Biochemical analysis of a papain-like protease isolated from the latex of Asclepias curassavica L.

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase （asclepain c-II） has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. （Asclepiadaceae）. Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography （FPLC）. Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantrollne, phenyimethanesulfonyl fluoride, and pepstatine. The N-terminal sequence （LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA） showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-α-CBZ-L-Gln-p-nitrophenyl ester as substrate： Km = 0.1634 mM, kcat = 121.48 s^-1, and kcat/Km = 7.4 ×10^5 s^-1/mM.     

Purification and Biochemical Characterization of Asclepain c I from the Latex of Asclepias curassavica L.

In this work we report the isolation, purification and characterization of a new protease from latex of Asclepias curassavica L. Crude extract (CE) was obtained by gathering latex on 0.1 M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-alpha-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (km and Kcat) were performed with PFLNA.     

Purification and Characterization of a New Plant Endopeptidase Isolated from Latex of Asclepias fruticosa L. (Asclepiadaceae)

Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 g of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45°C, but was quickly inactivated after 5 minutes at 80°C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (M_r = 23,652). The optimum pH range was achieved at 8.5–10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices of Asclepiadaceae even when a high degree of homology could be observed with other plant cysteine endopeptidases.     

Reproductive biology in Anagyris foetida L. (Leguminosae), an autumn–winter flowering and ornithophilous Mediterranean shrub

As most plants of the Mediterranean region bloom in spring, there have been few studies of the reproductive biology of species with autumn–winter flowering. In this study, we investigate the breeding system of Anagyris foetida , one of the few shrubs that blooms at this time. The floral, phenological, and reproductive aspects of two populations of this Mediterranean legume from south-west Spain were studied via field and laboratory experiments. The variability of fruit and seeds was studied in another 12 Iberian populations with respect to certain meteorological parameters (temperature and rainfall). Anagyris foetida shows cauliflory, marked floral longevity, and adichogamy. The peak of flowering is in January–February. It is self-compatible, with no clear advantage of cross- over self-pollination, and with virtually no autonomous self-pollination. This is because the stigma, like some other legumes, prevents the germination of pollen if its surface is not ruptured by pollinators. The number of seeds per fruit under natural pollination was positively correlated with the total rainfall during the fruiting period (from January to May), and significantly influenced the percentage of fruit weight represented by the pericarp, in the sense that the smaller the number of viable seeds in the fruit, the greater the percentage of pericarp weight.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 157 , 519–532.     

A comparison of levels of genetic polymorphism and self-compatibility in geographically restricted and widespread plant congeners

Summary Evolutionary theory predicts low levels of genetic polymorphism and high levels of self-compatibility in plant species with small ranges and few individuals. To test these predictions, I compared published data on electrophoretically detectable genetic variation and breeding systems for geographically restricted and widespread congeners in eleven genera. The restricted species exhibit significantly fewer polymorphic loci and alleles per polymorphic locus than do their widespread congeners. Although some rare species are genetically impoverished, others are nearly as polymorphic as their widespread congeners. The restricted species and their widespread congeners do not differ consistently with respect to breeding systems.     

Phylogenetic relationships and character evolution in Primula L.: The usefulness of ITS sequence data

ABSTRACT

The main goals of this research were to reconstruct the infrageneric phylogeny of the genus Primula based on both nuclear and chloroplast DNA sequences, and to use the resulting phylogenies to elucidate the evolution of breeding systems, morphological characters, chromosome number, and biogeographic distribution in the genus. In this paper, the results of a pilot study based on the nuclear ribosomal Internal Transcribed Spacer (ITS) region are described. ITS sequences from 21 taxa produced a number of variable characters sufficient to resolve relationships among sections. The resulting phylogeny confirmed the monophyly of sections Auricula and Aleuritia. Sections Armerina, Proliferae, Crystallophlomis, Parryi, and Auricula, with a base chromosome number of x = 11, and sect. Aleuritia, with a base chromosome number of x = 9, formed two well supported clades. The ITS topology also suggested that leaves with revolute vernation, previously believed to be a derived state, might represent the ancestral condition in Primula, with later reversals to the involute condition. Finally, this initial ITS tree provides preliminary support to the proposed role of the widespread, diploid and heterostylous P. mistassinica as having given origin to the polyploid, homostylous P. incana and P. laurentiana.     

元江芦荟与皂质芦荟的杂交育种及POD同工酶比较

以元江芦荟(AloeyuanjiangensisXiong,ZhengetLiu)和皂质芦荟(A.saponaria(Ait.)Haw.)为亲本进行了远缘有性杂交试验,获得了19株F1代植株。对F1代植株与亲本进行了外部形态比较;同时与亲本种及同属的华芦荟(中国芦荟)(A.chinensis(Haw.)Baker)和库拉索芦荟(A.veraL.)进行了过氧化物酶同工酶(POD)比较。其结果表明,F1代与亲本在外部形态上存在明显差异;POD同工酶酶谱显示,F1代与各个种间具有较高的相似程度,且各个种又具有各自特征酶带。这证实了F1是元江芦荟与皂质芦荟的杂种(A.yuanjiangensis×A.saponaria)。     

The effectiveness of some mechanisms of reproductive isolation in Arum maculatum and A. italicum (Araceae)

Mechanisms of reproductive isolation were studied in two species of Arum , A. italicum and A. maculatum , growing in England and the south of France. The study focused on three potential mechanisms for reproductive isolation: the effectiveness of dichogamy as a barrier to autogamy; the ability of self and outcrossed pollen to germinate on stigmas at different stages of anthesis and to effect pollination; and postzygotic barriers to selfing. Dichogamy was found to provide a very effective barrier to within-inflorescence selfing in these species, as no seeds were produced by spontaneous self-pollination (i.e. autogamy) in any population of either species. However, the study found that geitonogamy (cross-pollination between inflorescences of a same individual or clone) was possible, as genotypes frequently produced several inflorescences and stigmas were found to be receptive to pollen from before anthesis until their contraction at the end of the female phase of flowering. Hand pollination with self pollen from clone inflorescences produced as numerous and heavy seeds as outcross pollination. In addition, the germination and growth of geitonogamously produced seed was similar to that of outcrossed seed, suggesting that this potential postzygotic barrier is absent or weak. These findings suggest that geitonogamous seed production may be unrestricted by significant pre- or postzygotic barriers. The possible advantages of geitonogamy in Arum are discussed.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 323–328.     

Breeding systems and phylogeny in Poa,with special attention to Northeast Asia: The problem of Poa shumushuensis and sect. Nivicolae (Poaceae)

Poa sect. Poa subsect. Nivicolae (Prob.) Tzvelev was circumscribed to include four species of the Soviet Union: Poa shumushuensis, Poa caucasica, Poa irkutica, and Poa veresczaginii. Bayesian phylogenetic analyses of plastid and nuclear‐ribosomal DNA revealed that it is polyphyletic, none of these species are closely related. Poa shumushuensis, type of sect. Nivicolae, or its ancestor, likely displayed the nrDNA genotype characteristic of the higher polyploid P. sect. Malacanthae. Genotype codes are designated for each species: Hx, C c, Php, and Shp. Poa sect. Nivicolae s.s. is restricted to P. shumushuensis; P. sect. Irkuticae is restricted to P. irkutica; P. caucasica is moved to P. subg. & sect. Caucasicae nov.; and P. sect. Dschungaricae is resurrected for P. veresczaginii and two other species. Although diclinous breeding systems are known in many western hemisphere species of Poa, dicliny is infrequent and little studied in Asian Poa. Poa shumushuensis is judged to be either sequentially gynomonoecious or gynodioecious. A ratio of 2 perfect‐, to 2 mixed‐, to 1 pistillate‐flowered inflorescences from different plants in P. shumushuensis is suggestive of a recessive allele for stamen suppression, and this is associated with subtle sexual‐dimorphism. Poa irkutica is diclinous with a breeding system between simple gynomonoecy and sequential gynomonoecy; P. caucasica is perfectly flowered; and P. veresczaginii has infrequent abortive anthers, indicative of limited dicliny, or sterility for other reasons possibly related to its reticulate origin. In total, 23 Asian species are here reported to be diclinous and their breeding systems are characterized. A lectotype is designated for P. fauriei.