Radish Cotyledons Research Articles

RsNRAMP5, a major metal transporter, promotes cadmium influx and ROS accumulation in radish (Raphanus sativus L.)

Arable soil contamination with heavy metals (HMs) poses a great potential threat to vegetable crops and human health. Radish (Raphanus sativus L.), an economical and popular root vegetable crop, is easily absorbed HMs by its taproot. Although the Natural Resistance-Associated Macrophage Proteins (NRAMPs) were participated in transporting a number of HMs in plants, whether and how the NRAMP genes involved in cadmium (Cd) uptake and transport remains elusive in radish. In this study, a total of nine RsNRAMP gene members were identified, which were classified into three subgroups and dispersed on six radish chromosomes. Three RsNRAMPs (RsNRAMP3, RsNRAMP4 and RsNRAMP5) displayed high expression in the vascular cambium, and they exhibited obviously Cd-induced expression, among which the expression of RsNRAMP4 and RsNRAMP5 reached to the highest level at 24h. Moreover, the RsNRAMP5 was localized to the plasma membrane and its promoter activity was dramatically induced by Cd exposure. Heterologous expression analysis indicated that over-expression of RsNRAMP5 significantly promoted the uptake of Cd, lead (Pb), iron (Fe) and manganese (Mn) in yeast cells. In addition, the transient over-expression of RsNRAMP5 promoted Cd uptake and enhanced ROS (reactive oxygen species) accumulation in radish cotyledons. These findings would expedite unraveling the molecular mechanism underlying RsNRAMP5-mediated Cd uptake and transport in radish.

Establishing VIGS and CRISPR/Cas9 techniques to verify RsPDS function in radish

Virus-induced gene silencing (VIGS) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology. However, the investigation of gene silencing and editing in radish remains limited. In this study, a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus (TRV)- and turnip yellow mosaic virus (TYMV)-mediated gene silencing vectors. The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish. The expression level of RsPDS was significantly inhibited using VIGS in ‘NAU-067’ radish leaves. The rootless seedlings of ‘NAU-067’ were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences. Nine adventitious roots were blue with GUS staining, and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing. Furthermore, albino lines were generated with A. tumefaciens-mediated transformation of radish cotyledons. Five base substitutions and three base deletions occurred at target sequence 2 in Line 1, and three base insertions and three base substitutions occurred at target sequence 1 in Line 2. This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish, which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.

Discovery of N-benzyl-6-methylpicolinamide as a potential scaffold for bleaching herbicides.

In pesticide research, bleaching herbicides have always been a hot topic. Our previous research showed that N-(4-fluorobenzyl)-2-methoxybenzamide is an innovative lead compound for bleaching herbicides. A total of 40 derivatives of picolinamides were prepared and evaluated for their herbicidal activity by Petri dish tests and postemergence trials. The structure-activity relationship (SAR) revealed that introducing electron-withdrawing groups at the 3- or 4-positions of the benzyl significantly enhances herbicidal activity. Furthermore, ZI-04 induced similar symptoms such as bleaching effect in treated weeds and accumulation of biosynthetic precursors for carotenoids as observed with diflufenican. ZI-04 also exhibited significant cross-resistance to diflufenican and had a lower resistance risk than diflufenican. N-benzyl-6-methylpicolinamides were discovered as a novel scaffold for bleaching herbicides. The accumulation of phytoene, phytofluene and ζ-Carotene in radish cotyledons, and cross-resistance observed with diflufenican, showed that title compounds can interfere with carotenoid biosynthesis. © 2024 Society of Chemical Industry.

Genome‐wide characterization of homeodomain‐leucine zipper genes reveals RsHDZ17 enhances the heat tolerance in radish (Raphanus sativus L.)

Homeodomain-leucine zipper (HD-Zip) transcription factors are involved in various biological processes of plant growth, development, and abiotic stress response. However, how they regulate heat stress (HS) response remains largely unclear in plants. In this study, a total of 83 RsHD-Zip genes were firstly identified from the genome of Raphanus sativus. RNA-Seq, RT-qPCR and promoter activity assays revealed that RsHDZ17 from HD-Zip Class I was highly expressed under heat, salt, and Cd stresses. RsHDZ17 is a nuclear protein with transcriptional activity at the C-terminus. Ectopic overexpression (OE) of RsHDZ17 in Arabidopsis thaliana enhanced the HS tolerance by improving the survival rate, photosynthesis capacity, and scavenging for reactive oxygen species (ROS). In addition, transient OE of RsHDZ17 in radish cotyledons impeded cell injury and augmented ROS scavenging under HS. Moreover, yeast one-hybrid, dual-luciferase assay, and electrophoretic mobility shift assay revealed that RsHDZ17 could bind to the promoter of HSFA1e. Collectively, these pieces of evidence demonstrate that RsHDZ17 could play a positive role in thermotolerance, partially through up-regulation of the expression of HSFA1e in plants. These results provide novel insights into the role of HD-Zips in radish and facilitate genetical engineering and development of heat-tolerant radish in breeding programs.

RsGSTF12 Contributes to Anthocyanin Sequestration in Radish (Raphanus sativus L.).

Anthocyanins are water-soluble plant pigments mainly stored in the plant vacuoles. Glutathione S-transferases (GSTs) are a multifunctional enzyme family, which can regulate substance metabolism and biological and abiotic stresses in plants. However, few reports were focused on the involvement of GSTs in anthocyanin sequestration in red skin radish. Here, we identified a glutathione S-transferase gene RsGSTF12 that played roles in anthocyanin sequestration in radish. The bioinformatics analysis revealed that RsGSTF12 belonged to the phi (F) class of glutathione S-transferases and showed a high homology with AtGSTF12, followed by AtGSTF11. The subcellular localization assay showed that RsGSTF12 was located in the endoplasmic reticulum and tonoplast. Temporal and spatial gene expression-specific analyses uncovered a strong correlation of RsGSTF12 with anthocyanin accumulation in radish sprouts. The anthocyanin solubility assay found RsGSTF12 was capable of improving cyanidin water solubility in vitro. Transiently expressing RsGSTF12 in radish cotyledons was able to increase their anthocyanin sequestrations. Furthermore, the functional complementation and overexpression of the Arabidopsis thaliana tt19 mutant and wild type demonstrated that RsGSTF12 might play an indispensable role in anthocyanin accumulation in radish. Taken together, we provide compelling evidence that RsGSTF12 functions critically in how anthocyanins are sequestrated in radish, which may enrich our understanding of the mechanism of anthocyanin sequestration.

Loss of the R2R3 MYB Transcription Factor RsMYB1 Shapes Anthocyanin Biosynthesis and Accumulation in Raphanus sativus.

The red or purple color of radish (Raphanus sativus L.) taproots is due to anthocyanins, which have nutritional and aesthetic value, as well as antioxidant properties. Moreover, the varied patterns and levels of anthocyanin accumulation in radish roots make them an interesting system for studying the transcriptional regulation of anthocyanin biosynthesis. The R2R3 MYB transcription factor RsMYB1 is a key positive regulator of anthocyanin biosynthesis in radish. Here, we isolated an allele of RsMYB1, named RsMYB1Short, in radish cultivars with white taproots. The RsMYB1Short allele carried a 4 bp insertion in the first exon causing a frame-shift mutation of RsMYB1, generating a truncated protein with only a partial R2 domain at the N-terminus. Unlike RsMYB1Full, RsMYB1Short was localized to the nucleus and the cytoplasm and failed to interact with their cognate partner RsTT8. Transient expression of genomic or cDNA sequences for RsMYB1Short in radish cotyledons failed to induce anthocyanin accumulation, but that for RsMYB1Full activated it. Additionally, RsMYB1Short showed the lost ability to induce pigment accumulation and to enhance the transcript level of anthocyanin biosynthetic genes, while RsMYB1Full promoted both processes when co-expressed with RsTT8 in tobacco leaves. As the result of the transient assay, co-expressing RsTT8 and RsMYB1Full, but not RsMYB1Short, also enhanced the promoter activity of RsCHS and RsDFR. We designed a molecular marker for RsMYB1 genotyping, and revealed that the RsMYB1Short allele is common in white radish cultivars, underscoring the importance of variation at the RsMYB1 locus in anthocyanin biosynthesis in the radish taproot. Together, these results indicate that the nonsense mutation of RsMYB1 generated the truncated protein, RsMYB1Short, that had the loss of ability to regulate anthocyanin biosynthesis. Our findings highlight that the frame shift mutation of RsMYB1 plays a key role in anthocyanin biosynthesis in the radish taproot.

Zeocin-Induced DNA Double-Strand Breaks Affect Endoreduplication and Cell Size in Radish Cotyledon Epidermis

Zeocin-Induced DNA Double-Strand Breaks Affect Endoreduplication and Cell Size in Radish Cotyledon Epidermis

Differential anthocyanin accumulation in radish taproot: importance of RsMYB1 gene structure.

RsMYB1a was the crucial MYB, and RsbHLH4 is the essential partner in regulating the anthocyanin biosynthesis in radish. There are four color types of radish according to whether or not the anthocyanin accumulates in the skin and flesh of taproot. Red radishes accumulate a substantial amount of anthocyanins in both the skin and flesh. It is well known that the MYB-bHLH-WD40 transcription factor(s) complex regulates the biosynthesis of anthocyanin in plants. Here in, four candidate MYB and bHLH genes, RsMYB1a, RsMYB1b, RsbHLH2 and RsbHLH4, were isolated from red radish 'Hongxin 1'. The expression of RsbHLH4 and the two structural genes RsANS and RsUFGT was significantly positively correlated with anthocyanin contents. The expression of RsMYB1a was also highly correlated with anthocyanin accumulation, particularly when the white flesh sample of 'Hongxin 1-1' was excluded. The transient expression of RsMYB1a in the radish cotyledon and leaf induced anthocyanin accumulation with even stronger promoting role when expression in combination with RsbHLH4. These results suggested that RsMYB1a was the crucial MYB, and that RsbHLH4 is an essential partner in regulating the biosynthesis of anthocyanins in radish. The low or undetectable RsbHLH4 expression paralleled the lack of anthocyanin accumulation in the white flesh of 'Hongxin 1-1' and 'Shaguan 1'. Assays demonstrated that RsMYB1a interacted with RsbHLH4 and activated the expression of RsbHLH4. Notably, all the dark red radish cultivars have a longer RsMYB1a genomic DNA sequence, while the short and nonfunctional RsMYB1a is present in non-red cultivars. The length of the first intron and the presence of an early stop codon of RsMYB1 might underlie the differential anthocyanin accumulation in the radish taproot.

Characterization of Ethylene-mediated Curling of Japanese Radish (Raphanus sativus var. longipinnatus) Cotyledons

During the transport of vegetables, it is important to maintain quality. The cotyledons of Japanese radish (Raphanus sativus var. longipinnatus) sprouts curl during transport, lowering quality. It is known that ethylene causes the leaf curling of some true leaves by promoting cell growth on the adaxial side (epinasty); however, the mechanism of cotyledon curling is unknown. We investigated the effect of ethylene on cotyledon curling of Japanese radish sprouts. Curling was promoted by exogenous treatment with ethylene and repressed by treatment with 1-methylcyclopropene, an inhibitor of ethylene perception. Microscopic observation of ethylene-exposed curled cotyledons and normal cotyledons indicates that ethylene did not affect cell number but did inhibit transverse (lateral) cell growth on the abaxial side of the cotyledons, causing cotyledon curling through differential growth. Ethylene inhibition of cell growth on the abaxial side of leaves has not been reported before. We show a new mechanism responsible for curling.

Response of Plant Hormone bioassay to Paclobutrazol

Quantitative estimation of a known or suspected biologically active substance (such as a hormone or drug) by measuring its effect on a living organism in standard conditions is known as bioassay. The interaction of gibberellic acid (GA3) and paclobutrazol (PBZ) in lettuce hypocotyl bioassay and 6-benzyl aminopurine (BAP) and paclobutrazol in radish cotyledon enlargement test that paclobutrazol counteracts gibberellin activity when applied exogenously. The radish cotyledon enlargement at low concentration without counteracting BAP action. The reduction of growth to be counteracting of gibberellin activity by paclobutrazol as well as its cytokinin well activity at lower concentration which indicate concentration of gibberellins activity as evidence by their respective bioassay in the present investigation. Paclobutrazol slightly increased radish cotyledon growth at low concentration (up to 0.01 ppm) and could not counteract the growth caused by 1.0 ppm BAP at its 0.001 to 1.0 ppm concentrations suggesting independent effect of paclobutrazol to that of BAP. Furthermore, the activity of paclobutrazol was tested in corn root curvature test and it was found that PBZ concentrations from 0.001 to 10.0 ppm linearly increased the percentage of corn roots showing more than 90° curvature. Therefore, suggession that PBZ conutracted GA3, induced growih and also as IAA and cytokinin at lower concentration0.001 to 100ppm.

DNA double-strand breaks promote endoreduplication in radish cotyledon.

DSBs differently affect endoreduplication and organ size in radish cotyledons and hypocotyls in different light conditions, suggesting that DSBs-mediated endoreduplication varies based on different developmental and environmental cues. Endoreduplication induced by DNA double strand breaks (DSBs) in Arabidopsis thaliana roots and cultured cells has been reported in recent years. In this study, we investigated whether DSBs-mediated endoreduplication also occurs in other tissues, such as cotyledons and hypocotyls of radish (Raphanus sativus var. longipinnatus) plants. To induce DSBs, UV irradiation and Zeocin treatment were applied to in vitro-cultured radish seedlings, and ploidy distribution of the treated tissues was analyzed by flow cytometry. Consequently, frequencies of the higher ploidy (8C) cells and cycle values in the cotyledon tissues increased with increasing doses of UV irradiation and concentrations of Zeocin, irrespective of light conditions. UV-stimulated endoreduplication was also observed in four Brassica species. In hypocotyls, UV treatments decreased the frequencies of higher ploidy (32C) cells and cycle values in dark-grown seedlings, whereas Zeocin treatments increased the frequencies of higher ploidy (16C and 32C) cells and cycle values in light- and dark-grown seedlings. Among the treatments, organ sizes did not simply correlate with cycle values. The effects of treatments on endoreduplication and organ size differed based on organ and light conditions, indicating that DSBs-mediated endoreduplication may involve a multifaceted response to different developmental and environmental cues.

Synthesis of poly(fluorenyl-alt-p-phenyleneethynylene) with pendent carboxyl acid group and the sensing application

The present work focuses on the hydrophobic functionalization of water soluble celluloses methyl cellulose, hydroxyethyl cellulose and (hydroxypropyl)methyl cellulose with the anticancer steroid diosgenin, and two synthetic brassinosteroids (DI31 and S7) used as agrochemicals. Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopies confirmed the cellulose modification. Prepared amphiphilic steroid-cellulose conjugates can self-assemble in water as stable and almost neutral particles with micelle-like structure, as depicted using dynamic light scattering. Whereas scanning and transmission electron microscopies showed 50–300 nm almost spherical particles and aggregates in dried state, atomic force microscopy assessed particles aggregates with mean sizes of 220–355 nm. These cellulose particles showed sustained steroid release in acidic aqueous medium over 72 h, and good stimulatory agrochemical activity in radish cotyledons assay. Thus, the outlined synthesis of steroid-cellulose conjugates, which would be capable to form self-assembled particles in water for controlled release of agrochemicals, is envisioned as a promising strategy.

Self-assembled cellulose particles for agrochemical applications

The present work focuses on the hydrophobic functionalization of water soluble celluloses methyl cellulose, hydroxyethyl cellulose and (hydroxypropyl)methyl cellulose with the anticancer steroid diosgenin, and two synthetic brassinosteroids (DI31 and S7) used as agrochemicals. Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopies confirmed the cellulose modification. Prepared amphiphilic steroid-cellulose conjugates can self-assemble in water as stable and almost neutral particles with micelle-like structure, as depicted using dynamic light scattering. Whereas scanning and transmission electron microscopies showed 50–300nm almost spherical particles and aggregates in dried state, atomic force microscopy assessed particles aggregates with mean sizes of 220–355nm. These cellulose particles showed sustained steroid release in acidic aqueous medium over 72h, and good stimulatory agrochemical activity in radish cotyledons assay. Thus, the outlined synthesis of steroid-cellulose conjugates, which would be capable to form self-assembled particles in water for controlled release of agrochemicals, is envisioned as a promising strategy.

English

Information on microbial production of cytokinins and their effect on plant growth are limited. Therefore, the objective of the present study was to investigate cytokinins production by Pseudomonas sp., a major component of rhizobacteria with multiform and diverse activities, which enhance plant growth by synthesizing various secondary metabolites. In the present investigations, thirty Pseudomonas isolates were isolated from the rhizosphere of Pyrus and Malus and were screened for cytokinins production (50 to 210 μg/ml). Four strains viz PN-4-SAN, PN-10-SAN, AN-2-NAG and AN-4-NAG were selected on the basis of their higher cytokinins production. The maximum cytokinins production was observed at 72h incubation period in nutrient broth at pH 7.0 under shaken condition at 28°C. Cytokinins were extracted, purified and evaluated by thin layer chromatography and specific bioassay method. Key words: Cytokinins, plant growth promoting rhizobacteria (PGPR), Pseudomonas sp., thin layer chromatography, radish cotyledon bioassay.

Source-sink relationships in radish plant

The problem of source-sink relationships in di- and tetraploidal radish plants grown in. hydroponic cultures was investigated in two stages of their development: with intensively growing swollen hypocotyl and in the period of actively accumulating nutrients in the storage organ. It was found, that the proportion, between the mass of organs, their RGR and NAR was very similar in di- and tetraploidal populations, probably owing to a similar rate of photosynthesis and pattern of assimilates distribution. The high variability of swollen hypocotyls size is slightly correlated with the size of the whole aerial part and is not correlated with the rate of photosynthesis in leaves. Partial defoliation of radish plants did not affect the rate of photosynthesis of the remaining leaves. Only in the cotyledones the oldest donors of 14C-assimilates, a slight compensation of photosynthesis was reported. It may suggest, that the rate of photosynthesis in radish plants is not under the control of sink activity. The size of the storage organ have determined in some extent its attractive force and influenced the amount of 14C-assimilates exported from their donors. Translocation of photosynthates from the young, still growing leaves was conditioned mainly by their retention power. Therefore, in young radish plants cotyledons were the main donor of <sup>14</sup>C-assimilates.

Synthesis of brassinosteroids analogues from laxogenin and their plant growth promotion

Four steroid saponins (2–5) and three derivatives (6–8) were synthesised from laxogenin. Four of them were new compounds: (25R)-3β-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyloxy)-5α-spirostan-6-one (3), (25R)-3β-(β-d-galactopyranosyloxy)-5α-spirostan-6-one (5), 3β,16-diacetyl-26-hydroxy-5α-cholestan-6,22-dione (6) and 16-acetyl-3β,26-dihydroxy-5α-cholestan-6,22-dione (7). All the compounds showed plant growth-promoting activity in the radish hypocotyl elongation and cotyledon expansion bioassay. Above all, 2 and 6 were found to be more active.

Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination

Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination.

Isopentenyladenosine and cytokinin-like activity of different humic substances

Increasing attention has been devoted in the last few years to the mechanisms by which humic substances (HS) influence plant growth. Nevertheless, amongst the biologically active compounds that might be present in HS, only indoleacetic acid has been identified. An enzyme-linked immunosorbent assay (ELISA) was applied to detect the presence of isopentenyladenosine (IPA), a cytokinin, in two lignosulphonate-humates, leonardite humic acid and in a high molecular weight humic fraction extracted from earthworm faeces. The IPA concentration ranged from 34 to 145pmolmg−1 humic C and was estimated by using a linear dose–response curve between the carbon (C) concentration and percentage of binding of an anti-IPA monoclonal antibody. The cytokinin-like activity of HS was evaluated by weighing small radish (Raphanus sativus L.) cotyledon leaves and measuring chlorophyll and protein contents, the activities of ATP sulfurylase and O-acetylserine sulfhydrylase on maize plants. It is noteworthy that, amongst the four HS, those from earthworm faeces showed the highest amount of IPA and the strongest cytokinin-like activity in both plants. The presence of IPA confirmed that HS influence plant metabolism at different levels. Our results open new perspectives in the plant–HS relationship and shed new light on the active components of HS.

Effect of PGPR Fertilizer on Biological Characteristics in Cerasus pseudocerasus Rhizosphere

The aim of this research was to determine the effects of plant growth-promoting rhizobacteria(PGPR) fertilizer on the biological characteristics,root activity,growth and construction of Cerasus pseudocerasus(Lindl.) G.Don(sweet cherry).To prepare sweet cherry biological-fertilizer(YMF),dominant bacteria YT-3,a type of PGPR,was extracted from rhizosphere soil of C.pseudocerasus trees by keeping green method and radish cotyledon weight increase method.Sweet cherry biological-fertilizer was prepared by compounding YT-3 and decomposed chicken manure(DCM).The effects of YMF,normal biological fertilizer(NMF) and DCM on biological characteristics of C.pseudocerasus rhizosphere soil were studied in Yiyuan sweet cherry orchards.And the effects of YMF,DCM and NMF on the root activity,growth and construction of C.pseudocerasus were also investigated.The results showed that YMF increased the amount of bacteria and total microorganism amount in rhizosphere soil of C.pseudocerasus,but the fungus amount was decreased significantly.However,YMF had no influence on actinomycetes amount in rhizosphere soil.Root activity in YMF was significantly increased by 15.33%,22.49% and 13.25% than that in CK,NMF and DCM,respectively.YMF had great influence on the root growth and construction of C.pseudocerasus.Root weight,especially fine root weight in 0～40 soil profile of YMF was significantly increased.Rhizosphere soil pH value in YMF decreased by 8.61% than that in NMF.In addition,YMF significantly enhanced available phosphorus and potassium contents by 17.21% and 9.56%,respectively,compared to that in NMF.However,it had no effects on available nitrogen content in sweet cherry rhizosphere soil.Moreover,PGPR fertilizer increased the cation exchange capacity of rhizosphere soil,which benefited the nutrient retain and uptake of C.pseudocerasus.As a result,plant growth-promoting rhizobactera biological-fertilizer benefited the ecological environment of sweet cherry rhizosphere soil,improved nutrient availability and cation exchange capacity,increased the root activity and benefited the root growth,especially fine root growth in top soil(mainly 0～40 cm).

Identification of a plant aminopeptidase with preference for aromatic amino acid residues as a novel member of the prolyl oligopeptidase family of serine proteases

Genome analysis has indicated that plants, like animals, possess a variety of protease genes. However, bulk of putative proteases has not been characterized at the enzyme level. In this article, a novel enzyme that hydrolyses phenylalanyl-4-methylcoumaryl 7-amide (phenylalanyl-MCA) was purified from cotyledons of daikon radish by ammonium sulphate fractionation and successive chromatography with DEAE-cellulose, phenyl-Sepharose, Sephacryl S-200 and Mini-Q. The molecular mass of the enzyme was estimated to be 78 kDa by SDS-PAGE under reducing conditions and 74 kDa by gel filtration, indicating that the enzyme is a monomer. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that the enzyme is an orthologue of Arabidopsis unidentified protein, acylpeptide hydrolase-like protein (AHLP; UniProt ID: Q9FG66) belonging to the prolyl oligopeptidase (POP) family of a serine-type peptidase predicted from genetic information. Good substrates identified for the enzyme include phenylalanyl-MCA, tyrosyl-MCA and enkephalin. Neither acylamino acid-releasing activity nor endopeptidase activity was detected. The enzyme cleaved enkephalin (YGGFM, YGGFL), whereas, BAM-12 P (YGGFMRRVGRPE) and dynorphin A (YGGFLRRIRPKLK) were not digested. These results suggested that the enzyme possesses strict size selectivity of substrate. We propose the name 'tyrosyl aminopeptidase' for the uncharacterized protein AHLP.