Biomimetic natural biomaterials for tissue engineering and regenerative medicine:new biosynthesis methods,recent advances,and emerging applications

Biomimetic materials have emerged as attractive and competitive alternatives for tissue engineering(TE)and regenerative medicine.In contrast to conventional biomaterials or synthetic materials,biomimetic scaffolds based on natural biomaterial can offer cells a broad spectrum of biochemical and biophysical cues that mimic the in vivo extracellular matrix(ECM).Additionally,such materials have mechanical adaptability,micro-structure interconnectivity,and inherent bioactivity,making them ideal for the design of living implants for specific applications in TE and regenerative medicine.This paper provides an overview for recent progress of biomimetic natural biomaterials(BNBMs),including advances in their preparation,functionality,potential applications and future challenges.We highlight recent advances in the fabrication of BNBMs and outline general strategies for functionalizing and tailoring the BNBMs with various biological and physicochemical characteristics of native ECM.Moreover,we offer an overview of recent key advances in the functionalization and applications of versatile BNBMs for TE applications.Finally,we conclude by offering our perspective on open challenges and future developments in this rapidly-evolving field.

Chain Elongation Using Native Soil Inocula:Exceptional n-Caproate Biosynthesis Performance and Microbial Mechanisms

This study demonstrates the feasibility and effectiveness of utilizing native soils as a resource for inocula to produce n-caproate through the chain elongation(CE)platform,offering new insights into anaerobic soil processes.The results reveal that all five of the tested soil types exhibit CE activity when supplied with high concentrations of ethanol and acetate,highlighting the suitability of soil as an ideal source for n-caproate production.Compared with anaerobic sludge and pit mud,the native soil CE system exhibited higher selectivity(60.53%),specificity(82.32%),carbon distribution(60.00%),electron transfer efficiency(165.00%),and conductivity(0.59 ms∙cm^(-1)).Kinetic analysis further confirmed the superiority of soil in terms of a shorter lag time and higher yield.A microbial community analysis indicated a positive correlation between the relative abundances of Pseudomonas,Azotobacter,and Clostridium and n-caproate production.Moreover,metagenomics analysis revealed a higher abundance of functional genes in key microbial species,providing direct insights into the pathways involved in n-caproate formation,including in situ CO_(2)utilization,ethanol oxidation,fatty acid biosynthesis(FAB),and reverse beta-oxidation(RBO).The numerous functions in FAB and RBO are primarily associated with Pseudomonas,Clostridium,Rhodococcus,Stenotrophomonas,and Geobacter,suggesting that these genera may play roles that are involved or associated with the CE process.Overall,this innovative inoculation strategy offers an efficient microbial source for n-caproate production,underscoring the importance of considering CE activity in anaerobic soil microbial ecology and holding potential for significant economic and environmental benefits through soil consortia exploration.

Rational Engineering of Secondary Metabolic Pathways in a Heterologous Host to Enable the Biosynthesis of Hibarimicin Derivatives with Enhanced Anti-Melanomic Activity

A 61-kb biosynthetic gene cluster(BGC),which is accountable for the biosynthesis of hibarimicin(HBM)B from Microbispora rosea subsp.hibaria TP-A0121,was heterologously expressed in Streptomyces coelicolor M1154,which generated a trace of the target products but accumulated a large amount of shunt products.Based on rational analysis of the relevant secondary metabolism,directed engineering of the biosynthetic pathways resulted in the high production of HBM B,as well as new HBM derivates with improved antitumor activity.These results not only establish a biosynthetic system to effectively synthesize HBMs-a class of the largest and most complex Type-Ⅱpolyketides,with a unique pseudo-dimeric structure-but also set the stage for further engineering and deep investigation of this complex biosynthetic pathway toward potent anticancer drugs.

Transcriptome analysis reveals steroid hormones biosynthesis pathway involved in abdominal fat deposition in broilers

Excessive abdominal fat deposition reduces the feed efficiency and increase the cost of production in broilers.Therefore,it is an important task for poultry breeders to breed broilers with low abdominal fat.Abdominal fat deposition is a highly complex biological process,and its molecular basis remains elusive.In this study,we performed transcriptome analysis to compare gene expression profiles at different stages of abdominal fat deposition to identify the key genes and pathways involved in abdominal fat accumulation.We found that abdominal fat weight(AFW)increased gradually from day 35(D35)to 91(D91),and then decreased at day 119(D119).Accordingly,after detecting differentially expressed genes(DEGs)by comparing gene expression profiles at D35 vs.D63 and D35 vs.D91,and identifying gene modules associated with fat deposition by weighted gene co-expression network analysis(WGCNA),we performed intersection analysis of the detected DEGs and WGCNA gene modules and identified 394 and 435 intersecting genes,respectively.The results of the Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses showed that the steroid hormone biosynthesis and insulin signaling pathways were co-enriched in all intersecting genes,steroid hormones have been shown that regulated insulin signaling pathway,indicating the importance of the steroid hormone biosynthesis pathway in the development of broiler abdominal fat.We then identified 6 hub genes(ACTB,SOX9,RHOBTB2,PDLIM3,NEDD9,and DOCK4)related to abdominal fat deposition.Further analysis also revealed that there were direct interactions between 6 hub genes.SOX9 has been shown to bind to proteins required for steroid hormone receptor binding,and RHOBTB2 indirectly regulates the steroid hormones biosynthesis through cyclin factor,and ultimately affect fat deposition.Our results suggest that the genes RHOBTB2 and SOX9 play an important role in fat deposition in broilers,by regulating steroid hormone synthesis.These findings provide new targets and directions for further studies on the mechanisms of fat deposition in chicken.

Wetting alternating with partial drying during grain filling increases lysine biosynthesis in inferior rice grain

Lysine content is a criterion of the nutritional quality of rice.Understanding the process of lysine biosynthesis in early-flowering superior grain(SG)and late-flowering inferior grain(IG)of rice would advance breeding and cultivation to improve nutritional quality.However,little information is available on differences in lysine anabolism between SG and IG and the underlying mechanism,and whether and how irrigation regimes affect lysine anabolism in these grains.A japonica rice cultivar was grown in the field and two irrigation regimes,continuous flooding(CF)and wetting alternating with partial drying(WAPD),were imposed from heading to the mature stage.Lysine content and activities of key enzymes of lysine biosynthesis,and levels of brassinosteroids(BRs)were lower in the IG than in the SG at the early grainfilling stage but higher at middle and late grain-filling stages.WAPD increased activities of these key enzymes,BR levels,and contents of lysine and total amino acids in IG,but not SG relative to CF.Application of 2,4-epibrassinolide to rice panicles in CF during early grain filling reproduced the effects of WAPD,but neither treatment altered the activities of enzymes responsible for lysine catabolism in either SG or IG.WAPD and elevated BR levels during grain filling increased lysine biosynthesis in IG.Improvement in lysine biosynthesis in rice should focus on IG.

OsFK1 encodes C-14 sterol reductase,which is involved in sterol biosynthesis and affects premature aging of leaves in rice

The enzyme C-14 sterol reductase is involved in biosynthesis of brassinosteroids(BR)and sterols,as well as plant development.OsFK1,a member of the sterol biosynthesis pathway located in the endoplasmic reticulum(ER),encodes C-14 sterol reductase.However,there is little research on the function of C-14 sterol reductase in rice.Compared with the wild type,an osfk1 mutant showed dwarf phenotype and premature aging in the second leaf during the trefoil stage,and abnormal development of leaf veins during the tillering stage.The osfk1 mutant showed signs of aberrant PCD,as evidenced by TUNEL staining.This suggested that high ROS buildup caused DNA damage and ROS-mediated cell death in the mutant.The osfk1 mutant also showed decreased chlorophyll content and aberrant chloroplast structure.Sequencing of the osfk1 mutant allele revealed a non-synonymous G to A mutation in the final intron,leading to early termination.Here,we identified the OsFK1 allele,cloned it by Mutmap sequencing,and verified it by complementation.HPLC-MS/MS assays demonstrated that the osfk1 mutation caused lower phytosterol levels.These findings showed that the OsFK1 allele encoding C-14 sterol reductase is involved in phytosterol biosynthesis and mediates normal development of rice plants.

The biosynthesis of alarm pheromone in the wheat aphid Rhopalosiphum padi is regulated by hormones via fatty acid metabolism

Aphids are major insect pests in agriculture and forestry worldwide.Following attacks by natural enemies,many aphids release an alarm pheromone to protect their population.In most aphids,the main component of the aphid alarm pheromone(AAP)is the sesquiterpene hydrocarbon(E)-β-farnesene(EβF).However,the mechanisms behind its biosynthesis and regulation remain poorly understood.In this study,we used the bird cherry–oat aphid Rhopalosiphum padi,which is an important wheat aphid,to investigate the regulatory mechanisms of EβF biosynthesis.Our results showed that EβF biosynthesis occurs during the mature embryo period and the molting period of the 1st-and 2nd-instar nymphs.Triglycerides provide the prerequisite material for EβF production and release.Based on transcriptome sequencing,RNAi analysis,hormone treatments,and quantitative measurements,we found that the biosynthesis of EβF utilizes acetyl coenzyme A produced from fatty acid degradation,which can be suppressed by juvenile hormone but it is promoted by 20-hydroxyecdysone through the modulation of fatty acid metabolism.This is the first systemic study on the modulation of EβF production in aphids.The results of our study provide insights into the molecular regulatory mechanisms of AAP biosynthesis,as well as valuable information for designing potential aphid control strategies.

Identification and Molecular Characterization of the Alkaloid Biosynthesis Gene Family in Dendrobium catenatum

As one of the main active components of Dendrobium catenatum, alkaloids have high medicinal value. The physicochemicalproperties, conserved domains and motifs, phylogenetic analysis, and cis-acting elements of the genefamily members in the alkaloid biosynthesis pathway of D. catenatum were analyzed by bioinformatics, and theexpression of the genes in different years and tissues was analyzed by qRT-PCR. There are 16 gene families,including 25 genes, in the D. catenatum alkaloid biosynthesis pathway. The analysis of conserved domains andmotifs showed that the types, quantities, and orders of domains and motifs were similar among members ofthe same family, but there were significant differences among families. Phylogenetic analysis indicated that thegene family members showed some evolutionary conservation. Cis-acting element analysis revealed that therewere a large number of light-responsive elements and MYB (v-myb avian myeloblastosis viral oncogene homolog)-related elements in these genes. qRT-PCR showed that expressions of gene family members involved in alkaloidsynthesis were different in different years and tissues of D. catenatum. This study provides a theoretical basisfor further exploration of the regulatory mechanisms of these genes in the alkaloid biosynthesis of D. catenatum.

Research Progress on Functions and Biosynthesis of D-Psicose

D-Psicose,a naturally occurring rare sugar,exhibits a sweetness approximately 70%that of sucrose.It possesses high solubility,antioxidant activity,anti-inflammatory properties,and the ability to regulate cholesterol levels and enhance insulin sensitivity.However,D-psicose is relatively scarce in nature,making large-scale extraction and utilization impractical.Consequently,the development of cost-effective synthetic strategies for D-psicose is pivotal for its industrial application.In recent years,the Izumoring strategy has emerged as an efficient alternative to chemical synthesis for producing D-psicose.Nonetheless,limitations in the biotransformation of D-psicose,primarily governed by the conversion rate of D-psicose 3-epimerase(DPEase)and enzyme yield,continue to pose challenges in achieving economically viable production.Enzyme engineering and the establishment of high-level expression systems remain crucial avenues for reducing the overall biosynthesis costs.

Biosynthesis of Silver Nanoparticles with Clerodendron phlomoides Leave Extract:Particle Morphology,Antimicrobial Potential and Application

Silver nanoparticles are versatile nanomaterials that have found numerous applications in various fields.The use of plant extract for the synthesis of silver is a green and sustainable approach.Clerodendron phlomoides leaves extract has been found to contain various phytochemicals,such as phenols,flavonoids,tannins,and alkaloids,which possess reducing and stabilizing properties that can aid the production of silver particles.In this paper,morphological and topographical analyses were performed on silver nanoparticles.The biosynthesized silver nanoparticles showed antimicrobial potential against wound pathogens.SEM and TEM micrographs revealed that the particles were sphere and nanosized,which makes them suitable for various biomedical applications.

Systematic analysis of MYB transcription factors and the role of LuMYB216 in regulating anthocyanin biosynthesis in the flowers of flax(Linum usitatissimum L.)

Anthocyanin is an important pigment that affects plant color and nutritional quality.MYBs play an important role in plant anthocyanin synthesis and accumulation.However,the regulatory function of MYB transcription factors in anthocyanin synthesis in flax flowers is still unclear.In this study,402 MYB transcription factors were identified in the flax genome.These MYB members are unevenly distributed on 15 chromosomes.The R2R3-LuMYB members were divided into 32phylogenetic subfamilies.qRT-PCR analysis showed that seven R2R3-LuMYB genes in the adjacent subfamily of the evolutionary tree had similar expression patterns,among which Lu MYB216 was highly expressed in the petals of different colors.Moreover,gene editing of LuMYB216 in flax showed that the petal color,anther color and seed coat color of mutant plants were significantly lighter than those of wild-type plants,and the anthocyanin content of lumyb216 mutant plants was significantly reduced.Correlation analysis indicated that LuMYB216 was significantly positively correlated with the upstream regulator bHLH30.This study systematically analyzed the MYB gene family in flax,laying a foundation for studying the regulation of LuMYB216 in flax flower anthocyanin synthesis.

GA Associated Dwarf 5 encodes an ent-kaurenoic acid oxidase required for maize gibberellin biosynthesis and morphogenesis

Gibberellin(GA)functions in plant growth and development.However,genes involved in the biosynthesis and regulation of GA in crop plants are poorly understood.We isolated the mutant gad5-1(GAAssociated Dwarf 5),characterized by dwarfing,short internodes,and dark green and short leaves.Map-based gene cloning and allelic verification confirmed that ZmGAD5 encodes ent-kaurenoic acid oxidase(KAO),which catalyzes KA(ent-kaurenoic acid)to GA12 conversion during GA biosynthesis in maize.ZmGAD5 is localized to the endoplasmic reticulum and is present in multiple maize organs.In gad5-1,the expression of ZmGAD5 is severely reduced,and the levels of the direct substrate of KAO,KA,is increased,leading to a reduction in GA content.The abnormal phenotype of gad5-1 was restored by exogenous application of GA3.The biomass,plant height,and levels of GA12 and GA3 in transgenic Arabidopsis overexpressing ZmGAD5 were increased in comparison with the corresponding controls Col-0.These findings deepen our understanding of genes involved in GA biosynthesis,and could lead to the development of maize lines with improved architecture and higher planting-density tolerance.

The PcERF5 promotes anthocyanin biosynthesis in red-fleshed pear(Pyrus communis)through both activating and interacting with PcMYB transcription factors

As there is a strong interest in red-skinned pears,the molecular mechanism of anthocyanin regulation in red-skinned pears has been widely investigated;however,little is known about the molecular mechanism of anthocyanin regulation in red-fleshed pears due to limited availability of such germplasm,primarily found in European pears(Pyrus communis).In this study,based on transcriptomic analysis in red-fleshed and white-fleshed pears,we identified an ethylene response factor(ERF)from P.communis,PcERF5,of which expression level in fruit flesh was significantly correlated with anthocyanin content.We then verified the function of PcERF5 in regulating anthocyanin accumulation by genetic transformation in both pear skin and apple calli.PcERF5 regulated anthocyanin biosynthesis by different regulatory pathways.On the one hand,PcERF5 can activate the transcription of flavonoid biosynthetic genes(PcDFR,PcANS and PcUFGT)and two key transcription factors encoding genes PcMYB10 and PcMYB114.On the other hand,PcERF5 interacted with PcMYB10 to form the ERF5-MYB10 protein complex that enhanced the transcriptional activation of PcERF5 on its target genes.Our results suggested that PcERF5 functioned as a transcriptional activator in regulating anthocyanin biosynthesis,which provides new insights into the regulatory mechanism of anthocyanin biosynthesis.This new knowledge will provide guidance for molecular breeding of red-fleshed pear.

Metabolic and transcriptomic analyses elucidate a novel insight into the network for biosynthesis of carbohydrate and secondary metabolites in the stems of a medicinal orchid Dendrobium nobile

Dendrobium nobile is an important medicinal and nutraceutical herb.Although the ingredients of D.nobile have been identified as polysaccharides,alkaloids,amino acids,flavonoids and bibenzyls,our understanding of the metabolic pathways that regulate the synthesis of these compounds is limited.Here,we used transcriptomic and metabolic analyses to elucidate the genes and metabolites involved in the biosynthesis of carbohydrate and several secondary metabolites in the stems of D.nobile.A total of 1005 metabolites and 31,745 genes were detected in the stems of D.nobile.The majority of these metabolites and genes were involved in the metabolism of carbohydrates(fructose,mannose,glucose,xylulose and starch),while some were involved in the metabolism of secondary metabolites(alkaloids,β-tyrosine,ferulic acid,4-hydroxybenzoate and chrysin).Our predicted regulatory network indicated that five genes(AROG,PYK,DXS,ACEE and HMGCR) might play vital roles in the transition from carbohydrate to alkaloid synthesis.Correlation analysis identified that six genes(ALDO,PMM,BGLX,EGLC,XYLB and GLGA) were involved in carbohydrate metabolism,and two genes(ADT and CYP73A) were involved in secondary metabolite biosynthesis.Our analyses also indicated that phosphoenol-pyruvate(PEP) was a crucial bridge that connected carbohydrate to alkaloid biosynthesis.The regulatory network between carbohydrate and secondary metabolite biosynthesis established will provide important insights into the regulation of metabolites and biological systems in Dendrobium species.

The PcHY5 methylation is associated with anthocyanin biosynthesis and transport in ‘Max Red Bartlett' and ‘Bartlett' pears

The red coloring of pear fruits is mainly caused by anthocyanin accumulation. Red sport, represented by the green pear cultivar ‘Bartlett’(BL) and the red-skinned derivative ‘Max Red Bartlett’(MRB), is an ideal material for studying the molecular mechanism of anthocyanin accumulation in pear. Genetic analysis has previously revealed a quantitative trait locus(QTL) associated with red skin color in MRB. However, the key gene in the QTL and the associated regulatory mechanism remain unknown. In the present study, transcriptomic and methylomic analyses were performed using pear skin for comparisons between BL and MRB. These analyses revealed differential PcHY5 DNA methylation levels between the two cultivars;MRB had lower PcHY5 methylation than BL during fruit development, and PcHY5 was more highly expressed in MRB than in BL. These results indicated that PcHY5 is involved in the variations in skin color between BL and MRB. We further used dual luciferase assays to verify that PcHY5 activates the promoters of the anthocyanin biosynthesis and transport genes PcUFGT, PcGST, PcMYB10 and PcMYB114, confirming that PcHY5 not only regulates anthocyanin biosynthesis but also anthocyanin transport. Furthermore, we analyzed a key differentially methylated site between MRB and BL, and found that it was located in an intronic region of PcHY5. The lower methylation levels in this PcHY5 intron in MRB were associated with red fruit color during development, whereas the higher methylation levels at the same site in BL were associated with green fruit color. Based on the differential expression and methylation patterns in PcHY5 and gene functional verification, we hypothesize that PcHY5, which is regulated by methylation levels, affects anthocyanin biosynthesis and transport to cause the variations in skin color between BL and MRB.

Identification of key gene networks controlling monoterpene biosynthesis during grape ripening by integrating transcriptome and metabolite profiling

Monoterpenes are typical aroma components of muscat grape cultivars,providing pleasant floral and fruity aromas to grapes and wines.However,the molecular mechanism of monoterpene biosynthesis between muscat and non-muscat grape remains unclear.Here,the muscat grape cultivar‘Jumeigui’and the non-muscat grape cultivar‘Kyoho’were chosen as plant materials for a comprehensive transcriptome and metabolite analysis.The gas chromatography-mass spectrometry(GC-MS)analysis demonstrated that a total of 27 and 23 monoterpene compounds were identified and quantified in the‘Jumeigui’and‘Kyoho’grape,respectively.‘Jumeigui’grape accumulated significantly higher concentrations of monoterpenes than‘Kyoho’grape.Furthermore,geraniol,linalool,geranic acid,and β-citronellol might be important odorants contributing to the floral character of the‘Jumeigui’grape due to the high levels odor activity values(OAVs).Transcriptome analysis demonstrated that the expression profiles of VvDXS,VvGGPPS.SSU1,Vv TPS-b/g showed a positive correlation with monoterpene accumulation in grapes.In addition,the expression patterns of the genes involved in jasmonic acid(JA)synthesis and signal were also positively correlated with monoterpene accumulation.All these results will help guide the functional verification of candidate genes related to monoterpene biosynthesis,as well as identify the master transcriptional and hormonal regulators of this pathway in grapes.

VabHLH137 promotes proanthocyanidin and anthocyanin biosynthesis and enhances resistance to Colletotrichum gloeosporioides in grapevine

Proanthocyanidins(PAs)and anthocyanins are involved in the response of plants to various environmental stresses.However,the mechanism behind defense-induced PA biosynthetic regulation is still not completely elucidated,also in grapevine.This study performed a transcriptome sequencing analysis of grape berries infected with Colletotrichum gloeosporioides to highlight the induction of the VabHLH137 factor from the basic helix-loop-helix(bHLH)XII subfamily by the fungus,which appeared to be significantly co-expressedwith PA-related genes.The functional analysis of VabHLH137 overexpression and knockdownin transgenic grape calli showed that it positively regulated PA and anthocyanin biosynthesis.Moreover,VabHLH137 overexpression in the grape calli significantly increased resistance to C.gloeosporioides.A yeast one-hybrid and electrophoretic mobility shift assay revealed that VabHLH137 directly bound to the VaLAR2 promoter,enhancing its activity and interacting with VaMYBPAR,a transcriptional activator of PA biosynthesis.Furthermore,transient experiments showed that although the VabHLH137+VaMYBPAR complex activated VaLAR2 expression,it failed to further enhance VaLAR2 expression compared to VaMYBPAR alone.The findings indicated that VabHLH137 enhanced PA biosynthesis by activating of VaLAR2 expression,providing new insight into the transcriptional regulation of defense-induced PA biosynthesis in grapevine.

A comprehensive review of azadirachtin: physicochemical properties, bioactivities, production, and biosynthesis

Azadirachtin,a complex tetratriterpenoid limonin with potent insecticidal properties,is the most widely used biological pesticide worldwide.Its versatile pharmacological applications include the inhibition of tumor growth and anti-malarial,anti-bacterial,and anti-inflammatory properties.Azadirachtin plays a pivotal role in pest control and novel drug development.The primary source of azadirachtin is the neem tree(Azadirachta indica A.Juss),with an azadirachtin content ranging from 0.3%to 0.5%.Despite the market demand for botanical pesticides reaching approximately 100,000 tons per year,the annual neem production in China is only 1.14 tons.Although azadirachtin can be obtained through plant extraction or chemical synthesis,the quantity obtained does not meet the market demand in China.The sluggish pace of azadirachtin biosynthesis results from the limited availability of genetic information and the complexity of the synthetic pathway.Recent advancements in azadirachtin biosynthesis hold promise as an efficient collection method.In this study,we explored the physicochemical properties,biological activities,mechanisms of action,and acquisition methods of azadirachtin.We also delved into recent progress in azadirachtin biosynthesis and assessed potential future usage challenges.This study aims to establish a theoretical foundation for the scientific application and efficient synthesis of azadirachtin,offering valuable reference information to the industry.

EXPRESSION OF c－myc GENE AND BIOSYNTHESIS OF BIOLOGICAL MACROMOLECULES IN ANTISENSE TRANSFECTANT HL_（60）~R－9

The recombinant plasmid PGC was constructed for transcription unit of c-myc gene with diorientation in vitro, to make RNA probes for detection of c-myc mRNA and antisence RNA expression of tranfectant HL-9,which was obtained from HL60 cells transfected with inducible c-myc antisense RNA expression plasmid. The results from HL-9 cells induced by Cd2+ indicated that expression of c-myc antisense RNA increased with Cd2+ concentration and exposure time, while c-myc mRNA expression progressively reduced. Using immunohistochemical technique no c-myc P62 protein expression was detected. The incorporation of 3H-TdR, 3H-UR and 3H-Leu revealed significant suppression of DNA, RNA and protein biosynthesis. It is suggested that the reversion changes previously reported in malignant Phenotypes of HL-9 cells and the inhibition of macromolecular biosynthesis mentioned above were associated with the blockade of c-myc gene expression by its antisense RNA.

The Chlorophyll Biosynthesis in Lotus Embryo Is Light-dependent

Angiosperms need light to synthesize chlorophyll, but lotus (Nelumbo nucifera Gaertn.) embryo was suspected to have the ability to form chlorophyll in the dark because lotus embryo can turn into green under the coverage of four layers of integuments (cotyledon, seed coat, pericarp, lotus pod) which were thought impossible for light to pass through. The authors excluded this possibility based on two experimental results: First, enclosing the young lotus pod with aluminium foil, the growth of louts embryo continued, but the chlorophyll formation was seriously inhibited. A lot of protochlorophyllide, chlorophyll precursor, were accumulated, most of which were combined with LPOR (light dependent protochlorophyllide oxidoreductase). Second, DPOR (dark or light-independent protochlorophyllide oxidoreductase) was the enzyme necessary for chlorophyll synthesis in the dark. The genes encoding DPOR were conservative in many species, but no homologues could be found in lotus genome. Taken together, authers' results clearly demonstrated that lotus embryo synthesizes chlorophyll only through the light-dependent pathway.