Full-length transcriptome analysis of Phytolacca americana and its congener P. icosandra and gene expression normalization in three Phytolaccaceae species.

BACKGROUND: Phytolaccaceae species in China are not only ornamental plants but also perennial herbs that are closely related to human health. However, both large-scale full-length cDNA sequencing and reference gene validation of Phytolaccaceae members are still lacking. Therefore, single-molecule real-time sequencing technology was employed to generate full-length transcriptome in invasive Phytolacca americana and non-invasive exotic P. icosandra. Based on the transcriptome data, RT-qPCR was employed to evaluate the gene expression stability in the two plant species and another indigenous congener P. acinosa. RESULTS: Total of 19.96 Gb and 19.75 Gb clean reads of P. americana and P. icosandra were generated, including 200,857 and 208,865 full length non-chimeric (FLNC) reads, respectively. Transcript clustering analysis of FLNC reads identified 89,082 and 98,448 consensus isoforms, including 86,989 and 96,764 high-quality ones. After removing redundant reads, 46,369 and 50,220 transcripts were obtained. Based on structure analysis, total 1675 and 1908 alternative splicing variants, 25,641 and 31,800 simple sequence repeats (SSR) as well as 34,971 and 36,841 complete coding sequences were detected separately. Furthermore, 3574 and 3833 lncRNA were predicted and 41,676 and 45,050 transcripts were annotated respectively. Subsequently, seven reference genes in the two plant species and a native species P. acinosa were selected and evaluated by RT-qPCR for gene expression analysis. When tested in different tissues (leaves, stems, roots and flowers), 18S rRNA showed the highest stability in P. americana, whether infested by Spodoptera litura or not. EF2 had the most stable expression in P. icosandra, while EF1-α was the most appropriate one when attacked by S. litura. EF1-α showed the highest stability in P.acinosa, whereas GAPDH was recommended when infested by S. litura. Moreover, EF1-α was the most stable one among the three plant species whenever germinating seeds or flowers only were considered. CONCLUSION: Full-length transcriptome of P. americana and P. icosandra were produced individually. Based on the transcriptome data, the expression stability of seven candidate reference genes under different experimental conditions was evaluated. These results would facilitate further exploration of functional and comparative genomic studies in Phytolaccaceae and provide insights into invasion success of P. americana.

Novel Flavones from the Root of Phytolacca acinosa Roxb.

Two new flavones, 6,7-methylenedioxy-4-hydroxypeltogynan-7'-one (1), cochliophilin B (2), as well as two known ones, cochliophilin A (3) and 6-methoxy-7-hydroxy flavone (4), were isolated from the ethanol extract of the root of Phytolacca acinosa Roxb. Compound 1 is a flavanol framework with one δ-lactone unit, which is rather rare in nature. The structures of the new compounds were determined on the basis of extensive spectroscopic (IR, MS, 1D- and 2D-NMR) analyses, the absolute configuration of 1 was established by comparing experimental and calculated electronic circular dichroism spectra. The structures of known compounds were fixed by comparison with literatures data. Compounds 2 and 4 showed modest inhibitory activities against BEL-7402 cell line, with IC50 values of 28.22 and 39.16 µmol/L, respectively.

Sugar binding effects on the enzymatic reaction and conformation near the active site of pokeweed antiviral protein revealed by fluorescence spectroscopy.

In various trials for elucidating the physiological function of pokeweed antiviral protein (PAP), studies on the interaction with sugar are essential. The fluorescence titration curves showed that PAP retained the strong affinity against N-acetylglucosamine (NAG) and two sites in one PAP molecule co-operatively participated in the binding. In the complex of PAP with NAG, Trp208 located at the entrance lid site of substrate came closer to Tyr72 about 0.3 Å. Furthermore, the fluorescence anisotropy decay measurement demonstrated that the segmental rotation of Trp208 was enlarged by the binding of PAP with NAG. Such conformational changes around the active site closely correlate with the enzymatic activity of PAP. The N-glycosidase activity of PAP was enhanced more than two times in the presence of NAG. The obtained results consistently suggested the enzymatic activity of PAP would be regulated through the conformation change near the active site induced by the binding with NAG.

Structural and enzymatic properties of an in vivo proteolytic form of PD-S2, type 1 ribosome-inactivating protein from seeds of Phytolacca dioica L.

PD-S2, type 1 ribosome-inactivating protein from Phytolacca dioica L. seeds, is an N-ß-glycosidase likely involved in plant defence. In this work, we purified and characterized an in vivo proteolytic form of PD-S2, named cutPD-S2. Spectroscopic characterization of cutPD-S2 showed that the proteolytic cleavage between Asn195 and Arg196 does not alter the protein fold, but significantly affects its thermal stability. Most importantly, the proteolytic cleavage induces a 370-fold decrease of PD-S2 capacity of inhibiting in vitro protein biosynthesis. Our data catch the turning point from a typical role of PD-S2 as a defence protein to that of supplier of essential amino acids during seedling development.

Endophyte-assisted promotion of biomass production and metal-uptake of energy crop sweet sorghum by plant-growth-promoting endophyte Bacillus sp. SLS18.

The effects of Bacillus sp. SLS18, a plant-growth-promoting endophyte, on the biomass production and Mn/Cd uptake of sweet sorghum (Sorghum bicolor L.), Phytolacca acinosa Roxb., and Solanum nigrum L. were investigated. SLS18 displayed multiple heavy metals and antibiotics resistances. The strain also exhibited the capacity of producing indole-3-acetic acid, siderophores, and 1-aminocyclopropane-1-carboxylic acid deaminase. In pot experiments, SLS18 could not only infect plants effectively but also significantly increase the biomass of the three tested plants in the presence of Mn/Cd. The promoting effect order of SLS18 on the biomass of the tested plants was sweet sorghum > P. acinosa > S. nigrum L. In the presence of Mn (2,000 mg kg(-1)) and Cd (50 mg kg(-1)) in vermiculite, the total Mn/Cd uptakes in the aerial parts of sweet sorghum, P. acinosa, and S. nigrum L. were increased by 65.2%/40.0%, 55.2%/31.1%, and 18.6%/25.6%, respectively, compared to the uninoculated controls. This demonstrates that the symbiont of SLS18 and sweet sorghum has the potential of improving sweet sorghum biomass production and its total metal uptake on heavy metal-polluted marginal land. It offers the potential that heavy metal-polluted marginal land could be utilized in planting sweet sorghum as biofuel feedstock for ethanol production, which not only gives a promising phytoremediation strategy but also eases the competition for limited fertile farmland between energy crops and food crops.

Accumulation and fractionation of rare earth elements are conserved traits in the Phytolacca genus.

Rare earth elements (REEs) are now considered emerging pollutants in the environment. Phytolacca americana, an REE hyperaccumulating plant, has been proposed for the remediation of REE-contaminated soils. However, there is no REE-related information for other Phytolacca species. Here, we examined five species (P. americana, P. acinosa, P. clavigera, P. bogotensis, and P. icosandra) for their response to REEs. REE accumulation and fractionation traits both occurred on the same order of magnitude among the five species. Heavy REEs were preferentially transferred to leaves relative to light REEs. Regardless of the species, lateral root length and chlorophyll content decreased under REE exposure, and lateral roots and foliar anthocyanins increased. However, plants did not experience or only slightly experienced oxidative stress. Finally, REE exposure strongly modulated the ionome of roots and, to a lesser extent, that of leaves, with a negative correlation between REE and Mn contents. In conclusion, our study provides new data on the response of several Phytolacca species to REEs. Moreover, we highlighted that the REE accumulation trait was conserved among Phytolacca species. Thus, we provide valuable information for the phytoremediation of REE-contaminated sites since the most appropriate Phytolacca species could be selected depending on the climatic/pedological area to be remediated.

Primary structure and glycan moiety characterization of PD-Ss, type 1 ribosome-inactivating proteins from Phytolacca dioica L. seeds, by precursor ion discovery on a Q-TOF mass spectrometer.

Seeds from Phytolacca dioica L. contain at least three N-glycosylated PD-Ss, type 1 ribosome-inactivating proteins (RIPs), which were separated and purified to homogeneity by conventional chromatographic techniques. ESI-Q-TOF mass spectrometry provided the accurate M(r) of native PD-S1 and PD-S3 (30957.1 and 29785.1, respectively) and the major form PD-S2 (30753.8). As the amino acid sequence of PD-S2 was already known, its disulfide pairing was determined and found to be Cys34-Cys262 and Cys88-Cys110. Further structural characterization of PD-S1 and PD-S3 (N-terminal sequence determination up to residue 30, amino acid analysis and tryptic peptide mapping) showed that the three PD-Ss shared the entire protein sequence. To explain the different chromatographic behaviour, their glycosylation patterns were characterized by a fast and sensitive mass spectrometry-based approach, applying a precursor ion discovery mode on a Q-TOF mass spectrometer. A standard plant paucidomannosidic N-glycosylation pattern [Hex(3), HexNAc(2), deoxyhexose(1), pentose(1)] was found for PD-S1 and PD-S2 on Asn120. Furthermore, a glycosylation site carrying only a HexNAc residue was identified on Asn112 in PD-S1 and PD-S3. Finally, considering the two disulfide bridges and the glycan moieties, the experimental M(r) values were in agreement with the mass values calculated from the primary structure. The complete characterization of PD-Ss shows the high potential of mass spectrometry to rapidly characterize proteins, widespread in eukaryotes, differing only in their glycosylation motifs.

Xylem embolism alleviated by ion-mediated increase in hydraulic conductivity of functional xylem: insights from field measurements.

Recent studies have shown that, in some species, xylem hydraulic conductivity (K(h)) increases with increasing cation concentration of xylem sap. Evidence indicates that K(h) increases as a result of the de-swelling of pit membrane pectins caused by cation neutralization of polygalacturonanes. We tested whether this ionic effect partly compensates for the embolism-induced loss of stem hydraulic conductivity (PLC) by increasing K(h) of functioning conduits. We report changes in PLC, leaf water status and potassium concentration ([K(+)]) of xylem sap measured in April and July in two evergreens (Ceratonia siliqua L. and Phytolacca dioica L.) and one deciduous tree (Platanus orientalis L.) growing in the field in Sicily. In summer, Ceratonia siliqua and Phytolacca dioica showed similar native embolism (PLC = 30-40%) and [K(+)] of xylem sap (14 to 17 mM), and K(h) of stems perfused with 10 to 25 mM KCl increased by 15 to 18% compared with K(h) of stems perfused with a low concentration of a multi-ionic solution. In contrast, native [K(+)] of sap of Platanus orientalis was 50% of that in the two evergreens in summer, with a parallel lack of detectable changes in PLC that was below 10% in both spring and summer. The ionic effect was PLC-dependent: the enhancement of K(h) induced by 10 to 25 mM KCl changed from 15% for fully hydrated stems to 50-75% for stems with PLC = 50%. In Ceratonia siliqua, PLC was less than 10% in spring and about 40% in summer; concurrently, xylem sap [K(+)] increased from 3 to about 15 mM. This [K(+)] at the recorded PLC would cause an increase in residual K(h) of about 30%. Hence, the actual reduction in water transport capacity of Ceratonia siliqua stems in summer is about 20%. Similar calculations for Phytolacca dioica suggest that the actual loss of hydraulic conductivity in stems of this species in summer would be only about 10%, and not 30% as suggested by hydraulic measurements performed in the laboratory. We conclude that an increase in [K(+)] of xylem sap might be involved in the up-regulation of residual K(h), thus substantially alleviating the embolism-induced reduction in leaf water supply.

Isolation and characterization of heterotepalins, type 1 ribosome-inactivating proteins from Phytolacca heterotepala leaves.

Leaves from Phytolacca heterotepala H. Walter (Mexican pokeweed) contain at least 10 type 1 RIP isoforms, named heterotepalins. Their Mr values are included in the range 28,000-36,000, as shown by SDS-PAGE performed under reduced conditions and the pI values in the pH range 8.50-9.50. Some heterotepalins are glycosylated. ESI-QTOF mass spectrometry provides the accurate Mr of heterotepalin 4 (29,326.00) and heterotepalin 5b (30,477.00), two isoforms purified to homogeneity by conventional chromatographic techniques. The N-terminal sequences up to residue 35, show that heterotepalins exhibit an high percentage identity with other type 1 RIPs isolated from Phytolaccaceae. Some heterotepalins cross-react with antisera raised against RIPs isolated from Phytolacca dioica leaves. The complete amino acid sequence of heterotepalin 4 matches that of Phytolacca heterotepala anti-viral protein PAP (RIP1), deduced from the cDNA sequence of PhRIP1 gene (AC: AY327475), with one exception concerning residue 245 which, in the native protein, is Ile instead of Met. This substitution, found by mass spectrometry mapping, has been directly confirmed by Edman degradation sequencing of the C-terminal tryptic peptide 242-262. The results show the high potential of mass spectrometry and Edman degradation to verify and to uncover possible amino acid substitutions between native proteins and their cDNA deduced sequences.

Interaction of Fe-Mn plaque and Arthrobacter echigonensis MN1405 and uptake and translocation of Cd by Phytolacca acinosa Roxb.

In this study, hydroponic experiments were conducted to investigate the role of Fe-Mn plaque and Arthrobacter echigonensis MN1405 under different solution Cd levels (0, 2 and 50 mg L-1) on Cd uptake and translocation by Phytolacca acinosa Roxb. (P. acinosa). Results showed Cd accumulated by P.acinosa without plaque were mostly distributed in root surface, indicating that an exclusion strategy for Cd tolerance. The formation of Fe-Mn plaque could contribute to the increase in the Cd tolerance of P.acinosa, promotion of the growth, increase in the enhancement of Cd translocation. Among all the treatments, Fe-Mn plaque treatments inoculated with MN1405 accumulated the maximum DCB-Cd (46.61 ± 6.36 g kg-1) and had the highest value of TFaeria (2.14 ± 0.01) at 50 mg L-1 and 2 mg L-1 solution for Cd, respectively, demonstrating the greatest capacity to accumulate and translocate Cd. The uptake of Cd by P. acinosa in plaque treatments may result in the increase of soluble sugar and decrease of soluble protein synthesized from roots which involved in Cd detoxification and thus diminished the negative effects of Cd to some extent.

An investigation of cellular distribution of manganese in hyperaccumlator plant Phytolacca acinosa Roxb. using SRXRF analysis.

Phytolacca acinosa Roxb. (P. acinosa) is a recently discovered manganese hyperaccumulator plant from southern China. It is a good candidate for phytoremediation of manganese(Mn) polluted soil for its high biomass and fast growth. Knowledge of the tissue localization and identification of heavy metals can provide essential information on metal toxicity and bioaccumulation mechanisms. Synchrotron radiation X-ray fluorescence spectroscopy (SRXRF) microprobe was used in this study to investigate the cellular distributions of Mn and other elements in root, stem, leaf, petiole and midrib of P. acinosa. The highest Mn content was found in the vascular tissues of root, stem, petiole and midrib. Cortex in root played a key role in Mn absorption and Mn was limited in the vascular bundle during the process of transportation in stem. Moreover, Mn content in leaf epidermis was higher than that in mesophyll, which suggested that the sequestration of Mn in leaf epidermis might be one of the detoxification mechanisms of P. acinosa. The significance of other elemental (such as P, S, K, Ca, Fe, Zn and Cu) distribution patterns and the correlation with Mn were also discussed.

Isolation and characterization of pokeweed mitogen-like phytomitogens from Shoriku, Phytolacca esculenta.

From saline extracts of Phytolacca esculenta (shoriku) roots, two phytomitogenes were isolated by salting out with (NH4),SO4 and chromatography on DEAE-cellulose and Sephadex G-100 columns. Both fractions were homogeneous on disc electrophoresis and on immunoelectrophoresis. One of these (Fraction E-2) was shown to be similar to pokeweed mitogen in respect to mol. wt (32,000) and amino acid composition. The other (Fraction E-3) was a protein of 18,000 mol. wt. Both fractions had similar biological activities to pokeweed mitogen in their ability to stimulate pig blood lymphocytes in vitro to incorporate tritiated thymidine, and to induce blastoid transformation. Both fractions contained an unusually large amount of cystine, i.e., 18 half-cystine residues % for Fraction E-2 and 22 residues % for Fraction E-3. Although these mitogens were resistant to deproteinizing procedures such as perchloric acid treatment and Sevag's procedure, the DNA synthesis-stimulating activity was inactivated by digestion with Pronase E and Nagarse, but resistant to trypsin, chymotrypsin, deoxyribonuclease, ribonuclease, lysozyme and neuraminidase. The activity was stable at acidic and neutral pH (4-7) but unstable at alkaline pH. The activity at pH 7.3 was stabilized by the addition of Ca2+ or Mg2+. On the addition of more than 2 mM of Ca2+, precipitation of mitogen occurred. From the above results the molecular basis of the mitogenic activity of shoriku mitogen is discussed.

Biotransformation of 6beta-santonin by Phytolacca acinosa cell suspension cultures.

AIM: To obtain more valuable derivatives for the further structural modification of 6beta-santonin (1) via biotransformation by using cell suspension cultures of Phytolacca acinosa. METHODS: The substrate 1 was incubated with cell suspension cultures of P. acinosa, the products were obtained by chromatography, and identified on the basis of their physical and spectral data (HRMS, 1D NMR, 2D NMR, NOE and IR). RESULTS: After incubation with cell suspension cultures of P. acinosa, 1 was converted into five products. Among them, 3 is a new compound. CONCLUSION: 6beta-santonin could be selectively reduced and hydroxylated by the cell suspension cultures of P. acinosa, which would provide valuable intermediates for its further structural modification.

Manganese uptake and accumulation by the hyperaccumulator plant Phytolacca acinosa Roxb. (Phytolaccaceae).

The perennial herb Phytolacca acinosa Roxb. (Phytolaccaceae), which occurs in Southern China, has been found to be a new manganese hyperaccumulator by means of field surveys on Mn-rich soils and by glasshouse experiments. This species not only has remarkable tolerance to Mn but also has extraordinary uptake and accumulation capacity for this element. The maximum Mn concentration in the leaf dry matter was 19,300 microg/g on Xiangtan Mn tailings wastelands, with a mean of 14,480 microg/g. Under nutrient solution culture conditions, P. acinosa could grow normally with Mn supplied at a concentration of 8000 micromol/l, although with less biomass than in control samples supplied with Mn at 5 micromol/l. Manganese concentration in the shoots increased with increasing external Mn levels, but the total mass of Mn accumulated in the shoots first increased and then decreased. At an Mn concentration of 5000 micromol/l in the culture solution, the Mn accumulation in the shoot dry matter was highest (258 mg/plant). However, the Mn concentration in the leaves reached its highest value (36,380 microg/g) at an Mn supply level of 12,000 micromol/l. These results confirm that P. acinosa is an Mn hyperaccumulator which grows rapidly, has substantial biomass, wide distribution and a broad ecological amplitude. This species provides a new plant resource for exploring the mechanism of Mn hyperaccumulation, and has potential for use in the phytoremediation of Mn-contaminated soils.

Manganese-mitigation of cadmium toxicity to seedling growth of Phytolacca acinosa Roxb. is controlled by the manganese/cadmium molar ratio under hydroponic conditions.

Manganese (Mn) can interact with cadmium (Cd) in environments and influence the toxic effect of Cd on plants. However, few studies have investigated the relationship between the Mn/Cd ratio and plant Cd-toxicity along Cd concentrations. In this paper, we studied the effects of external Mn/Cd molar ratios (0, 10, 30, 50 and 60) on Cd toxicity in the Mn hyperaccumulator and Cd tolerant plant, Phytolacca acinosa Roxb., at three Cd levels (50, 100 and 200 µM) under hydroponic conditions. Our result showed that seedling growth (y) under Cd stress was strongly positively related to the solution Mn/Cd molar ratio (SMCR). The relationship between the two variables under solution Cd concentrations was well explained by the linear regression model y=a+b1 (SMCR)+b2 (Solution-Cd). Increasing SMCR significantly reduced the Cd concentration and increased the Mn concentration in plant tissues. However, seedling growth was consistent with the shoot Mn/Cd molar ratio rather than with the Mn or Cd concentrations in plant tissues. At low levels of SMCR (e.g. 0 and 10), elevation of Mn distribution in shoot tissues might be a mechanism in P. acinosa seedlings to defend against Cd-toxicity. In comparison with low levels of SMCR, high levels of SMCR (e.g. 50 and 60) greatly alleviated lipid peroxidation and plant water-loss, and enhanced photosynthesis. However, the alleviated lipid peroxidation in the Mn-mitigation of Cd toxicity was likely to be the secondary effect resulting from the antagonism between Mn and Cd in the plant.

Pokeweed antiviral protein increases HIV-1 particle infectivity by activating the cellular mitogen activated protein kinase pathway.

Pokeweed antiviral protein (PAP) is a plant-derived N-glycosidase that exhibits antiviral activity against several viruses. The enzyme removes purine bases from the messenger RNAs of the retroviruses Human immunodeficiency virus-1 and Human T-cell leukemia virus-1. This depurination reduces viral protein synthesis by stalling elongating ribosomes at nucleotides with a missing base. Here, we transiently expressed PAP in cells with a proviral clone of HIV-1 to examine the effect of the protein on virus production and quality. PAP reduced virus production by approximately 450-fold, as measured by p24 ELISA of media containing virions, which correlated with a substantial decline in virus protein synthesis in cells. However, particles released from PAP-expressing cells were approximately 7-fold more infectious, as determined by single-cycle infection of 1G5 cells and productive infection of MT2 cells. This increase in infectivity was not likely due to changes in the processing of HIV-1 polyproteins, RNA packaging efficiency or maturation of virus. Rather, expression of PAP activated the ERK1/2 MAPK pathway to a limited extent, resulting in increased phosphorylation of viral p17 matrix protein. The increase in infectivity of HIV-1 particles produced from PAP-expressing cells was compensated by the reduction in virus number; that is, virus production decreased upon de novo infection of cells over time. However, our findings emphasize the importance of investigating the influence of heterologous protein expression upon host cells when assessing their potential for antiviral applications.

The role of the glycan moiety on the structure-function relationships of PD-L1, type 1 ribosome-inactivating protein from P. dioica leaves.

N-glycosylation is one of the major naturally occurring covalent co-translational modifications of proteins in plants, being involved in proteins structure, folding, stability and biological activity. In the present work the influence of carbohydrate moieties on the structure-function relationships of type 1 ribosome-inactivating proteins (RIPs) was investigated. To this aim, PD-Ls, RIPs isolated from Phytolacca dioica L. leaves, differing for their glycosylation degree, were used as an experimental system. In particular, comparative structural and biological analyses were performed using native and unglycosylated recombinant PD-L1, the most glycosylated P. dioica RIP isoform. The glycans influence on protein synthesis inhibition and adenine polynucleotide glycosidase activity was investigated. The interaction with adenine, the product of the de-adenylation reaction, was also investigated for native and recombinant PD-L1 by fluorescence spectroscopy. Furthermore, the crystal structure of PD-L1 in complex with adenine was determined. Our data confirm that the absence of glycan moieties did not affect the biological activity in terms of protein synthesis inhibition. However, the removal of carbohydrate chains significantly increased the deadenylation capability, likely as a consequence of the increased accessibility of substrates to the active site pocket. Furthermore, preliminary data on cellular uptake showed that all PD-L isoforms were internalized and, for the first time, that the vesicular distribution within cells could be influenced by the protein glycosylation degree.

Structural characterization and comparative modeling of PD-Ls 1-3, type 1 ribosome-inactivating proteins from summer leaves of Phytolacca dioica L.

The amino acid sequence and glycan structure of PD-L1, PD-L2 and PD-L3, type 1 ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves, were determined using a combined approach based on peptide mapping, Edman degradation and ESI-Q-TOF MS in precursor ion discovery mode. The comparative analysis of the 261 amino acid residue sequences showed that PD-L1 and PD-L2 have identical primary structure, as it is the case of PD-L3 and PD-L4. Furthermore, the primary structure of PD-Ls 1-2 and PD-Ls 3-4 have 81.6% identity (85.1% similarity). The ESI-Q-TOF MS analysis confirmed that PD-Ls 1-3 were glycosylated at different sites. In particular, PD-L1 contained three glycidic chains with the well known paucidomannosidic structure (Man)(3) (GlcNAc)(2) (Fuc)(1) (Xyl)(1) linked to Asn10, Asn43 and Asn255. PD-L2 was glycosylated at Asn10 and Asn43, and PD-L3 was glycosylated only at Asn10. PD-L4 was confirmed to be not glycosylated. Despite an overall high structural similarity, the comparative modeling of PD-L1, PD-L2, PD-L3 and PD-L4 has shown potential influences of the glycidic chains on their adenine polynucleotide glycosylase activity on different substrates.

Crystal structure of PD-L1, a ribosome inactivating protein from Phytolacca dioica L. leaves with the property to induce DNA cleavage.

The structure of the highly glycosylated type 1 ribosome inactivating protein PD-L1 was determined by X-ray crystallography. This protein belongs to a group of four PD-Ls (PD-L1-4) expressed in Phytolacca dioica leaves. Of these, PD-L1 and PD-L2 are endowed with the ability to cleave double strand DNA, a property which is not shared by the other two components of the family. Single crystals of native PD-L1, the most glycosylated, were obtained using seeding techniques and phase determination was achieved using molecular replacement. To investigate the role of glycosylation in the different functionality of these proteins, we performed DNA cleavage assays on the E. coli plasmid pBR322. These experiments revealed that DNA cleaving ability does not depend on the level of glycosylation of PD-L1, since there is no difference in the activities displayed by native PD-L1 and a recombinant non-glycosylated form. Besides, confirming that DNA cleavage by PD-L1 cannot be attributed to contaminations, these data unambiguously show that functional changes between PD-L1 and PD-L4 are solely to be attributed to their sequence differences. On the basis of the comparison of PD-L1 and PD-L4 crystal structures, we propose possible structural determinants responsible for their different functional behavior.

Type 1 ribosome-inactivating proteins from Phytolacca dioica L. leaves: differential seasonal and age expression, and cellular localization.

The expression of type 1 ribosome-inactivating proteins (RIPs) in Phytolacca dioica L. leaves was investigated. Fully expanded leaves of young P. dioica plants (up to 3 years old) expressed two novel RIPs, dioicin 1 and dioicin 2. The former was also found in developing leaves from adult P. dioica within about two and a half weeks after leaf development, and the latter continuously synthesized, with no seasonal or ontogenetic constraint. Fully expanded leaves from adult P. dioica expressed four RIPs (PD-Ls1-4) exhibiting seasonal variation. RIPs were localized in the extracellular space, in the vacuole and in the Golgi apparatus of mesophyll cells. Dioicin 1 and dioicin 2 showed rRNA N-beta-glycosidase activity and displayed the following properties, respectively: (1) Mr values of 30,047.00 and 29,910.00, (2) pIs of 8.74 and 9.37, and (3) IC(50) values of 19.74 (0.658 nM) and 6.85 ng/mL (0.229 nM). Furthermore, they showed adenine polynucleotide glycosylase activity and nicked pBR322 dsDNA. The amino acid sequence of dioicin 2 had 266 amino acid residues, and the highest percentage identity (81.6%) and similarity (84.6%) with PAP-II from Phytolacca americana, while its identity with other RIPs from Phytolaccaceae was around 40%.