Establishment of a high-efficiency transformation and genome editing method for an essential vegetable and medicine Solanum nigrum.

Solanum nigrum, which belongs to the Solanaceae family, is an essential plant for food and medicine. It has many important secondary compounds, including glycoproteins, glycoalkaloids, polyphenolics, and anthocyanin-rich purple berries, as well as many ideal characteristics such as self-fertilization, a short life cycle and a small genome size that make it a potential model plant for the study of secondary metabolism and fruit development. In this study, we report a highly efficient and convenient tissue culture, transformation and genome editing method for S. nigrum using leaf segments after 8 weeks of tissue culture, with a required period from transformation initiation to harvest of about 3.5 months. Our results also show multi-shoot regeneration per leaf segment and a 100% shoot regeneration efficiency in a shoot regeneration medium. Moreover, over 82% of kanamycin-resistant plants exhibited strong green fluorescence marker protein expression, with genetic integration confirmed by PCR results and green fluorescence protein expression in their T1 progeny. Furthermore, we successfully applied this transformation method to achieve an average of 83% genome editing efficiency of SnMYB1, a gene involved in regulating the anthocyanin biosynthetic pathway of S. nigrum in response to missing nutrients. Taken together, the combination of highly efficient tissue culture, transformation and genome editing systems can provide a powerful platform for supporting fundamental research on the molecular mechanisms of secondary metabolism, fruit development, and production of important compounds by biotechnology.

Down, then up: non-parallel genome size changes and a descending chromosome series in a recent radiation of the Australian allotetraploid plant species, Nicotiana section Suaveolentes (Solanaceae).

BACKGROUND AND AIMS: The extent to which genome size and chromosome numbers evolve in concert is little understood, particularly after polyploidy (whole-genome duplication), when a genome returns to a diploid-like condition (diploidization). We study this phenomenon in 46 species of allotetraploid Nicotiana section Suaveolentes (Solanaceae), which formed <6 million years ago and radiated in the arid centre of Australia. METHODS: We analysed newly assessed genome sizes and chromosome numbers within the context of a restriction site-associated nuclear DNA (RADseq) phylogenetic framework. KEY RESULTS: RADseq generated a well-supported phylogenetic tree, in which multiple accessions from each species formed unique genetic clusters. Chromosome numbers and genome sizes vary from n = 2x = 15 to 24 and 2.7 to 5.8 pg/1C nucleus, respectively. Decreases in both genome size and chromosome number occur, although neither consistently nor in parallel. Species with the lowest chromosome numbers (n = 15-18) do not possess the smallest genome sizes and, although N. heterantha has retained the ancestral chromosome complement, n = 2x = 24, it nonetheless has the smallest genome size, even smaller than that of the modern representatives of ancestral diploids. CONCLUSIONS: The results indicate that decreases in genome size and chromosome number occur in parallel down to a chromosome number threshold, n = 20, below which genome size increases, a phenomenon potentially explained by decreasing rates of recombination over fewer chromosomes. We hypothesize that, more generally in plants, major decreases in genome size post-polyploidization take place while chromosome numbers are still high because in these stages elimination of retrotransposons and other repetitive elements is more efficient. Once such major genome size change has been accomplished, then dysploid chromosome reductions take place to reorganize these smaller genomes, producing species with small genomes and low chromosome numbers such as those observed in many annual angiosperms, including Arabidopsis.

RGB color analysis of formaldehyde in vegetables based on DNA functionalized gold nanoparticles and triplex DNA.

A highly sensitive and selective RGB color analysis for the detection of formaldehyde (FA) was developed by using a DNA functionalized gold nanoparticle (AuNPs-DNA) probe. When complementary oligonucleotides (oligo 2 and oligo 3) and a silver ion (Ag+) were added to the AuNPs-DNA solution, triplex DNA was formed, resulting in the aggregation of AuNPs, and accompanied by a solution color change from red to purple. With the addition of formaldehyde, it reacted with Ag+, decreased the stability of triplex DNA between AuNPs-DNA, induced the dispersion of AuNPs, and the color of AuNPs recovered to red. Therefore, the formaldehyde concentration could be estimated with the RGB (red, green, blue) values of the AuNP solution by using a smartphone application (APP). The R value of the system was proportional to the concentration of formaldehyde within the range of 0.23-4.50 mg L-1, with a detection limit of 0.14 mg L-1. The method has been successfully applied to detect the residues of formaldehyde in vegetable samples and has the potential of the on-site determination of formaldehyde.

Capsicum SIZ1 contributes to ABA-induced SUMOylation in pepper.

Abiotic and biotic stresses are the major factors limiting plant growth. Arabidopsis E3 SUMO ligase SIZ1 plays an essential role in plant stress tolerance. Herein, we identified a SIZ/PAIS-type protein in pepper (Capsicum annuum), namely CaSIZ1, which shares 60 % sequence identity with AtSIZ1. The stems and flowers of pepper had a relatively higher expression of CaSIZ1 than the fruits, leaves, and roots. ABA and NaCl treatments induced CaSIZ1. CaSIZ1 protein was localized in the nucleus and partially rescued the dwarf and ABA-sensitive phenotypes of Atsiz1-2, suggesting the functional replacement of CaSIZ1 with AtSIZ1. We found that CaSIZ1 interacted with CaABI5, and ABA promoted the accumulation of SUMO conjugates in pepper. CaSIZ1 knockdown did not only reduce ABA-induced SUMOylation, but also attenuated the salt tolerance of pepper. Overall, the results of this study suggest that CaSIZ1 has a significant role in ABA-induced SUMOylation and stress response.

Varied Expression of Senescence-Associated and Ethylene-Related Genes during Postharvest Storage of Brassica Vegetables.

The genus Brassica comprises a highly diverse range of vegetable crops varying in morphology, harvestable crop product, and postharvest shelf-life that has arisen through domestication, artificial selection and plant breeding. Previous postharvest studies on the shelf-life of Brassica species has mainly focused on the variable rates of physiological changes including respiration and transpiration. Therefore, further understanding of the molecular basis of postharvest senescence in Brassica vegetables is needed to understand its progression in improving their postharvest shelf-life. The aim of this study was to better understand the trajectory of molecular responses in senescence-associated genes but not induced by ethylene and ethylene-induced genes towards altered postharvest storage conditions. After storage at different temperatures, the expression levels of the key senescence-associated genes (SAGs) and the ethylene biosynthesis, perception, and signaling genes were quantitatively analyzed in cabbage, broccoli and kale. The expression levels of these genes were tightly linked to storage temperature and phase of senescence. Expression of ORE15, SAG12, and NAC29 were continuously increased during the twelve days of postharvest storage at room temperature. Prolonged exposure of these three vegetables to cold temperature reduced the variation in the expression levels of ORE15 and SAG12, observed as mostly decreased which resulted in limiting senescence. The transcript levels of the ethylene receptor were also decreased at lower temperature, further suggesting that decreased ethylene biosynthesis and signaling in cabbage during postharvest storage would delay the senescence mechanism. These results enhanced our understanding of the transcriptional changes in ethylene-independent SAGs and ethylene-related genes in postharvest senescence, as well as the timing and temperature sensitive molecular events associated with senescence in cabbage, broccoli and kale and this knowledge can potentially be used for the improvement of postharvest storage in Brassica vegetables.

Development of nucleic acid isolation by non-silica-based nanoparticles and real-time PCR kit for edible vegetable oil traceability.

For efficient extraction of amplifiable DNA from edible vegetable oils, we developed a novel DNA extraction approach based on the non-silica-based dipolar nanocomposites. The nanoparticle comprises a hydrophilic polymethyl methacrylate core with abundant capillaries, hydrophilic vesicles decorated with molecules having DNA affinity and a coating hydrophobic polystyrene layer. The nanoparticles are soluble in oil, adsorb the DNA from the aqueous phase and gave a high DNA recovery ratio. All DNA extracts from fully refined vegetable oil soybean, peanut, rapeseed, and cottonseed oils, including their blends, were sufficiently pure to be amplified by real-time PCR targeting the chloroplast ribulose-1,5-bisphosphate gene (rbcL), therefore, the species of origin and their ratios in mixed vegetable oils blended from two or three oil-species could be determined. These results indicate that the novel DNA isolation and real-time PCR kit is a simple, sensitive and efficient tool for the species identification and traceability in refined vegetable oils.

Differences in the uptake and bioconcentration of dichlorodiphenyltrichloroethane by eight vegetable cultivars and their health risk assessments.

Dichlorodiphenyltrichloroethane (DDT) is not easily degraded in soils, which will pose a threat to human health. We investigated the differences of eight vegetables' capacity to take up DDT, removing DDT from soil, and tolerating DDT (monitoring the responses of growth, root morphology and photosynthesis of vegetables to DDT). These vegetables included Chinese mustard (two genotypes, B.jf and B,jm), napa cabbage (two genotypes, B.coz and B.coc) and Bok choy (four genotypes, B.cz, B.cq, B.cs and B.chg). The results demonstrated that 5 mg kg-1 DDT did not display significant effects on the growth of most vegetables in this study. As compared to the control, 5 mg kg-1 DDT significantly increased the shoot and root biomass, the fine root numbers, and the fine root ratio for the genotype of B.chg. However, 5 mg kg-1 DDT exposure showed a negative effect on the shoot growth of two genotypes of napa cabbage. In general, 5 mg kg-1 DDT did not significantly affect the photosynthesis and root morphology of most vegetables in this study. Consuming these vegetables had a low non-cancer health risk, but showed a high cancer health risk. In addition, among the eight vegetables, B.chg accumulated less DDT in the edible parts and had low values of HRnon-cancer and HRcancer for consuming these vegetables containing DDT. Planting these vegetables might promote the degradation of DDT reducing its residual amount in soil.

Productivity and Antioxidant Activity of Wild, Reconstituted, and Hybrid Strains of the Pink Oyster Mushroom, Pleurotus djamor (Agaricomycetes), from Mexico.

The genus Pleurotus is the third most commonly produced edible fungi in the world. In addition, species of genus Pleurotus have functional properties such as anticancer, antiviral, antimicrobial, anti-inflammatory, and antioxidant activities, which are mainly attributed to phenolic compounds. For these reasons, this study evaluated the productivity and antioxidant activity (AA) of 2 wild strains (white and pink), 2 reconstituted strains (called "BB" and "RR"), and 4 hybrid strains (H1, H2, H3, and H4) of P. djamor from monokaryotic components (neohaplonts). The results showed that the white wild-type strain and the reconstituted strains exhibited the best production potential, expressed as biological efficiency and mycelial growth rate. The carpophores of hybrid strains H1 and H3 had the greatest AA, as evaluated with DPPH radical scavenging and reducing power assays, respectively. The H3 strain had the highest total phenol (TP) content. Pearson correlations led us to conclude that the mycelial growth rate has a regular inverse correlation with TP and a regular direct correlation with AA of methanolic extracts from carpophores and myce-lia. This is, to our knowledge, the first report in the literature about the effect of Pleurotus strain hybridization through a chemical de-dikaryotization process on TP content.

Identification and expression analysis of glucosinolate biosynthetic genes and estimation of glucosinolate contents in edible organs of Brassica oleracea subspecies.

Glucosinolates are anti-carcinogenic, anti-oxidative biochemical compounds that defend plants from insect and microbial attack. Glucosinolates are abundant in all cruciferous crops, including all vegetable and oilseed Brassica species. Here, we studied the expression of glucosinolate biosynthesis genes and determined glucosinolate contents in the edible organs of a total of 12 genotypes of Brassica oleracea: three genotypes each from cabbage, kale, kohlrabi and cauliflower subspecies. Among the 81 genes analyzed by RT-PCR, 19 are transcription factor-related, two different sets of 25 genes are involved in aliphatic and indolic biosynthesis pathways and the rest are breakdown-related. The expression of glucosinolate-related genes in the stems of kohlrabi was remarkably different compared to leaves of cabbage and kale and florets of cauliflower as only eight genes out of 81 were expressed in the stem tissues of kohlrabi. In the stem tissue of kohlrabi, only one aliphatic transcription factor-related gene, Bol036286 (MYB28) and one indolic transcription factor-related gene, Bol030761 (MYB51), were expressed. The results indicated the expression of all genes is not essential for glucosinolate biosynthesis. Using HPLC analysis, a total of 16 different types of glucosinolates were identified in four subspecies, nine of them were aliphatic, four of them were indolic and one was aromatic. Cauliflower florets measured the highest number of 14 glucosinolates. Among the aliphatic glucosinolates, only gluconapin was found in the florets of cauliflower. Glucoiberverin and glucobrassicanapin contents were the highest in the stems of kohlrabi. The indolic methoxyglucobrassicin and aromatic gluconasturtiin accounted for the highest content in the florets of cauliflower. A further detailed investigation and analyses is required to discern the precise roles of each of the genes for aliphatic and indolic glucosinolate biosynthesis in the edible organs.

Determination of multiple pesticides in fruits and vegetables using a modified quick, easy, cheap, effective, rugged and safe method with magnetic nanoparticles and gas chromatography tandem mass spectrometry.

Based on a modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation method with Fe3O4 magnetic nanoparticles (MNPs) as the adsorbing material and gas chromatography-tandem mass spectrometry (GC-MS/MS) determination in multiple reaction monitoring (MRM) mode, we established a new method for the determination of multiple pesticides in vegetables and fruits. It was determined that bare MNPs have excellent function as adsorbent when purified, and it is better to be separated from the extract. The amount of MNPs influenced the clean-up performance and recoveries. To achieve the optimum performance of modified QuEChERS towards the target analytes, several parameters including the amount of the adsorbents and purification time were investigated. Under the optimum conditions, recoveries were evaluated in four representative matrices (tomato, cucumber, orange and apple) with the spiked concentrations of 10 µg kg(-1), 50 µg kg(-1)and 200 µg kg(-1) in all cases. The results showed that the recovery of 101 pesticides ranged between 71.5 and 111.7%, and the relative standard deviation was less than 10.5%. The optimum clean-up system improved the purification efficiency and simultaneously obtained satisfactory recoveries of multiple pesticides, including planar-ring pesticides. In short, the modified QuEChERS method in addition to MNPs used for removing impurities improved the speed of sample pre-treatment and exhibited an enhanced performance and purifying effect.

Iron bioavailability of maize hemoglobin in a Caco-2 cell culture model.

Maize ( Zea mays ) is an important staple crop in many parts of the world but has low iron bioavailability, in part due to its high phytate content. Hemoglobin is a form of iron that is highly bioavailable, and its bioavailability is not inhibited by phytate. It was hypothesized that maize hemoglobin is a highly bioavailable iron source and that biofortification of maize with iron can be accomplished by overexpression of maize globin in the endosperm. Maize was transformed with a gene construct encoding a translational fusion of maize globin and green fluorescent protein under transcriptional control of the maize 27 kDa γ-zein promoter. Iron bioavailability of maize hemoglobin produced in Escherichia coli and of stably transformed seeds expressing the maize globin-GFP fusion was determined using an in vitro Caco-2 cell culture model. Maize flour fortified with maize hemoglobin was found to have iron bioavailability that is not significantly different from that of flour fortified with ferrous sulfate or bovine hemoglobin but is significantly higher than unfortified flour. Transformed maize grain expressing maize globin was found to have iron bioavailability similar to that of untransformed seeds. These results suggest that maize globin produced in E. coli may be an effective iron fortificant, but overexpressing maize globin in maize endosperm may require a different strategy to increase bioavailable iron content in maize.

Intact glucosinolates modulate hepatic cytochrome P450 and phase II conjugation activities and may contribute directly to the chemopreventive activity of cruciferous vegetables.

The currently accepted view is that the chemopreventive activity of glucosinolates is exclusively mediated by their degradation products, such as isothiocyanates. In the present study, evidence is presented for the first time that intact glucosinolates can modulate carcinogen-metabolising enzyme systems. The glucosinolates glucoraphanin and glucoerucin were isolated from cruciferous vegetables and incubated with precision-cut rat liver slices. Both glucosinolates elevated the O-dealkylations of methoxy- and ethoxyresorufin, markers for CYP1 activity; supplementation of the incubation medium with myrosinase, the enzyme that converts glucosinolates to their corresponding isothiocyanates, abolished these effects. Moreover, both glucoerucin and glucoraphanin increased the apoprotein levels of microsomal CYP1A1, CYP1A2 and CYP1B1. At higher concentrations, both glucosinolates enhanced quinone reductase activity, whereas glucoraphanin also elevated glutathione S-transferase; in this instance, however, supplementation of the incubation medium with myrosinase exacerbated the inductive effect. Finally, both glucosinolates increased modestly cytosolic quinone reductase, GSTα and GSTµ protein levels, which became more pronounced when myrosinase was added to the incubations with the glucosinolate. It may be inferred that intact glucosinolates can modulate the activity of hepatic carcinogen-metabolising enzyme systems and this is likely to impact on the chemopreventive activity linked to cruciferous vegetable consumption.

Novel self-compatible lines of Brassica rapa L. isolated from the Japanese bulk-populations.

Self-incompatibility (SI) in Brassicaceae is sporophytically controlled by a single S-locus with multi allelic variety. The male S determinant, SP11/SCR (S-locus protein 11/S-locus cysteine-rich protein), is a small cysteine-rich protein, and the female S determinant, SRK (S-locus receptor kinase), functions as a receptor for SP11 at the surface of stigma papilla cells. Although a few of the following downstream factors in the SP11-SRK signaling cascade have been identified, a comprehensive understanding of the SI mechanism still remains unexplained in Brassicaceae. Analysis of self-compatible (SC) mutants is significant for understanding the molecular mechanism in SI reactions, thus we screened SC lines from a variety of Japanese bulk-populations of B. rapa vegetables. Two lines, TSC4 and TSC28, seem to have disruptions in the SI signaling cascade, while the other line, TSC2, seems to have a deficiency in a female S determinant, SRK. In TSC4 and TSC28, known SI-related factors, i.e. SRK, SP11, MLPK (M-locus protein kinase), THL (thioredoxin-h-like), and ARC1 (arm repeat containing 1), were expressed normally, and their expression levels were comparable with those in SI lines. On a B. rapa genetic linkage map, potential SC genes in TSC4 and TSC28 were mapped on linkage groups A3 and A1, respectively, whereas MLPK, ARC1, and THL were mapped on A3, A4, and A6, respectively. Although potential SC genes of TSC4 and MLPK were on the same linkage group, their positions were apparently independent. These results indicate that the SC genes of TSC4 and TSC28 are independent from the S-locus or known SI-related genes. Thus, the SC lines selected here have mutations in novel factors of the SI signaling cascade, and they will contribute to fill pieces in a signal transduction pathway of the SI system in Brassicaceae.

Antimutagenic and antigenotoxic effects of vegetable matrices on the activity of pesticides.

Two in vitro tests, one to detect bacterial mutagenicity (Ames test) on Salmonella typhimurium TA98, TA100, and TA1535 and the other the primary DNA damage (SOS Chromotest) on Escherichia coli PQ37, were applied to determine the overall genotoxic activity of 12 pesticides (azinphos methyl, chlorothalonil, chlorphyriphos ethyl, chlorphyriphos methyl, lambda-cyhalothrin, cypermethrin, cyprodinil, fenazaquin, fludioxonil, indoxacarb, iprodione and penconazol). These were detected by gas chromatography (GC) analysis with electron capture (ECD) and nitrogen phosphorus detection (NPD) in 18 samples of vegetables. Some extracts of vegetables, found positive for pesticides with GC, were subjected to the Ames test and SOS Chromotest to evaluate the possible antimutagenic and/or antigenotoxic effects of vegetable matrices. The same bioassays were also performed on the mixtures of pesticides found in these samples to evaluate whether interactions could occur between pesticides and be responsible for the possible antimutagenic and/or antigenotoxic effects of the contaminated matrices. Experiments were also carried out to compare the results found for contaminated vegetables with their content of antioxidant components. Significant differences in mutagenicity and genotoxicity were found among the pesticides selected for this study. Of the 12 pesticides tested, only azinphos methyl, cyprodinil, fludioxonil and iprodione were found to be positive for both S. typhimurium and E. coli. No mutagenic/genotoxic activity was found in the extracts of vegetables contaminated by pesticides. S. typhimurium TA1535 showed a strong positive mutagenic effect for the mixtures of pesticides while they were not able to induce the SOS system. The data concerning the content of polyphenols and the total reducing activity of the contaminated vegetables indicated high amounts of antioxidants that could explain the inhibitory effect on the activity of pesticides shown by vegetables.

Accumulation and bioavailability of dietary carotenoids in vegetable crops.

Carotenoids are lipid-soluble pigments found in many vegetable crops that are reported to have the health benefits of cancer and eye disease reduction when consumed in the diet. Research shows that environmental and genetic factors can significantly influence carotenoid concentrations in vegetable crops, and that changing cultural management strategies could be advantageous, resulting in increased vegetable carotenoid concentrations. Improvements in vegetable carotenoid levels have been achieved using traditional breeding methods and molecular transformations to stimulate biosynthetic pathways. Postharvest and processing activities can alter carotenoid chemistry, and ultimately affect bioavailability. Bioavailability data emphasize the importance of carotenoid enhancement in vegetable crops and the need to characterize potential changes in carotenoid composition during cultivation, storage and processing before consumer purchase.

Paradoxical effects of chemicals in the diet on health.

In 1992, Block et al. published a summary of 200 epidemiological investigations which indicated that a diet that was high in fruit and vegetables cut cancer risks approximately in half. These investigations used conventionally farmed produce that contained traces of synthetic pesticides and mycotoxins as well as an estimated 10,000 secondary products (i.e. natural pesticides). Dietary consumption of fruits and vegetables also reduces risks of cardiovascular disease, cataracts and brain dysfunction. Before genetic manipulation is undertaken to elevate or diminish any individual constituent of fruits and vegetables, the contribution of each of these constituents to health must be better understood, as in many cases their effects on health can be paradoxical.

[Genotoxic effects of pesticide-treated vegetable extracts using the Allium cepa chromosome aberration and micronucleus tests]. / Valutazione della genotossicità di frutta e verdura contaminata da pesticidi mediante il test dell'Allium cepa.

The presence of chemical residues in vegetables and fruit is a source of human exposure to toxic and genotoxic chemicals. The mutagenic and carcinogenic action of herbicides, insecticides and fungicides on experimental animals is already known. Several studies have shown that chronic exposure to low levels of pesticides can cause adverse health effects and that many pesticides are mutagenic/carcinogenic. In the present research we monitored concurrently the presence of pesticides and genotoxic compounds extracted from 21 treated vegetables and 8 types of grapes sampled from the markets of a region in Southern Italy. The extracts were analysed for pesticides by gas-chromatography and HPLC, and for genotoxicity with two plant tests in Allium cepa roots: the micronucleus test and the chromosomal aberration test. We found 33 pesticides, some of which are outlawed. Genotoxicity was found in some of the vegetables and grapes tested. Allium cepa tests were sensitive for monitoring genotoxicity in food extracts. The micronucleus test in interphase cells gave much higher mutagenicity than the chromosomal aberration test in anaphase-telophase cells.

CENP-B is a conserved gene among vegetal species

To explore the CENP-B centromere protein in beans, carrots, onions and potatoes, total RNA was isolated and reverse transcribed by PCR, and the cDNA encoding the CENP-B amino terminus domain amplified using CENP-B oligonucleotides. Blots containing PCR products were hybridized with a nick-translated pG/CNPB probe containing a complete human CENP-B gene. In all the plant species, anti-CENP-B antibodies recognized an 80-kDa protein. A 360-bp sequence encoding for the amino terminus region of the CENP-B protein was amplified by PCR in all the species and the nick translated pG/CNPB probe hybridized with the PCR products. Apparently the CENP-B centromere protein or an equivalent protein is widely distributed in the vegetal kingdom.

Identification of mammalian-like purple acid phosphatases in a wide range of plants.

Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal approximately 35kDa, plant approximately 55kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.