterine, lung, ovarian, gastric and brain cancers [As the JAK-STAT pathway is crucial for growth and survival of immune cells, gain-of-function mutations in fibrosis ,146,147.eukaemia ,149,150.ukaemias ,152. STA cancers .MplW515L in bone marrow cells from Gata1s/Ts1Rhr mice causes thrombocytosis and intense bone marrow fibrosis, leading to lethal leukaemia in recipient mice [CSF2RB, a coreceptor of IL-3, IL-5 and GM-CSF, was detected in 4.7% of ML-DS patients [Missense and non-frameshift indel mutations usually affecting the pseudokinase domain of JAK1, JAK2 and JAK3 are frequently found in ML-DS and in some TMD cases ,161,162.ent mice . In addipatients . CSF2RB patients ,165. Mecpatients . There apatients . FurtherRAS proteins belong to a superfamily of low molecular weight GTP-binding proteins that are involved in signalling transduction and control of cell proliferation and survival. RAS proteins are active when they are bound to GTP and inactive when bound to GDP . ActivatRAS genes are present in 20% of all human tumours, most frequently in KRAS (85%), NRAS (15%) and HRAS (fewer than 1% of cases) [RAS genes compromise the hydrolysis of GTP, and consequently RAS accumulates in the GTP-bound, active form. KRAS is predominantly mutated in solid tumours  [KRAS, NRAS and NF1 are found in 14% of patients. The majority are gain-of-function as they involve the GTP-binding domain [NRAS, KRAS and PTPN11 (15.7%) [Oncogenic mutations in f cases) . The maiarcinoma , 42% of arcinoma  and 33% arcinoma ), whereaia (15%) . The RASia (15%) ,176. RASia (15%) . There aia (15%) . In MDS,ia (15%) . In ML-Dg domain ,21. Paed (15.7%) .G12C in non-small cell lung cancer (NSCLC) [EZH2 show increased dependency on RAS signalling, which renders tumour cells more sensitive to MEK inhibitors [The development of drugs targeting mutant RAS proteins has yielded almost no molecules available for clinical use . However (NSCLC) . An alte (NSCLC) . This cohibitors .GATA1, and secondary lesions in transcriptional regulators and signalling proteins. While the aberrant foetal haematopoietic differentiation of trisomy 21 may provide a basis for the selective advantage of clones with GATA1 mutations, it is yet unclear why mutations in cohesin and CTCF are so abundant in ML-DS. In particular, several questions are still unanswered: How is the 3D genome organization affected by cohesin and CTCF mutations in ML-DS? What genes and pathways are deregulated? Do they cooperate or synergize with chromosome 21 genes or GATA1s? Do cohesin and CTCF mutations cause the same oncogenic effects, or alternatively, do they promote ML-DS through different mechanisms? Cellular and animal models that incorporate all the elements of this disease will be key in providing answers to these questions.Three main genetic elements characterize ML-DS: trisomy 21, mutations in The well-defined sequence of events that leads to ML-DS represents a suitable model to understand the stepwise acquisition of mutations that is thought to occur during the clonal expansion leading to AML. Moreover, most mutations found in ML-DS are also found in AML, albeit at different frequencies. Current data indicate that the specific combination and order in which mutations are acquired is critical for AML development ,183,184.


We report on the development of nephrotic proteinuria and microhematuria, with histological features of renal thrombotic microangiopathy (TMA), following the first dose of BNT162b2 COVID-19 vaccine (Pfizer-BioNTech) and COVID-19 diagnosis. A 35-year-old previously healthy man was admitted at our hospital due to the onset of foamy urine. Previously, 40 days earlier, he had received the first injection of the vaccine, and 33 days earlier, the RT-PCR for SARS-CoV-2 tested positive. Laboratory tests showed nephrotic proteinuria (7.9 gr/day), microhematuria, serum creatinine 0.91 mg/dL. Kidney biopsy revealed ultrastructural evidence of severe endothelial cell injury suggestive of a starting phase of TMA. After high-dose steroid treatment administration, complete remission of proteinuria was achieved in a few weeks. The association of COVID-19 with renal TMA has been previously described only in patients with acute renal injury. Besides, the correlation with COVID-19 vaccine has not been reported so far. The close temporal proximity (7 days) between the two events opens the question whether the histological findings should be ascribed to COVID-19 itself or to vaccine injection. Until the end of June 2021, more than 182 million confirmed SARS-CoV-2 infections had been reported in over 200 different countries with 3.9 million deaths. Up to now, about 4.21 billion doses of vaccine have been administered worldwide. The potential adverse effects of COVID-19 vaccines represent a hot topic of current research. Major side-effects appear to be uncommon, although some cases of new onset or relapsing minimal change disease  have recWe describe here a case of nephrotic proteinuria and microhematuria, with histological features of renal thrombotic microangiopathy (TMA), without AKI, starting 40 days after the first injection of the BNT162b2 COVID-19 vaccine (Pfizer-BioNTech) and 33 days after SARS-CoV-2 positivity detection through PCR test.This is a case report of renal TMA following COVID-19 infection and the first dose of Pfizer-BioNTech vaccination. A written informed consent was obtained from the patient to publish this paper. Approval from local ethics committee was waived, as it is not required at our institution for a single case report.A 35-year-old previously healthy man was admitted at our nephrology center following the onset of foamy urine. Previously, 40 days earlier, he had received the first injection of Pfizer-BioNTech vaccine and 33 days earlier the RT-PCR test for SARS-CoV-2, required for the presence of anosmia, was positive. He described moderate fatigue the day after vaccination and anosmia the day before SARS-CoV-2 test. During the first 3 weeks, he reported intermittent intake of paracetamol. The subsequent clinical symptoms were dysgeusia, myalgia, pharyngodynia. It is also possible that proteinuria was already present before the patient noticed foamy urine. The timeline of clinical events is shown in 3/µL, platelets 226 × 103/µL. HBsAg, HCV and HIV Ab were negative, C3 and C4 levels were also unaltered, and ANCA, anti-GBM Ab, ANA and anti-PLA2R Ab were negative. PCR test for COVID-19 was negative. The antibody titer of IgG against spike protein subunits S1 and S2 (S1/S2-IgG), measured using an indirect chemiluminescence immunoassay , was 62 AU/mL. Spot urinalysis revealed a moderate presence of proteins (300 mg/dL). Urinary sediment showed 476 red blood cells PHPF, 24-hr urinary protein excretion was 7.9 grams. At renal ultrasound, kidneys appeared of normal size with no evidence of urinary tract obstruction. Blood pressure and heart rate were regular. Physical examination did not reveal edema. Biochemistry and hematology tests were within the normal range: serum creatinine 0.91 mg/dL, albumin 4.5 g/L, cholesterol 170 mg/dL, triglycerides 77 mg/dL, hemoglobin 14.5 g/dL, white blood cell count 4.35 × 10Percutaneous kidney biopsy detected 44 glomeruli: at light microscopy A,B, 2 ofIn summary, light microscopy and immunofluorescence seemed to be consistent with minimal change disease, but electron microscopy excluded this possibility, rather showing some ultrastructural alterations suggestive of an initial phase of renal TMA. Other renal diseases, such as focal and segmental glomerulosclerosis, post-infectious glomerulonephritis, membranoproliferative glomerulonephritis, have been described to trigger endothelial swelling and lamina rara widening, but the histological features of light microscopy and the specific viral and bacterial tests ruled out these hypotheses. High-dose steroid treatment was initiated with 1 gram methylprednisolone for 3 days, followed by prednisone 60 mg/day and then reduction to 10 mg every 4 weeks. The patient was discharged after nine days of hospitalization. A total of 2 weeks later, proteinuria decreased to 0.8 grams/day with microhematuria still present, and then in the following 4 weeks, complete remission of proteinuria (0.07 gr/day) and microhematuria was achieved.To the best of our knowledge, we describe here the first case of urine abnormalities with histological features of initial renal TMA without renal failure, showing an interesting temporal correlation with both positive PCR test for COVID-19 of mild severity and first dose of BNT162b2 COVID-19 vaccine (Pfizer-BioNTech). Several indirect pathways of COVID-related kidney injury have been suggested, mainly attributed to the combined effects of virus-induced cytokine storm with complement and coagulation cascades ,8. SARS-In previous reports, COVID-19 patients who underwent kidney biopsy showed heterogeneous kidney histologic features, including acute tubular injury, collapsing glomerulopathy, focal segmental glomerulosclerosis, tubulointerstitial nephritis, thrombotic microangiopathy ,10,11. AIn a retrospective study of 110 hospitalized COVID-19 patients, the urinalysis data of proteinuria and hematuria were strongly associated with the development of AKI [Up to now, the association of COVID-19 with renal TMA has been described only in patients with AKI ,6. HowevIn our patient, the close temporal proximity (7 days) between the administration of the vaccine dose and SARS-CoV-2 positivity detection prevents us from drawing a causal inference of the histological findings with either the vaccine or the disease itself. Another hypothesis to be evaluated is that, given the strict timeframe between the two events, the combination of both may have triggered the renal disease, although this possibility has been never speculated in the literature. Alternatively, we cannot exclude that the mild clinical and histological features of renal TMA could result from a protective effect of the vaccine, which might have mitigated thromboinflammation, endothelial injury and COVID-induced complement activation.


The review focuses on sulfated steroids that have been isolated from seaweeds, marine sponges, soft corals, ascidians, starfish, and other marine invertebrates. Sulfur-containing steroids and triterpenoids are sourced from sedentary marine coelenterates, plants, marine sediments, crude oil, and other geological deposits. The review presents the pharmacological profile of sulfated steroids, sulfur-containing steroids, and triterpenoids, which is based on data obtained using the PASS program. In addition, several semi-synthetic and synthetic epithio steroids, which represent a rare group of bioactive lipids that have not yet been found in nature, but possess a high level of antitumor activity, were included in this review for the comparative pharmacological characterization of this class of compounds. About 140 steroids and triterpenoids are presented in this review, which demonstrate a wide range of biological activities. Therefore, out of 71 sulfated steroids, thirteen show strong antitumor activity with a confidence level of more than 90%, out of 50 sulfur-containing steroids, only four show strong antitumor activity with a confidence level of more than 93%, and out of eighteen epithio steroids, thirteen steroids show strong antitumor activity with a confidence level of 91% to 97.4%. Sulfur is one of the most abundant chemical elements in nature ,2. In adSulfated compounds, including steroids, are widely distributed in nature ,5,6,7,8.Studies of the last decades of marine sediments have shown that extracts from these geological formations contain a wide variety of steroids, terpenoids, and other lipids ,27,28,29This review focuses on sulfated steroids and triterpenoids that have been found and isolated from marine sources such as algae, sponges, and other marine invertebrates. The review also presents sulfur-containing steroids and triterpenoids isolated from soft corals, marine sediments, and crude oil and other geological deposits. In addition, we have included in this review several semi-synthetic and synthetic epithio steroids, which represent a rare group of bioactive lipids and have not yet been found in nature but exhibit a high level of antitumor activity. For all natural, semi-synthetic and synthetic compounds included in this review, their pharmacological profile and specific diversity of biological activity are presented.It is known that sulfated steroids are of potential interest for practical and clinical medicine, due to the fact that many of them exhibit a wide range of biological activities; however, the activity of many sulfated steroids has not been determined ,18,21,461, for for structure see 2), were obtained from a lipophilic extract of an undescribed bryozoan species in the genus Calyptotheca. Both isolated metabolites exhibited moderate cytotoxicity at IC50 54 and 30 µM, respectively, against the rat bladder carcinoma epithelial NBT-T2 cell line [Two sulfated steroids, (ell line .3) was isolated from the Arctic starfish Asterias microdiscus. The 28-sulfooxy-24-methylcholestane side chain in (3) has been found among starfish steroid metabolites for the first time [4 and 5) and an unusual glucoside (6) were isolated from the starfish Archaster typicus collected in shallow waters of the Quang Ninh province (Vietnam). Isolated compounds showed moderate toxic effects in the sperm and 8-blastomere tests on embryonal development of the sea urchin Strongylocentrotus intermedius [Several sulfated steroids have been found and isolated from the marine starfish. Thus, sulfated polyhydroxysteroid named microdiscusol G  and D (8) were isolated from the marine sponge Haliclona gracilis. Both compounds inhibited the expression of prostate-specific antigen in a human prostate carcinoma epithelial tumor cell lines (22Rv1) [9), belonging to the group of bi- and monoglycosides of polyhydroxysteroids, respectively, was isolated from the ethanolic extract of the starfish Choriaster granulatus. The obtained compound showed immunomodulatory properties, increasing the intracellular ROS (reactive oxygen species) level in peritoneal murine macrophages by 20% and decreasing intracellular ROS level by 21% in pre-treated with endotoxic lipopolysaccharide from E. coli peritoneal macrophages [Polyoxygenated steroids belonging to a new structural group of sponge steroids called gracilosulfates B ( (22Rv1) . Steroidrophages .10), C (11), J (12), and H (13) sulfated steroid glucuronides, were isolated from the starfish Henricia downeyae (family Echinasteridae) collected in the northern Gulf of Mexico [Several starfish polar steroids, downeyosides A  and cholestanol sulphate (15) were identified as the major components among the 34 different sterol sulfates of the sea cucumber Eupentacta fraudatrix [Cholesterol sulphate  was detected in the diatomic microalga Nitzchia alba more than 50 years ago [17) with an unusual side chain was found in the haptophytic microalga Hymenomonas sp. [14), 24-methylcholest-5-en-3β ol sulfate (18), and 5-sitosten-3β-ol sulphate (19) were produced by the diatom Skeletonema marinoi [20) was found in the methanol extract of the sea urchin Diadema savignyi [The 24-methylidene-cholesterol sulfate  were isolated from the tropical marine cucumber Holothuria sp. [23 and 24) and minor sterols (25–29) were detected in the Far Eastern holothurian Eupentacta fraudatrix [Two derivatives of cholesterol sulfate  [31) was isolated from the Pacific deep-water sponge Poecilastra laminaris, and this compound showed a β-1,3-glucanase inhibitor [32, for structure see Toxadocia zumi [33) was isolated from the sponge Echinoclathria suhispida collected from the Japan Sea near the coast of Japan [Haliclona sp. from an Indo-Pacific has yielded haliclostanone sulphate (34) [35) containing a sulfate group in 6α-position was found in the sponge Dysidea fragilis collected from the lagoon of Venice, Italy [36) was isolated from a Far Eastern sponge Aphelasterias japonica [37) was found in the sponge Acanthodendrilla sp. containing sulfate group in 6-position [An Australian marine sponge ton (30) , and annnhibitor . Polyhydcia zumi . Rare moof Japan . The Malate (34) . Cytotoxe, Italy . A sulfajaponica , and acaposition .38) containing a sulfate group in 16-position was found in starfish Luidia clathrata (family Luidiidae) [39) was isolated from Far Eastern starfish Luidia quinaria (Japan Sea) [40–42) were isolated from the visceral extract of the cone snail Conus pulicarius. The three new compounds exhibited significant in vitro cytotoxicity  against the K562 human leukemia cell line [43–45), and a sulfated polyhydroxylated steroid (33) were isolated from the alcoholic extract of the Far Eastern starfish Leptasterias ochotensis [43) demonstrated inhibition of T-47D cell colony formation in a soft agar clonogenic assay at nontoxic doses. In addition, this compound decreased the epidermal growth factor induced colony formation of mouse epidermal JB6-Cl41 cells. The cancer preventive action of (43) is realized through regulation of the mitogen-activated protein kinase signaling pathway.Rare mono sulfate (idiidae) , althougpan Sea) . Three sell line . Three shotensis . Leptaoc47), was isolated from the Mediterranean ascidian Phallusia fumigate [48, for structure see Patiria (Asterina) pectinifera [A rare sulfated steroid at position 5, named phallusiasterol A  was detected in marine sponges and echinoderms. Thus, starfish, Styracaster caroli, which was collected at a depth of 2000 m between the islands of Thio and Lifou , contained unique polyhydroxylated steroids in water–acetone extract (49 and 50) [49–52 and 54) were found in the sponge Polymastia boletiformis [53), has been isolated from the ethanolic extract of the starfish Anasterias minuta. This xyloside exhibited antifungal activity against Cladosporium cucumerinum and Aspergillus flavus [A series of sulfated steroid-containing amide fragments ( and 50) . The samtiformis ,80. Sulfs flavus .55) showed antiviral activity was found in the marine sponge Petrosia weinbergi [56), a sulphated tetrahydroxy steroid with an unprecedented cyclopropane-containing side chain, was isolated from the sponge Petrosia weinbergi. This compound is active in vitro against feline leukemia virus and active in vitro against HIV [Natural di- and poly-sulfates of steroids represent a rare group of bioactive lipids. Their total content in marine organisms is two to three times less than that of steroids containing one sulfate group ,16,18,43einbergi . Weinberinst HIV .57) was isolated from the starfish Tremaster novaecaledonia [Aphelasterias japonica [58) was first discovered in brittle star Ophioderma longicaudum [59) containing an additional hydroxy group at C12 was isolated from O. longicaudum from the Mediterranean Sea [60) containing three functional groups in the ring A was isolated from the Antarctic brittle star Ophiosparte gigas [Astroclades exiguus and Amphiophiura ponderosa [62), containing sulfate groups in 2β- and 21α-positions, was detected in extracts from the starfish of the starfish Pteraster tesselatus [Several steroid disulfates were found in starfish and ophiuroids. Thus, compound , halistanol sulfate I sodium (64) was isolated from a marine sponge Halichondria sp. collected at Hachijo-jima Island. The obtained steroid showed inhibitory activity against SIRT 1-3 with IC50 of 45.9, 18.9, and 21.8 μM, respectively [Trisulfated polyhydroxysteroids are rare and typical metabolites that are produced by marine sponges and echinoderms ,18,19,93-workers . Analog ectively .65) has been isolated from a marine sponge Topsentia sp. collected from the South China Sea. The isolated compound exhibited cytotoxicity against human gastric carcinoma cell line SGC-7901 with an IC50 value of 8.0 μM [66) was found in extracts of the sponge Spheciospongia sp., collected in the Philippines. This compound inhibited PKCzeta with an IC50 value of 1.21 µM, and in a cell-based assay also inhibited NF-kappa B activation with EC50 value of 12 µM [A polyhydroxylated sterol derivative called topsensterol B  is isolated from Ophiura sarsi, and another steroid (68) is isolated from Ophiorachna incrassate [Representatives of Echinodermata’s and other marine and freshwater invertebrates contain very interesting lipid molecules, such as polar lipids and fatty aldehydes ,103,104.crassate .69) and B (70) were isolated from a Lissodendoryx (Acanthodoryx) fibrosa sponge from Coron Island Palawan, Philippines [50 values of 16.4 and 5.6 μM, respectively [71) was isolated from different sponge species [69) was found in Topsentia ophiraphidites [Rare sulfated sterol dimers called fibrosterol sulphates A  has been found and isolated in the human urine by Karim and Brown in 1972 [The 3--propionic acid γ-lactone called spironolactone , was present in the ethanol extract of the whole plant of Andrographis echioides [75), and 3β-24-methyl-5α-cholestane (76) were isolated from extracts from Marrakech activated sludge, which is formed from the leaves and the date palm of the date palm [5α-Androstan-16-one, cyclic ethylene mercaptole  were isolated from the polar fractions of the organic matter of both oils and sediment extracts, which were collected in various parts of the world, in Europe, North America, and China [Four thio-5α-cholestane derivatives , and their unusual chemical structures have been established [80,88–92, for structure see Many authors have attempted to investigate the organic matter generated in hypersaline environments, including both sea and salt seas, and a warm eutrophic salt lake in Southern California, where sulfur-containing compounds have been found and isolated, including unusual thiosterols  have been identified [96), has been identified in a sediment extract from the Miocene Northern Apennines marl  [97) was isolated from the polar fractions of sediment extracts and a crude oil taken from a Jurfed Darawish oil shale Jordan [Several series of organic sulfur compounds have been identified in several oils and sediment extracts including Rozel Point Oil . Series of isoprenoid thiophenes, isoprenoid thiolanes, isoprenoid bithiophenes, isoprenoid thienylthiolanes, isoprenoid benzothiophenes, 2,5-di-n-alkylthiolanes, 2,6-di-n-alkylthianes and 2,4-di-n-alkyl-benzo(b)thiophenes, and several thiophene and thiolane steranes  . Anothere Jordan . C27–C2986, 92, 94, 97, 99, 102, and 104) were isolated from water–methanol extracts of the organic phase of solutions obtained from treatment with microbial mats, which were deposited in a Lagoona setting, revealing three lithofacie, and limestones with fine-scale parallel laminations, limestones with undulated (stromatolite-type) laminations, and massive limestones. These different lithofacies refer to The Upper Jurassic Calcaires en plaquettes Formation, which is located on the southern Jura in France [105) was identified from organic matter in the Holocene and latest Pleistocene sediments of the Cariaco Basin, Venezuela [Several sulfur-containing compounds  was detected in petroleum from Alberta province, Western Canada [A series of sulfur-containing sterols  and methylthio-steroids (110–113) were identified by GC-MS in saturate hydrocarbon fractions of heavy oil with a high sulfur content in the Jinxian Sag, Bohai Bay Basin, North China [91, 106, and 117), have been identified in immature sediments [Sulfur-containing steroids  have been identified in sediments of the Miocene Monterey Formation  and in the Turonian Tarfaya basin (Morocco). Their carbon skeletons were unusual in the sense that the alkyl sidechains at C-3 are almost exclusively isopentyl, 3-methylpentyl, and 2,3-dimethylbutyl moieties, whilst n-alkyl (pentyl or hexyl) moieties are almost absent [A series of thiophenes with C-3 alkylated steroid carbon skeletons (t absent .119–121) that were present in the polar fractions of six immature samples (both crude oils and sediment extracts) have been analyzed using S-selective chemolysis methods and analytical pyrolysis [2α-, 3α-, and 4α-Methylthio-steroids , and 5α,6α-epithio cholestan-3β-ol (135), 7α,8β-epithio-3β-cholesterol (136), 11β,12β-epithio- (137), 17α,18α-epithio- (138) steroids, and an interesting thiirane-containing steroid (139) have been synthesized in different laboratories; however, they had one goal of obtaining a steroid with specific biological activities, for example, an inhibitor of estrogen synthetase, gonadotropin inhibitors, or agents that demonstrate antiseptic, germicidal, fungicidal, or antitumor activities [2,3-Epithio steroids , 2 (92.7%), and 21 (92.8%) exhibit hemostatic properties with more than 92% confidence, and other steroids 29 (93.3%), 32 (93.3%), 34 (96.8%), 48 (92.4%), 65 (91.5%), 68 (91.5%), 69 (91.8%), 70 (93.4%), and 71 (93.4%) show hepatoprotective properties  excreted from human urine is of interest. According to the PASS data, this steroid is multifunctional and demonstrates seventeen different activities. Sulfur-containing steroids are an interesting class of natural lipids, but most of these compounds exhibit weak or moderate antitumor activity. Some steroids have no antitumor activity, and only four steroids have strong antitumor activity. Most epithio steroids are classified as anabolic steroids and are used in bodybuilding diets, as well as stimulants by athletes or a specific category of people during physical or physiological stress ,179,180.Clostridium difficile infection, and others [Several epithio steroids shown in d others ,182,183.122, 123, 129, and 138 exhibit properties to increase the efficiency and improve the contraction of the heart muscle, i.e., are typical cardiotonic agents with a Pa confidence of 70% to 93.6%.As shown by the PASS data, the four epithio steroids The review focuses on the interesting topic of sulfated steroids, which have been isolated from algae, sea sponges, soft corals, ascidians, starfish, and other marine invertebrates. In addition, sulfur-containing steroids and triterpenoids that are isolated from sedentary marine coelenterates, plants, marine sediments, crude oil, and other geological deposits are also presented in this review. Additionally, several semi-synthetic and synthetic epithiosteroids, which are a rare group of bioactive lipids that have not yet been found in nature but have a high level of antitumor activity, were included in this review for the comparative pharmacological characterization of this class of compounds.This review presents 139 steroids and triterpenoids that exhibit a broad spectrum of biological activity. The data obtained with the PASS program show that thirteen sulfated steroids exhibit strong antitumor activity with a confidence level of more than 90%. Among the sulfur-containing steroids, only four show strong antitumor activity with a confidence level of more than 93%, although most of the presented epitosteroids show strong antitumor activity with a confidence level of 91% to 97.4%. These data are of great interest to pharmacologists and clinical physicians.


Trichoderma can induce plant hormone signal pathways mediating plant defenses, resulting in broad-spectrum resistance to phytopathogens. Herein, Malus sieversii seedlings were treated with Trichoderma biofertilizer and/or Alternaria alternata f. sp. mali, and transcriptome analysis revealed significant differential expression. There was a high similarity between the transcriptome expression profiles of Trichoderma-induced and A. alternata-infected M. sieversii samples for genes related to jasmonic acid (JA), ethylene, and salicylic acid (SA) signaling pathways. Additionally, Trichoderma biofertilizer activated numerous disease-resistant genes  and defense response genes . Among transcription factors, members of the ERF family were the most differentially expressed (18 ERFs), indicating that they may be closely related to defense responses. Among ERFs, differential expression of MsERF105 was the most significant (upregulated 27.6-fold compared to controls). MsERF105 was heterologously expressed in PdPap poplar , and following infection with A. alternata , transgenic PdPap-MsERF105s plants displayed lower malondialdehyde (downregulated 41.4%) and reactive oxygen species (ROSs) levels, and higher reductase activities, especially superoxide dismutase . Furthermore, the lesion areas of PdPap-MsERF105s leaves were significantly smaller (0.2%) than those of PdPap-ROK2 leaves (∼26.0%), and the cell membrane integrity was superior for PdPap-MsERF105s leaves. Thus, MsERF105 enhanced the resistance of PaPap poplar to Aal, presumably because MsERF105 activates the expression of PR1 and PDF1.2. In conclusion, Trichoderma biofertilizer modulated the differential expression of numerous disease resistance genes and defense response genes in M. sieversii in response to pathogen attack, and MsERF105 played important roles in this process. Trichoderma to establish themselves in the rhizosphere and thereby facilitate the evolution of positive interactions with plants  related to defense responses (Trichoderma koningii induces systemic resistance in tobacco against tobacco mosaic virus (TMV), stimulates the activities of pathogenesis-related (PR) enzymes [superoxide dismutase (SOD), peroxidase (POD), and polyphenol oxidase), and upregulates the expression of defense-related genes   .Trichoderma on plants. Trichoderma-induced plant transcriptome expression profiles display high similarity to those induced by pathogen infection (Trichoderma-induced plant resistance against pathogens is mainly mediated by jasmonic acid (JA), ethylene (ET), and salicylic acid (SA) signaling pathways  and the JA/ET pathway  , suggesting that the fungus contributes to the priming of plant responses (Trichoderma rossicum T7 (TroT7) and T. harzianum T2 (ThaT2) can enhance the resistance of Malus sieversii to F. oxysporum and Alternaria alternata f. sp. mali   . In addiopersici . Plants esponses . Besidesi  . Thus, TERF genes are involved in responses to biotic stresses through plant hormone signaling pathways , and TMV (ZmERF105 can improve the resistance of maize to northern leaf blight (Exserohilum turcicum), while the erf105 mutant showed the opposite phenotype. In addition, the activities of SOD and POD in ZmERF105 overexpression lines were significantly higher than those in wild-type plants  were analyzed by RNA sequencing (RNA-Seq). Gene ontology (GO) function classification, Clusters of Orthologous Groups (COG) function classification, and Kyoto Encyclopedia of Genes and Genomes (KEGG) classification analyses were conducted. In addition, differential expression of transcription factors was explored. The M. sieversii MsERF105 gene was heterologously expressed in Populus davidiana × Populus alba var. pyramidalis Louche (PdPap poplar), and PdPap seedlings were infected with Aal. The antioxidant ability of transgenic PdPap-MsERF105s plants was evaluated by determining malondialdehyde (MDA) and reactive oxygen species (ROSs) content, reductase  activities, resistance to Aal, relative lesion area, and cell membrane permeability. Differential expression of defense response genes (PR1 and PDF1.2) was assessed. Based on the above results, the functions of ERFs in defense responses in M. sieversii were evaluated.In the present study, Malus sieversii seeds from a Xinjiang wild fruit forest were germinated in the laboratory (temperature 28°C and humidity 70%) to obtain 2-month-old seedlings. Apple-spotted defoliation pathogen Aalm was obtained from the Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography. Poplar leaf blight pathogen Aal was obtained from the State Key Laboratory of Tree Genetics and Breeding . Agrobacterium tumefaciens EHA105 and the pROK2 vector were used for the genetic transformation of PdPap poplar. PdPap poplar seedlings were cultured aseptically in a liquid rooting medium at 25°C  and mixed. The solid fermentation medium for Trichoderma comprised rice husk (19.9% w/w), wheat bran (5% w/w), (NH4)2SO4 (0.1% w/w), and H2O (75% w/w). The tray was then sealed with preservative film. TroT7 and ThaT2 were cultured at 28°C for 1 week, spores of both Trichoderma strains were mixed (1:1) as Trichoderma biofertilizer, and the concentration of conidia in the biofertilizer was 108 spores/ml.Spores of TroT7 and ThaT2 were separately inoculated into potato dextrose agar (PDA) medium and cultured at 28°C for 1 week. Both us study . Spores M. sieversii seedlings were subjected to four different treatments, three biological replicates each treatment were set. The samples treated without Trichodema biofertilizer and Aalm, as negative control, were collected at 0 h, the other samples were collected at 6 and 48 h post-induction (hpi), and all samples were stored at −80°C for RNA-Seq. For the Tri treatment, seedlings were irrigated with 50 ml Trichodema biofertilizer (108 spores/ml), one leaf of each seedling was stabbed, and inoculated without Aalm mycelium. For the Aalm treatment, seedlings were irrigated with 50 ml sterilized water, and one leaf of each seedling was stabbed and inoculated with 0.6 cm disks containing Aalm mycelium, which was cultured in PDA medium at 28°C for 10 days, and 0.6 cm disks were cut with a hole punch. For the Tri + Aalm treatment, seedlings were irrigated with 50 ml Trichodema biofertilizer (108 spores/ml), and one leaf of each seedling was stabbed and inoculated with 0.6 cm disks containing Aalm mycelium. For the negative control, seedlings were irrigated with 50 ml sterilized water, one leaf of each seedling was stabbed and inoculated without Aalm mycelium.Two-month-old Total RNA was extracted using the cetyltrimethylammonium bromide (CTAB) method , followiBased on sequencing by synthesis technology, an Illumina hiseq high-throughput sequencing platform was used to sequence the cDNA library to produce a large number of high-quality reads (raw data). This was filtered to remove joined and low-quality reads to obtain high-quality clean data, which was assembled using Trinity software . Then, GMsActin (MZ605395) in M. sieversii was used as an internal reference. Three technical replicates were performed for each cDNA sample. Primers for RT-qPCR  at 28°C and 70% humidity, the treatment of samples was the same as that described for RNA-Seq, three plants  each treatment were set. Leaf samples were collected at 6 and 48 hpi, and untreated seedlings served as controls, leaf samples were collected at 0 hpi. Total RNA was extracted using the CTAB method, and reverse-transcribed into cDNA for quantitative real-time polymerase chain reaction (RT-qPCR). The gene-encoding  RT-qPCR  were desThe method is the same as given in the section “Expression of genes involved in plant hormone signal pathway using RT-qPCR.” Following the three treatments  at 28°C and 70% humidity, three plants  each treatment were set. Leaf samples were collected at 0, 3, 6, 12, and 24 hpi, and untreated seedlings served as controls. Three technical replicates were performed for each cDNA sample. Primers for RT-qPCR  were desMsERF105 gene was cloned by PCR using a sense primer (5′-ATCGTCTAGAGAGTTTCTAAATATGGCATCAGAAGC-3′) containing an XbaI  site (underlined) and an antisense primer (5′-CGATGGTACCTCAAATAACCATGAGCGGAGGATATC-3′) containing a KpnI  site (underlined). The resulting MsERF105 PCR product and the pROK2 vector were double digested with XbaI and KpnI at 37°C for 4 h, and ligated using T4 DNA ligase  at 4°C for 12 h, to generate the pROK2-MsERF105 construct. MsERF105 was transferred into PdPap poplar using the A. tumefaciens-mediated transformation system, and empty vector pROK2 was transferred in PdPap to generate the PdPap-ROK2 control. The method was described in detail in our previous study . First, infected leaves were scanned to obtain digital images, the software accurately determined the location and profile of disease spots, and the relative area of disease spots was evaluated .g for 10 min, three biological replicates each treatment were set. The supernatant was used to determine the MDA content using an MDA-2-Y kit . The activities of SOD, POD, and catalase (CAT) were determined using dedicated kits , three biological replicates each treatment were set.Following treatment of PdPap-MsERF105s and the control PdPap-ROK2 plants with 0.6 cm disks containing Aal mycelia, leaves were collected at 0, 3, 6, 12, 24, 48, and 72 hpi, 0.1 g was weighed using an analytical balance, ground in a pestle and mortar with liquid nitrogen, and the leaf powder was added to extraction solution (from the kit mentioned below) and centrifuged at 1,100 × Evans blue and nitrotetrazolium blue chloride (NBT) were dissolved in 10 mM phosphate-buffered saline (pH 7.8), and diaminobenzidine (DAB) were dissolved in 50 mM Tris-acetate buffer (pH 5.0) , and thePR1 genes and two PDF1.2 genes were determined by RT-qPCR, three biological replicates and technical replicates each treatment were set, respectively. The gene encoding PdActin in PdPap poplar served as an internal reference. The method was the same as described above for RT-qPCR analysis of ERF family genes. All primers are listed in Following treatment of PdPap-MsERF105s and the control PdPap-ROK2 plants with 0.6 cm disks containing Aal mycelia, leaves were collected at 0, 3, 6, 12, 24, 48, and 72 hpi, and the expression levels of two p ≤ 0.05 was considered significant.Statistical analysis was performed using SPSS 17.0 . Data are expressed as the mean of three or more independent replicates ± SD. Analysis of variance was conducted using Duncan’s test, and Trichoderma and/or Aalm, the gene expression pattern of M. sieversii was significantly different from that of controls, and the three replicate treatments yielded highly similar results, especially the three control replicates, which shared > 98% similarity  was significantly lower than that of controls (18.7 nmol/g at 72 hpi) after 24 hpi, but the MDA content was not significantly different between PdPap-MsERF105s and control plants before 12 hpi .Furthermore, the activities of SOD, POD, and CAT were determined. The results showed that following treatment with Aal, the activities of SOD and CAT were significantly increased in PdPap-MsERF105s plants compared to controls after 6 hpi, and SOD activity peaked at 48 hpi. Additionally, values were > 105.1 nmol/min/g for all the three replicates, compared with only 64.2 nmol/min/g for controls . POD actTo further verify the resistance ability of PdPap-MsERF105 plants, leaves were inoculated with Aal mycelium disks. The results revealed no lesions on the leaves of all the three PdPap-MsERF105s replicates at 5 days postinfection (dpi), but there were large lesions on leaves of the controls at 5 dpi, and the relative lesion area was 26% . In addiPR1s (PR1-1 and PR1-2) and two PDF1.2 proteins (PDF1.2-1 and PDF1.2-2) were determined. The results showed that following infection by Aal, expression levels of PR1-1 were significantly higher than the controls, with an increase after 12 hpi and a peak at 48 hpi. For Pap-MsERF105-3, the increase was 7.41 (22.89)-folds  in PdPap-MsERF105-2 (increased by 18-fold compared with the control PdPap-ROK2 (PDF1.2-2 were the highest (24.8-fold) in PdPap-MsERF105-2, an increase of ∼28-fold compared with the control PdPap-ROK2 -folds . PR1-2 er 24 hpi . The expPap-ROK2 . ExpressPap-ROK2 .Trichoderma genus, the widely used biocontrol agents, can induce plant hormone signal pathways, and thereby endow plants with broad-spectrum resistance to phytopathogens  and transcription factor genes , many of which are responsible for activating downstream defense response genes, such as DRP, ABC, and HSP. Previous studies showed that JA and SA induced increased expression of OsMSRPK1 (a serine/threonine protein kinase) in Oryza sativa (Nicotiana repanda NrSTK (a serine/threonine protein kinase) in the susceptible tobacco variety Honghuadajinyuan, which significantly enhanced resistance to the black shank . Similarly, under cold stress, when Cynodon dactylon CdERF1 was heterologously expressed in Arabidopsis plants, the MDA content was reduced, and the activities of SOD and POD were elevated , and increased Arabidopsis resistance to the pathogens Pseudomonas syringae pv. tomato DC3000 and B. cinerea (Overexpression of ZmERF105 in soybean enhanced the expression of several PR genes, including ZmPR1a, ZmPR2, ZmPR5, ZmPR10.1, and ZmPR10.2, following infection with E. turcicum (MsERF105 plays a positive modulatory role in response to pathogen infection in M. sieversii.Following treatment with Aal mycelia, lesions on PdPap-MsERF105s leaves were significantly smaller than those on PdPap-ROK2 leaves, and cell membrane integrity in PdPap-MsERF105s leaves was better than in PdPap-ROK2 leaves. This suggests that  cinerea . Overexpturcicum . These rTrichoderma biofertilizer not only activated JA and SA signaling pathways but also induced the differential expression of numerous disease resistance genes and defense response genes, especially ERF transcription factor family members. Heterologous expression of MsERF105 significantly enhanced the antioxidant and antipathogen abilities of transgenic PdPap poplar. The findings suggest that MsERF105 is crucial for the response to pathogen attack in M. sieversii.In conclusion, SRR16148375, SRR16148374, and SRR16148363, the accession numbers of T6 transcriptome data are SRR16148361, SRR16148360, and SRR16148359, the accession numbers of A6 transcriptome data are SRR16148358, SRR16148357, and SRR16148356, the accession numbers of TA6 transcriptome data are SRR16148373, SRR16148372, and SRR16148355, the accession numbers of T48 transcriptome data are SRR16148371, SRR16148370, and SRR16148369, the accession numbers of A48 transcriptome data are SRR16148368, SRR16148367, and SRR16148366, and the accession numbers of TA48 transcriptome data are SRR16148365, SRR16148364, and SRR16148362. And the transcriptome assembly data have been deposited in the SRA database, and the accession number is GJMU 00000000. BioProject number is PRJNA 736527, and BioSample number is SAMN 19651934.The original contributions presented in the study are publicly available. The raw transcriptome data have been deposited in the SRA database. The accession numbers of Con transcriptome data are SJ: conceptualization, formal analysis, investigation, methodology, and roles/writing – original draft. ZL: conceptualization, data curation, formal analysis, funding acquisition, methodology, supervision, and writing – review and editing. YW: conceptualization, data curation, funding acquisition, methodology, project administration, supervision, and writing – review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


SPL module can participate in the regulation of anthocyanin synthesis in plants. However, little is known about how this module responds to hormonal signals manipulating this process in grapes. In this study, exogenous GA, ABA, MeJA, and NAA were used to treat the ‘Wink’ grape berries before color conversion, anthocyanin and other related quality physiological indexes  were determined, and spatio-temporal expression patterns of related genes were analyzed. The results showed that the expression levels of VvmiR156b/c/d showed a gradually rising trend with the ripening and color formation of grape berries, and the highest expression levels were detected at day 28 after treatment, while the expression level of VvSPL9 exhibited an opposite trend as a whole, which further verifies that VvmiR156b/c/d can negatively regulate VvSPL9. Besides, VvmiR156b/c/d was positively correlated with anthocyanin content and related genes levels, while the expression pattern of VvSPL9 showed a negative correlation. Analysis of promoter cis-elements and GUS staining showed that VvmiR156b/c/d contained a large number of hormone response cis-elements  and were involved in hormone regulation. Exogenous ABA and MeJA treatments significantly upregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early stage of color conversion and made grape berries quickly colored. Interestingly, GA treatment downregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early color-change period, but significantly upregulated in the middle color-change and ripening stages, therefore GA mainly modulated grape berry coloring in the middle- and late-ripening stages. Furthermore, NAA treatment downregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes and delayed the peak expression of genes. Meanwhile, to further recognize the potential functions of VvmiR156b/c/d, the mature tomato transient trangenetic system was utilized in this work. Results showed that transient overexpression of VvmiR156b/c/d in tomato promoted fruit coloring and overexpression of VvSPL9 inhibited fruit coloration. Finally, a regulatory network of the VvmiR156b/c/d-VvSPL9 module responsive to hormones modulating anthocyanin synthesis was developed. In conclusion, VvmiR156b/c/d-mediated VvSPL9 participated in the formation of grape color in response to multi-hormone signals.In recent years, more and more reports have shown that the miR156- Grape berry ripening is an extremely complex physiological and biochemical process, accompanied by the formation of fruit color, flavor, and aroma. Among them, the color of grape berry is an important indicator of fruit quality. As grapes color, sugar and aroma accumulate in the berries. Therefore, the berry color and its related qualities above-mentioned directly affect its price and market competitiveness. Anthocyanin is the main pigment in grapes including cyanidin, delphinidin, peonidin, petunidin, and malvidin. Fruit color development is a very complex metabolic process, which is not only affected by genotype, climatic conditions, and cultivation techniques, but also by plant hormones. In the process of fruit coloring, plant hormones are key regulators and play important roles. A variety of hormones interact to co-regulate the formation of colors such as abscisic acid (ABA), gibberellin (GA), ethylene (ETH), auxin (AUX), and jasmonic acid (JA). The application of exogenous hormones can cause changes in endogenous hormones modulating the color formation. For example, ABA, ETH, and JA treatments promote fruit coloring and ripening; conversely, AUX applications inhibit the corresponding processes ,2,3,4. TArabidopsis thaliana, tomato, apple, and grape, miR828 regulates the accumulation of anthocyanin and flavonol by targeting MYB transcription factors such as PAP1/MYB75, PAP2/MYB90, and MYB113 [NLA [MYB12 are also involved in the regulation of anthocyanin biosynthesis [Arabidopsis thaliana, miR156 regulates anthocyanin synthesis by negatively regulating target gene SPL through interacting with the PAP1 protein; SPL9 affects the transcriptional activation activity of the MYB-BHLH-WD40 complex, which inhibits the expression of anthocyanin synthesis ANS and DFR genes, and thus decreases the accumulation of anthocyanin [SPL9 may directly manipulate anthocyanin biosynthesis through PAP1 and DFR, while plants can mediate anthocyanin metabolism through miR156-SPLs-DFR pathways [Litchi chinensis Sonn.), miR156a is inversely correlated with the expression profiles of its target genes LcSPL1/2 and LcCHI, and the increased expression of miR156a decreases the transcription level of LCSPL1, which downregulates anthocyanin biosynthesis via interaction with LcMYB1 [MicroRNAs (miRNAs), widely distributed in plants, are very important non-coding RNA regulators with a length of 19–24 nucleotides (nt). They play critical roles in plant growth and development, signal transduction, and the response to stress, mainly via their action in causing either complementarity-dependent cleavage or translational inhibition of the target mRNA molecules at the post-transcriptional level ,6. In red MYB113 ,11,12,13113 [NLA ,15; and ynthesis ,17,18. Fynthesis ,21. In Ahocyanin . Under spathways . In litcVvSPL might respond to the hormone to manipulate grape berry and ripening. However, it is not clear whether the mechanism of miR156-SPL response hormone signals regulate grape color formation. To elucidate this, we treated ‘Wink’ (Vitis vinifera L.) with exogenous GA, ABA, MeJA, and NAA before the color transition period, and measured the physiological indicators associated with quality . Then, based on the report of miR156-SPL [In our previous study, we confirmed that VvSPL9 is the target gene of VvmiR156b/c/d, which is involved in the regulation of grape berry development and maturation; and miR156s could be in response to gibberellin ,26,27. TR156-SPL ,23,26,27Vitis vinifera L.) were used as the plant material. Based on the results of the preliminary experiments and horticultural practices, grape berry clusters were treated by using 50 mg/L gibberellin (GA), 100 mg/L abscisic acid (ABA), 100 mg/L methyl jasmonate (MeJA), 200 mg/L naphthalene acetic acid (NAA), and 0.01% Tween-80 solution (CK), respectively, one week before color conversion (16 July). Berries were sprayed to a completely wet state and samples were collected at 0 days after treatment , seven days after treatment , 14 days after treatment , 21 days after treatment , 28 days after treatment , 35 days after treatment . Ten clusters were randomly selected from each group of the control and treated plants, and three berry grains were randomly selected from the top, middle, and lower parts of each panicle. After weighing, berries were separated into skin, flesh, and seed, then immediately frozen in liquid nitrogen and stored at −80 °C until use.The experiment was conducted at Jiangsu Academy of Agricultural Sciences, Nanjing, China from July 2019 to August 2019. A 6-year-old grapevine cv. ‘Wink’ , abscisic acid (ABA), methyl jasmonate (MeJA), naphthalene acetic acid (NAA), and 0.1% Tween-80 Solution (CK). Collected leaves were sampled at 1 h, 3 h, 5 h, and 7 h after treatment and five leaves were repeated for each treatment period. Then, the samples of leaves were immediately placed into liquid nitrogen.v/v) and (B) acetonitrile. The gradient for buffer B was 5% for 2 min, 5~95% for 15 min, and 95% for 2 min. Anthocyanin were monitored with a flow rate of 1.00 mL/min, a column temperature of 25 °C, and a detection wavelength of 530 nm. The chromatographic peaks were identified by comparison with authentic standards of cyanidin-3-O-glucoside chloride (A sample of 0.5 g of grape skin freeze-dried powder (ground with liquid nitrogen) was accurately weighed into a 15 mL Eppendorf tube, and 10 mL of 0.1% methanolic hydrochloric acid solution was added, and placed in a refrigerator at 4 °C for 24 h in the dark. Then, the supernatant was collected and filtered with a 0.45 μm nylon filter. Extracts were analyzed by ultra-performance liquid chromatography . Separation was achieved with a Zorbax SB-C18 column , and a binary solvent system of (A) 1% aqueous formic acid , and 2 μL octanal ethanolic solution  and placed them into a 20 mL clean capped vial. ,29. A 50RT Reagent Kit  and stored at −40 °C following the manufacturer’s instructions.The total RNA and DNA were isolated from 200 mg of the grapevine plant materials using the modified cetyltrimethylammonium bromide (CTAB) method. The concentration of RNA and DNA samples were measured by a UV-1800 spectrophotometer and the quality and quantity of RNA and DNA samples were assessed by 1.5% agarose gel electrophoresis. The cDNA was synthesized from total RNA using the PrimeScriptTMhttp://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/, accessed on 14 April 2021). Promoter cis-elements were further analyzed by PlantCARE software , which provided a basis for exploring the biological functions of VvMIR156b/c/d.We obtained promoter sequences (approximately 2000 bp upstream of genes) of VvMIR156b/c/d from the Grape Genome Browse  and left to stand at room temperature for 3~4 h.According to the VvmiR156b/c/d promoter sequences , the upsNicotiana benthamiana) leaves were selected for Agrobacterium tumefaciens infection, and the transient expression of the target protein in tobacco was carried out. The injected tobacco was kept in the dark for two days and the leaves were treated with 100 μM GA, ABA, MeJA, and NAA. On day 3, leaves were collected and each treatment was repeated with five leaves and the experiment was repeated three times.Six-week-old wild tobacco (The β-glucuronidase (GUS) gene is a commonly used reporter gene, which could reflect promoter expression levels. GUS histochemical staining was performed on tobacco leaves using the GUS Staining Kit . First, tobacco leaves were punched and soaked in GUS staining solution. The vacuum pump was used for 30 min under a 0.085 kPa condition, then stained overnight at 37 °C. Finally, a 75% ethanol solution was used to decolorize. GUS expression was observed and photographed under a microscope . Moreover, the quantification of GUS activity was determined using the fluorometric 4-methylumbelliferyl-b-D-glucuronide (MUG) method. One unit of GUS activity was defined as 1 nM of 4-methylumbelliferon (4-MU) generated per milligram of soluble protein per minute. Three leaves were infiltrated for each construct in each independent experiment and then combined to detect GUS activity.http://www.mirbase.org/, accessed on 14 April 2021), specific primers containing NcolⅠ and SpeⅠ restriction sites were designed for PCR amplification  and kanamycin (100 μg/mL). After OD600 = 0.8, the bacteria were pelleted and resuspended in suspension buffer  and left to stand at room temperature for 3~4 h. Finally, a syringe was used to inject the Agrobacterium liquid into the tomato before the color change [In transgenic research, grape transgenic technology has not yet been perfected and matured, while tomato is a model plant and has formed a series of mature technical systems. Therefore, instant injection of tomato can be used to quickly verify the function of genes. Each r change . Ten unihttp://bioinfo.ut.ee/primer3-0.4.0/primer3/, accessed on 14 April 2021). Using cDNA as a template, VvActin (Accession number: XM_002273532) was used as a reference gene for qRT-PCR. qRT-PCR was conducted following the instructions of the SYBR Premix Ex TapTM Kit . The amplification system was 10 μL: cDNA 1 μL, upstream and downstream primer 0.3 μL each, TransStart® Tip Green qPCR SuperMix 5 μL, dd H2O 3.4 μL. The reaction procedure was 95 °C for 300 s, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. The relative expression level was calculated with the formula 2−ΔΔCT = normalized expression ratio. Each PCR assay was carried out by three biological replicates, and each replicate corresponded to three repeats of separate experiments.According to the principle of fluorescent quantitative PCR primer design, qRT-PCR primers  were desp < 0.05; ** p < 0.01) as determined by the Student’s t-test.Data processing, difference significance analysis, and correlation analysis were done using Excel and SPSS 16.0. Asterisks indicated statistically significant differences at  was used to evaluate the appearance and color of berries. CIRG range: CIRG < 2 is green, 2 < CIRG < 4 is pink, 4 < CIRG < 5 is red, 5 < CIRG < 6 is purple, CIRG > 6 is blue-black or purple-black. The results of the CIRG value evaluation of fruit appearance and color C were coThe volatile compound identification and relative contents were based on data including the retention time, BP area, and BP height as obtained from GC-MS . A varieAs shown in Hormone-related motifs in their promoter regions were scanned and it was found that almost all VvmiR156b/c/d had similar hormones-related motifs including the ABA, GA, and MeJA responsive motifs . FurtherThe promoter vectors (pBI121-VvmiR156b/c/d-35SCaMV) were conducted as shown in VvSPL9, the treated grape leaves with GA, ABA, MeJA, and NAA were used to detect the expression of VvmiR156b/c/d and VvSPL9. As VvSPL9. Furthermore, we revealed that among the four hormone treatments, GA significantly upregulated the expressions of VvmiR156b/c/d at 7HAT and decreased VvSPL9 at the corresponding stage, thereby enhancing their negative correlation at the expression levels, which suggests that GA promoted the negative regulatory roles of VvmiR156b/c/d on VvSPL9. Although the other three hormone treatments nearly downregulated the expression levels of VvmiR156b/c/d and VvSPL9 at all times, the expression levels of VvmiR156b/c/d and VvSPL9 under the ABA, MeJA, and Auxin hormones treatments could still maintain a negative correlation, implying that these hormones mediate the negative regulatory roles of VvmiR156b/c/d on VvSPL9. Meanwhile, the results suggest that these modules had a stronger response to GA than the remaining three hormones ABA, MeJA, and Auxin, similar to the GUS stain results above , upregulated the expression level of VvmiR156b/c/d in the middle color-change period (21DAT), and then downregulated the expression level of VvmiR156b/c/d in the berry mature period (28DAT-35DAT); in contrast, GA treatment increased the expression of VvSPL9 in the early color-change period (0DAT–14DAT) and berry mature period (28DAT), suggesting that GA treatment significantly enhanced the negative correlation of vmiR156b/c/d-VvSPL9, and mainly regulated berry coloring in the middle color-change period. ABA and MeJA treatments significantly increased the expression of VvmiR156b/c/d in the early stage of color conversion (7DAT) and downregulated the expression of VvmiR156b/c/d in the late stage of color conversion (21DAT-35DAT). Furthermore, VvSPL9 had a stronger regulatory effect in the early stage of color conversion (7DAT), which indicated that ABA and MeJA treatments promoted berry coloring in the early color-change period. Moreover, NAA was a negative regulator of VvmiR156b/c/d. NAA significantly downregulated the expression of VvmiR156b/c/d in berry mature period (28DAT), and NAA showed a gentle upregulated effect on VvSPL9. In conclusion, we preliminarily concluded that VvmiR156b/c/d had the earliest response to ABA and MeJA, followed by GA, while NAA had the latest response to VvmiR156b/c/d.Interestingly, different hormone treatments exhibited three regulatory modes for VvmiR156b/c/d-VvC4H, VvCHS, VvCHI, VvF3H, VvDFR, VvLDOX, and VvOMT were highly expressed in grape roots, stems, buds, and rachis; VvCHS, VvCHI, VvF3H, and VvLDOX were also strongly expressed in flower and seed; moreover, VvPAL, VvF3H, VvDFR, and VvLDOX were also strongly expressed in the berry, while VvUFGT was only specifically highly expressed in the berry skin and pericarp. In addition, we found that in flesh, most of the anthocyanin synthesis-related genes were highly expressed in the berry flesh ripening period. In pericarp, most of the anthocyanin synthesis-related genes were highly expressed in the berry pericarp mid-ripening period. In the peel, most of the anthocyanin synthesis-related genes were highly expressed in the berry skin veraison period.The biosynthesis of anthocyanins is controlled by structural and regulatory genes. In order to investigate the temporal and spatial expression patterns of anthocyanin structural genes, we analyzed the expression patterns of 12 anthocyanin structural genes based on the transcriptional mapping data of 54 tissues, organs, and development stages . In the VvPAL, VvC4L, VvCHS, VvCHI, VvF3H, VvF3’H, VvF3’5’H, VvDFR, VvUFGT, and VvLDOX) peaked at 28DAT, then their expression levels were downregulated , which indicates that GA is mainly involved in the formation of berry color in the middle stages of color conversion. ABA and MeJA treatments significantly upregulated the expression of anthocyanin structural genes in the early color-change period (7DAT–14DAT). After exogenous ABA and MeJA treatment, the expression levels of most anthocyanin structural genes reached the maximum at 7DAT and 14DAT, and the expression of anthocyanin structural genes was higher in ABA treatment than in MeJA treatment. Furthermore, we found that NAA treatment generally downregulated the expression level of anthocyanin structural genes and delayed the peak expression of anthocyanin structural genes to 35DAT, indicating that NAA inhibits the expression level of anthocyanin structural genes and is mainly involved in the formation of berry color in the mature stage , while the correlation between VvmiR156b and anthocyanin synthesis-related genes is poor. VvmiR156b was only positively correlated with VvLDOX and VvUFGT (r = 0.3451~−0.4372), but VvSPL9 was negatively correlated with the expression of anthocyanin synthesis-related genes including VvPAL, VvF3’H, VvF3’5 ‘H, VvLDOX, and VvUFGT (r = −0.3352~−0.6396). GA and ABA treatment enhanced the positive correlation between VvmiR156b and VvPAL, VvC4H, Vv4CL, boosted the positive correlation between VvmiR156c/d and VvC4H, VvLDOX, and weakened the positive correlation between VvmiR156c/d and VvCHS, VvCHI, VvDFR, VvUFGT. While GA treatment enhanced the negative correlation between VvSPL9, VvPAL, VvCHI, and VvUFGT. In addition, ABA treatment significantly boosted the positive correlation between VvmiR156b, VvF3’H, VvF3’5’H, VvLDOX, and VvUFGT (r = 0.8592~0.9423), while MeJA enhanced the positive correlation between VvmiR156b and VvC4L, VvF3’H, VvF3’5’H, and boosted the positive correlation between VvmiR156c/d and VvDFR. NAA promoted the positive correlation between VvmiR156b and VvDFR and between VvmiR156c/d and VvLDOX, VvUFGT, while the negative correlation between VvSPL9 and VvDFR was enhanced by NAA treatment. In summary, the correlation analysis of temporal and spatial expression showed that VvmiR156b/c/d can negatively regulate VvSPL9 expression by responding to different hormone signals, modulated the expression levels of genes related to anthocyanin synthesis, and thereby participated in the formation of grape color.By comparing the expression levels of VvmiR156b/c/d-ed genes , we founVvSPL9-OE inhibited tomato coloring. The qRT-PCR results showed that in VvmiR156b/c/d-OE fruits, the expression of SlSPL9 was inhibited to varying degrees, while in VvSPL9-OE fruits, the expression of VvSPL9 was significantly upregulated  was further observed A. Comparegulated B. In addnhibited C. These PAL, CHS, CHI, F3H, DFR, LDOX, and UFGT directly encode enzymes in anthocyanin biosynthesis pathways and determine the types of anthocyanin synthesis. Regulating genes encoding transcription factors can activate or inhibit the expression patterns and levels of structural genes, thus regulating the strength of anthocyanin synthesis. At present, the regulatory genes of anthocyanin biosynthesis mainly include R2R3-MYB, bHLH, and WD40 transcription factors, and MYB transcription factors interact with bHLH and WD40 to form the WBM complex to regulate the expression of related structural genes [PIF3, HY5, COP1, WRKY, WIP, MADS-box, NAC, SPL are also involved in the transcriptional regulation of anthocyanin synthesis [MYB111/MYB12, miR828-MYB75/MYB113, miR827-NLA, and miR408-SPL7/HY5 [Anthocyanin belongs to the flavonoid class. Genes involved in anthocyanin biosynthesis are divided into structural and regulatory genes ,11,33. Sal genes . Moreoveynthesis . FurtherSPL7/HY5 ,22,36. TVvSPL9 showed a negative correlation. These findings indicate that VvmiR156b/c/d could promote the expression of anthocyanin synthetic genes by negative regulatory VvSPL9 expression, thus promoting coloring.It is generally believed that the expression of genes involved in anthocyanin synthesis increases with the maturation of grape and the deepening of pericarp color. In this study, with the ripening and color formation of grape berries, the expression levels of most anthocyanin structural genes reached their peak at 28DAT, then the expression levels were downregulated. VvmiR156b/c/d showed similar expression trends with anthocyanin structural genes, and the expression pattern of VvmiR156b/c/d was positively correlated with anthocyanin contents and anthocyanin synthesis related gene expression, and the expression pattern of VvSPL9 to further verify their functions. However, since grape transgenic technology is not perfect and mature, while tomato is a model transgenic plant system and is thus widely used in the verification of gene functions. Therefore, we utilized transient transformation of injecting tomato fruit for determination of gene functions in this work. Interestingly, four days after injection, all VvmiR156-OE fruits showed a redder color, but VvSPL9-OE fruits had inhibited coloring. Meanwhile, in VvmiR156-OE fruits, the expression levels of anthocyanin metabolism-related genes had a higher expression, in particular, SlPAL, SlDFR, Sl3GT, and SlANS were significantly upregulated, but the expression of SlPAL and Sl3GT was inhibited in VvSPL9-OE fruits. Similarly, the regulation of anthocyanin synthesis by the miR156-SPL pathway also exists in Arabidopsis thaliana, apple, litchi, and rice [SPL on plant-specific metabolites is highly conserved in different species, which also provides an idea for the functional study of miR156-SPL in other species.In addition, we constructed the overexpression vectors of VvmiR156 and and rice ,24,25, iCHS, F3H, LDOX, and UFGT), and increase the content of anthocyanin [Arabidopsis thaliana, JA has synergistic regulatory effects on sucrose-induced DFR gene expression and anthocyanin synthesis [GL3, EGL3, TT8) and R2R3-MYBS  transcription factors in the MBW complex to inhibit anthocyanin synthesis [CHS, CHI, F3H, F3 ‘5’ H, DFR, LDOX, UFGT) in grape skins were lower than that in the control group, indicating that exogenous NAA treatment inhibited anthocyanin synthesis [GAI gene) is a negative regulator of GA signal. gai mutants do not affect GA synthesis but affect GA signal transduction. The expression level of the DFR gene in seedlings and leaves of the gai mutant was much higher than that of the wild-type, indicating that GA regulates anthocyanin biosynthesis through the DELLA protein [Hormone is one of the most important factors affecting anthocyanin synthesis ,38. Studhocyanin . Jasmoniynthesis . JAZ proynthesis . Auxin iynthesis . There a protein .VvSPL9 response hormones. Interestingly, GA treatment downregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early color-change period, but significantly upregulated the expression in the middle color-change and ripening stages. In addition, GA upregulated the expression levels of anthocyanin structural genes in the middle color-change stage and ripening stage, so GA mainly promoted grape berry coloring in the middle- and late-ripening stages. After ABA and MeJA treatment, grape berries can quickly color. ABA and MeJA significantly upregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early stage of color conversion (7DAT and 14DAT), and ABA and MeJA are mainly involved in the formation of color in the early color-change period. NAA treatment downregulated the expression of VvmiR156b/c/d and anthocyanin structural genes; NAA delayed the peak expression of genes to 35DAT and is mainly involved in the formation of color in the mature stage. Thus, we conclude that VvmiR156b/c/d-VvSPL9 can respond to different hormone signals to participate in the formation of anthocyanin of different periods , GA , MeJA , and auxin (TGA-element) responsive cis-elements. Of these hormone cis-acting elements, ABA and MeJA had the most cis-acting elements, followed by GA, while NAA had the least cis-acting elements. Based on this, ABA, GA, MeJA, and NAA were used to treat grape leaves and fruits, respectively, to verify the short-term and long-term effects of VvmiR156b/c/d and  periods , but the


JAZ gene families have been discovered across several plants, our knowledge about the JAZ gene family remains limited in the economically and medicinally important Chinese herb Mentha canadensis L. Here, seven non-redundant JAZ genes named McJAZ1–McJAZ7 were identified from our reported M. canadensis transcriptome data. Structural, amino acid composition, and phylogenetic analysis showed that seven McJAZ proteins contained the typical zinc-finger inflorescence meristem (ZIM) domain and JA-associated (Jas) domain as conserved as those in other plants, and they were clustered into four groups (A-D) and distributed into five subgroups . Quantitative real-time PCR (qRT-PCR) analysis showed that seven McJAZ genes displayed differential expression patterns in M. canadensis tissues, and preferentially expressed in flowers. Furthermore, the McJAZ genes expression was differentially induced after Methyl jasmonate (MeJA) treatment, and their transcripts were variable and up- or down-regulated under abscisic acid (ABA), drought, and salt treatments. Subcellular localization analysis revealed that McJAZ proteins are localized in the nucleus or cytoplasm. Yeast two-hybrid (Y2H) assays demonstrated that McJAZ1-5 interacted with McCOI1a, a homolog of Arabidopsis JA receptor AtCOI1, in a coronatine-dependent manner, and most of McJAZ proteins could also form homo- or heterodimers. This present study provides valuable basis for functional analysis and exploitation of the potential candidate McJAZ genes for developing efficient strategies for genetic improvement of M. canadensis.Jasmonate ZIM-domain (JAZ) proteins are the crucial transcriptional repressors in the jasmonic acid (JA) signaling process, and they play pervasive roles in plant development, defense, and plant specialized metabolism. Although numerous  Jasmonates (JAs), are lipid-derived natural phytohormones that act as ubiquitous moderators of a broad range of biological processes including plant growth ,4,5,6,7,Arabidopsis and rice Mentha case to SA , high tese to SA , and thise to SA ,62,63.M. canadensis are scant due to a lack of sequence data and mainly focused on chemical composition and medicinal usage [M. canadensis. Recently, transcriptome sequencing of M. canadensis under Methyl jasmonate (MeJA) treatment uncovered that JA was involved in promoting EO biosynthesis [M. canadensis provides basic information to analyze the key genes involved in regulating different aspects of M. canadensis including EO biosynthesis. As mentioned, JAZ proteins play an important role in JA signaling and JA-mediated secondary metabolism. Thus, based on the reported transcriptome sequencing data of M. canadensis, we first identified McJAZ family genes, analyzed their sequence properties, structural features, phylogenetic relationships, amino acid composition, and expression profiles in different tissues or under JA, ABA, and abiotic stress treatments. In addition, our results showed that McJAZ proteins were localized to the nucleus or cytoplasm, and most of them interacted with McCOI1a in a coronatine-dependent manner, and also formed homo- or heterodimers in M. canadensis. Thus, the current results enrich our knowledge of JAZ family genes and provide a valuable foundation for functional analysis and exploitation of the candidate McJAZ genes for genetic improvement of M. canadensis.By contrast, previous genetic studies of al usage , plant gal usage , and theal usage . Howeverynthesis , similarynthesis , and theMcJAZ family genes in M. canadensis, in total, twelve well-known JAZ protein sequences from A. thaliana [A. annua [McJAZ genes in the M. canadensis transcriptome database. Next, we obtained seven amplicons corresponding to seven distinct full-length McJAZ genes through PCR amplification with gene specific primers. Subsequently, the purified amplicons were ligated with pClone007 Blunt Simple vector, and the resulting products were transformed into Escherichia coli DH5α and sequenced, respectively. After this, we successfully cloned seven full-length McJAZ genes, and denoted them as McJAZ1-McJAZ7  with the default parameters  ranged from 372 (McJAZ7) to 1041 (McJAZ3) base pairs (bp), which was similar in length to the known AtJAZ CDs ranging from 396 (AtJAZ8) to 1059 (AtJAZ3) bp in A. thaliana [McJAZ genes encoded predicted products which varied in length from 123 (McJAZ7) to 346 (McJAZ3) amino acids (aa) residues, with molecular weights (MW) ranging from 13.87 kilodalton (kDa) (McJAZ7) to 36.89 kDa (McJAZ3), and the isoelectric point (pI) ranged from 6.12 (McJAZ2) to 9.96 (McJAZ6)  . NotablyJAZ family genes between M. canadensis and other known species including A. thaliana, O. sativa, and A. annua, a phylogenetic tree was constructed based on an unrooted neighbor-joining (NJ) method using twelve AtJAZ proteins from A. thaliana, fifteen OsJAZ proteins from O. sativa, nine AaJAZ proteins from A. annua, and seven McJAZ proteins from M. canadensis. The results showed that all the JAZ proteins were clustered into four groups (Groups A–D) and further classified into A1, A2, B1–B3, C, and D subgroups according to the reported phylogenetic analysis of AtJAZ proteins in Arabidopsis [To understand the evolutionary and phylogenetic relationships of the bidopsis . Among tM. canadensis  after prolonged MeJA exposure (24 h). In contrast, McJAZ3, McJAZ4, and McJAZ5 expression increased at 1 h after MeJA exposure, and significantly increased until a peak after 3 h, then gradually decreased after prolonged MeJA exposure   A.McJAZ4, the transcripts of McJAZ genes were also dynamically affected by exogenous ABA treatment. Among them, McJAZ2, McJAZ5, and McJAZ6 transcript levels were significantly up-regulated at 1, 3, and 24 h for McJAZ2, at 1, 12, and 24 h for McJAZ5, and at 1 and 24 h for McJAZ6 after ABA exposure  at 1 h respectively. Additionally, the expression of McJAZ1 was slightly up-regulated at 1 h, but down-regulated subsequently , and reached its lowest level at 6 h after ABA exposure. The transcript level of McJAZ3 was not obvious different at 1, 3, and 24 h, but was down-regulated at 6 and 12 h, compared with control (CK 0 h). McJAZ7 expression was slightly decreased after 1, 3, 6, and 12 h of ABA exposure, and then returned to a level comparable to that seen at the control time point (CK 0 h). These results revealed that McJAZ genes expression responds to JA and ABA, and may be involved in JA or ABA signaling pathway in M. canadensis.In addition, we found that except for exposure B. In parMcJAZ genes in response to abiotic stresses, qRT-PCR assays were employed to investigate the expression patterns of McJAZ genes in M. canadensis treated with drought and salt stress. The results showed that, under drought stress treatment, the expression levels of the seven McJAZ genes were significantly up-regulated at the individual time point, and expression levels varied by gene  at 24 h after drought treatment. On the other hand, the transcripts of McJAZ5 and McJAZ6 were also gradually induced, and reached their highest levels at nearly 8-fold and 39-fold at 24 h under drought treatment compared with control   at 12 h or 6 h, respectively. In addition, the transcript levels of McJAZ5 and McJAZ6 were up-regulated, and peaked at more than 7-fold or 9-fold compared with the control at 3 h, gradually decreased after prolonged salt treatment (6 and 12 h), then returned to levels similar to control (CK 0 h) at 24 h after salt treatment. These results implied that most of the McJAZ genes were sensitive to drought and salt stress treatments, and are likely involved in drought and salt stress related signaling networks in M. canadensis.In addition, seven reatment B. Among (CK 0 h) B. McJAZ3http://cello.life.nctu.edu.tw/, accessed on 30 April 2021). Preliminary results showed that seven McJAZ proteins were predicted to be targeted to nuclei (McJAZs-GFP (Green Fluorescence Protein) fusion expression vector under control of the CaMV 35S promoter, and subsequently these fusion proteins or the GFP control were transiently expressed in Nicotiana benthamiana leaf cells. The subcellular localization results showed that, in contrast to GFP, which was localized uniformly throughout the cell, most of the McJAZ proteins were exclusively located in the nucleus marked with the red arrow in the leaves of N. benthamiana  assays. As shown in M. canadensis, and then examined the interaction between McCOI1a and McJAZ proteins. As expected, interaction was also not detected for McCOI1a and all seven McJAZ proteins under selection medium (QDO) in the absence of COR  in a JA-Ile- or coronatine -dependent manner rather than a JA- or MeJA-dependent manner ,23,71,72e of COR B. In cone of COR C, suggesM. canadensis.Our previous results showed that all of the McJAZ proteins have a conserved TIFY domain , and preA. thaliana [T. aestivum [H. brasiliensis [L. aurea [JAZ gene family has not yet been reported in the economically and medicinally important plant species M. canadensis. Based on the available transcriptome data [M. canadensis and further investigated their sequence characteristics and protein structure, evolutionary relationships, expression profiles, subcellular locations, and protein-protein interactions. This work provided a basis for subsequent functional analysis of JAZ genes to deepen our understanding of JA signaling in the Lamiaceae family, especially in M. canadensis.JAs play diverse functions across the JA signaling pathway in plants . Notablythaliana , T. aestaestivum , H. brasiliensis , and L. L. aurea . Howeverome data , we perfMcJAZ genes from M. canadensis, and found that the length of CDs in the McJAZ gene family ranged from 372 to 1041 bp , but our results showed that McJAZ2 has a truncated Jas motif (DLPIARKNSLARFLEKRRDR) without the X5PY sequence  lacking LVSTSPY (X5PY) sequence in the whole Jas domain of AtJAZ10.1 [We isolated seven  1041 bp , which we family . Moreovesinensis . In geneterminus . Here, aterminus . The Jasterminus , and is terminus . The EARterminus . Given tsequence . PrevioutJAZ10.1 . Thus, wJAZ family genes were constitutively and differentially expressed in different tissues of various plants such as wheat [L. aurea [JAZ genes in different plant tissues is involved in regulating plant growth and development [McJAZ genes were constitutively expressed in the tested tissues, and showed a variable expression profile . For in vitro culture of N. benthamiana, seeds were sown on soil and 10-day-old seedlings were transplanted into plastic pots containing a mixture of nutrient soil and vermiculite . Next, M. canadensis and N. benthamiana plants were grown in a light chamber at 23 ± 2 °C with the desired light regime, and a 16-h light/8-h dark photoperiod.Province , for useM. canadensis seedlings were sprayed with 100 μM MeJA (Sigma-Aldrich (Shanghai) Trading Co. Ltd., Shanghai, China), 20 μM ABA (Sigma-Aldrich (Shanghai) Trading Co. Ltd., Shanghai, China), and 150 mM NaCl for 1, 3, 6, 12, and 24 hours (h) according to methods adapted from previous studies [M. canadensis seedlings were exposed to dry filter paper for 1, 3, 6, 12, and 24 h at room temperature according to methods adapted from previous studies [For hormone and salt stress treatments, two-week-old  studies . For dro studies . UntreatM. canadensis McJAZ genes were performed as described previously [M. canadensis was downloaded from the National Center for Biotechnology Information . The well-known 12 A. thaliana [A. annua JAZ protein sequences were downloaded from TAIR  or the annotated A. annua genome [M. canadensis protein databases by the local BLASTP method with an e-value of 1 × 10−5. The screened sequences were submitted to ORF Finder  to gain the complete coding sequences, and the candidate sequences were subjected to SMART  tools with default parameters to substantiate the existence of the conserved TIFY and Jas domains [McJAZ gene sequences with both TIFY and Jas domains were selected for further analysis.The isolation of eviously . In shorthaliana , and 9 Aa genome , respect domains , and detMcJAZ genes, the young leaves of two-week-old M. canadensis were collected for RNA extraction using the reagent Trizol  Co., Ltd., Beijing, China), and cDNA was synthesized using PrimeScript RT reagent Kit with gDNA Eraser  Co., Ltd., Beijing, China). The candidate McJAZ genes were amplified using PCR with defined primers . Subsequently, the full-length coding sequences of McJAZ genes were gel-purified using Gel DNA Mini Purification Kit , and the purified amplicons were ligated with pClone007 Blunt Simple vector , and then the resulting products were transformed into Escherichia coli DH5α and sequenced in TSINGKE Biotechnology Co., Ltd .To clone the  primers  in a 25 http://us.expasy.org/tools, accessed on 3 May 2021) [MW), and theoretical protein isoelectric points (pIs). The CELLO v.2.5 tool  was employed to predict the subcellular localization of candidate McJAZ proteins.The sequence and physio-chemical properties of candidate McJAZ proteins were predicted using the online ExPASy server (ay 2021) , includihttp://meme-suite.org/tools/meme, accessed on 8 May 2021) using the default settings. The composition and position of the conserved motifs, and the sequence logos of the TIFY and Jas domains were created by online MEME tools. Next, to check out the evolutionary relationships of McJAZ family proteins, the JAZ protein sequences including 12 A. thaliana [O. sativa [A. annua [https://phytozome.jgi.doe.gov/, accessed on 20 January 2021) and NCBI, respectively. The MEGA 5.0 program was adopted to construct the phylogenetic tree using a neighbor-joining (NJ) approach with the following parameters: Pairwise deletion and 2000 bootstrap replicates [Multiple sequence alignment analysis of the seven McJAZ proteins was performed to analyze the characteristics of the conserved sequences using Clustal X2 software with default settings . Meanwhi. sativa  and 9 A.A. annua  homologoplicates .McJAZ gene without its stop codon was PCR-amplified using gene-specific primers  used as internal control in the mint family in the previous study [−∆∆Ct method [To study tissue expression patterns, different tissues of 3-month-old us study , and thet method . All priMcJAZ genes were cloned into the prey vector pGADT7  or bait vector pGBKT7  , respectively, and the full-length coding sequences of McCOI1a and AtCOI1 were amplified using gene-specific primers  Co., Ltd., Beijing, China), and bait-only or prey-only was tested with empty AD or BD as negative controls.Yeast two-hybrid assays were used to test the paired physical interactions among McJAZ proteins, or the interactions between McJAZ proteins and AtCOI1, or its homolog McCOI1a. The full-length coding sequences of  primers , and insPseudomonas syringae according to previous studies [To investigate protein-protein interactions, the transformed AH109 yeast cells were first cultivated in liquid control medium SD/-Leu/-Trp (DDO) over 24 h, and then 5 μL of each yeast suspension was taken to inoculate the solid control medium SD/-Leu/-Trp (DDO) and selection medium SD/-Leu/-Trp/-Ade/-His (QDO) with or without 35 μM coronatine (COR), a structural and functional mimic of JA-Ile obtained from several strains of  studies ,72,95. Tt-test using GraphPad Prism 5 software. The values are mean ± standard error (SE) for replicates in each group. * p values ≤ 0.05 or ** p values ≤ 0.01 were considered as significant or extremely significant.All experiments were conducted with at least three biological replicates. Significant differences between the control versus each treatment were analyzed by Student’s The gene sequences data mentioned in this article can be found in the NCBI database under the accession numbers: McJAZ1 (MZ229995), McJAZ2 (MZ229996), McJAZ3 (MZ229997), McJAZ4 (MZ229998), McJAZ5 (MZ229999), McJAZ6 (MZ230000), McJAZ7 (MZ230001), and AtCOI1 (AT2G39940).