宝山堇菜（Viola baoshanensis）、紫花地丁(V. yedoensis)光合异质性比较

利用荧光成像技术,研究了特定和不同光合有效辐射下,宝山堇菜（Viola baoshanensis）、紫花地丁 (V. yedoensis)不同叶龄（幼叶和成熟叶片）叶片纵向（叶基、叶中部、叶尖）间的光合异质性特征。特定光化光照射下,两种堇菜不同叶龄的F_v/F_m、ΦpsⅡ、qP、PS/50和Abs在叶尖、叶中部、叶基间呈依次降低趋势,NPQ/4和qN变化趋势与之相反。F_v/F_m、ΦpsⅡ和Abs在两种堇菜不同叶龄的叶片纵向间均没有显著性差异,NPQ/4和qN均显示宝山堇菜不同叶龄的叶片纵向间存在显著差异;但NPQ/4和qN分别显示紫花地丁成熟叶和幼叶叶片的叶尖和叶基处差异显著。qP的显著差异只存在于宝山堇菜幼叶的叶尖和叶基处,PS/50在两种植物幼叶纵向间均有显著差异。快速光曲线的变化中,两种堇菜α由叶尖向叶基下降幅度不明显,幼叶纵向间P_m差异显著,成熟叶叶尖处P_m显著高于叶中部和叶基。两种堇菜成熟叶叶尖处I_k显著高于叶中部和叶基,宝山堇菜幼叶纵向间I_k差异显著,而紫花地丁幼叶纵向间I_k差异不显著。以上结果反映出两种堇菜叶片纵向间F_v/F_m和ΦpsⅡ具有较高的均质性,F_v/F_m和ΦpsⅡ的下降受到NPQ/4、qN、qP、PS/50和Abs的综合影响,但F_v/F_m和ΦpsⅡ的变化与NPQ/4、qN、qP、PS/50的显著变化并不一致。叶龄对两种堇菜叶片纵向间α影响不显著,对P_m影响显著,α的小幅下降反映两种堇菜叶片纵向间捕光能力基本相同。叶片纵向间I_k的显著差异受叶龄和植物种类的综合影响。     

宝山堇菜(Viola baoshanensis)镉胁迫下的光合作用

利用荧光成像技术研究了自然生长状态下宝山堇菜、紫花地丁镉含量与光合作用间的关系。野外调查表明,宝山堇菜、紫花地丁镉含量差异显著,地上部分镉含量分别为867mg／kg和2mg／kg,相差近450倍;镉转运系数、富集系数,宝山堇菜均大于1,紫花地丁均小于1。叶绿素荧光参数,宝山堇菜Fm、Ik、Pm均显著低于紫花地丁（p=0．05）;两种植物间Fo、Fv/Fm、α均无显著差异。紫花地丁地上部分镉含量与叶绿素荧光参数的相关性较低,宝山堇菜地上部分镉含量与Fm、Pm、Ik达到极显著负相关。Fv／Fm,宝山堇菜、紫花地丁均在0．8左右,与镉的相关性较低,表明其光合作用没有受到镉胁迫。但是,宝山堇菜Fm显著低于紫花地丁,而且与镉呈负相关,表明宝山堇菜叶片Fm会随镉含量的增加而降低,预示镉可能会造成宝山堇菜光系统Ⅱ叶绿素结构破坏或者抑制其电子传递。参数Ik和Pm在两种植物间差异显著及其与宝山堇菜地上部分镉含量显著负相关反映宝山堇菜光耐受能力和电子传递能力在镉胁迫下逐渐减弱。初始斜率α始终维持在较高水平,说明镉胁迫并未对宝山堇菜捕光系统造成显著伤害。结合Fm值的变化和快速光曲线中荧光参数的变化推断镉最有可能会造成宝山堇菜电子传递能力的减弱。     

The alpine violet, Viola biflora, is a rich source of cyclotides with potent cytotoxicity

The cyclotides are currently the largest known family of head-to-tail cyclic proteins. The complex structure of these small plant proteins, which consist of approximately 30 amino acid residues, contains both a circular peptide backbone and a cystine knot, the combination of which produces the cyclic cystine knot motif. To date, cyclotides have been found in plants from the Rubiaceae, Violaceace and Cucurbitaceae families, and are believed to be part of the host defence system. In addition to their insecticidal effect, cyclotides have also been shown to be cytotoxic, anti-HIV, antimicrobial and haemolytic agents. In this study, we show that the alpine violet Viola biflora (Violaceae) is a rich source of cyclotides. The sequences of 11 cyclotides, vibi A-K, were determined by isolation and MS/MS sequencing of proteins and screening of a cDNA library of V. biflora in parallel. For the cDNA screening, a degenerate primer against a conserved (AAFALPA) motif in the cyclotide precursor ER signal sequence yielded a series of predicted cyclotide sequences that were correlated to those of the isolated proteins. There was an apparent discrepancy between the results of the two strategies as only one of the isolated proteins could be identified as a cDNA clone. Finally, to correlate amino acid sequence to cytotoxic potency, vibi D, E, G and H were analysed using a fluorometric microculture cytotoxicity assay using a lymphoma cell line. The IC(50)-values of the bracelet cyclotides vibi E, G and H ranged between 0.96 and 5.0 microM while the M?bius cyclotide vibi D was not cytotoxic at 30 microM.     

Metalliferous and non-metalliferous populations of Viola tricolor represent similar mode of antioxidative response

Heavy metal-contaminated sites are excellent areas to examine the antioxidative machinery responsible for physiological adaptations of many plant species.Superoxide dismutase (SOD), guaiacol peroxide (GPX), ascorbate peroxide (APX), catalase (CAT) activity and hydrogen peroxide (H₂O₂) content were analyzed in leaves and roots of Viola tricolor (Viola) from contaminated soils (‘Bukowno’, ‘Saturn’, ‘Warpie’ heaps), and non-contaminated soil (‘Zakopane meadow’) to examine the level of oxidative stress and antioxidative response.In leaves, six isoforms of SOD were recognized. Roots possessed two additional bands, named manganese superoxide dismutase (MnSOD)-like form (MnSODI) and Cu/ZnSOD-like form (Cu/ZnSODIV). The H₂O₂ content in leaves ranged from 554 to 5 098 μmol H₂O₂/g f.w. and was negatively correlated with CAT activity. The non-contaminated population was characterized by the lowest CAT activity combined with the highest H₂O₂ concentration. Two isoforms of CAT, CAT-1 and CAT-2, were recognized in leaves of plants from non-contaminated and contaminated sites, respectively. In roots of individuals from two heaps (‘Warpie’ and ‘Saturn’), two distinct bands for each CAT isoform were observed. A slower migrating band may be an aggregate, exhibiting CAT and MnSODs activities. Both peroxidases (APX and GPX) presented the same pattern of activity, depending on the organ, indicating that in leaves and roots APX and GPX were regulated in parallel.Differences in enzyme activities and H₂O₂ content between plants from different contaminated sites were statistically significant, but were tightly maintained at a very similar level. Prolonged and permanent heavy metal stress evoked a very similar mode of antioxidative response in specimens of analyzed metalliferous populations not causing measurable oxidative stress. Thus, our results clearly indicate that V. tricolor is a taxon well adapted to heavy metal-contaminated soils, and that differences in enzyme activities and H₂O₂ content result from adjustment of plants to a variety of conditions.     

Isolation and characterization of cytotoxic cyclotides from Viola philippica

Cyclotides are a large family of plant peptides characterized by a macrocyclic backbone and knotted arrangement of three disulfide bonds. This unique structure renders cyclotides exceptionally stable to thermal, chemical and enzymatic treatments. They exhibit a variety of bioactivities, including uterotonic, anti-HIV, cytotoxic and hemolytic activity and it is these properties that make cyclotides an interesting peptide scaffold for drug design. In this study, eight new cyclotides (Viphi A-H), along with eight known cyclotides, were isolated from Viola philippica, a plant from the Violaceae family. In addition, Viba 17 and Mram 8 were isolated for the first time as peptides. The sequences of these cyclotides were elucidated primarily by using a strategy involving reduction, enzymatic digestion and tandem mass spectroscopy sequencing. Several of the cyclotides showed cytotoxic activities against the cancer cell lines MM96L, HeLa and BGC-823. The novel cyclotides reported here: (1) enhance the known sequence variation observed for cyclotides; (2) extend the number of species known to contain cyclotides; (3) provide interesting structure-activity relationships that delineate residues important for cytotoxic activity. In addition, this study provides insights into the potential active ingredients of traditional Chinese medicines.     

Discovery and characterization of a linear cyclotide from Viola odorata: implications for the processing of circular proteins

Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule.     

Allozyme diversity in natural populations of Viola palmensis Webb & Berth. (Violaceae) from La Palma (Canary Islands): implications for conservation genetics

Genetic diversity was measured by allozyme electrophoresis in eight natural populations of the threatened Canarian endemic Viola palmensis Webb & Berth. (Violaceae). Nineteen alleles corresponding to 11 gene loci were detected. High levels of genetic diversity were found, ranging from 36.3 to 45.4 % for the percentage of polymorphic loci (P), from 1.45 to 1.60 for the average number of alleles per locus (A) and from 0.128 to 0.200 for the expected heterozygosity (H(e)). Between 85.5 and 96.6 % of genetic variability was apportioned within populations. As a whole, populations were not at Hardy-Weinberg equilibrium, with a deficit of heterozygous individuals attributable to the existence of genetic structuring in the populations analysed. The levels of interpopulation genetic differentiation were low (mean F(ST) = 0.100), while genetic identity pair-wise comparisons were high (mean I = 0.973) suggesting considerable levels of gene flow among populations. No relationship was detected between genetic differentiation and geographical distances between populations. An outcrossing insect-mediated breeding system might contribute to pollen dispersion of this species. For conservation genetics we suggest in situ preservation areas are defined that are free of disturbance and that include populations with the highest genetic diversity.     

Spread of violets in polluted pine forests: morphological and molecular evidence for the ecological importance of interspecific hybridization

Hybridization between plant species occurs frequently but hybrids are often restricted to ecotones or disturbed habitats. In this study we show that introgressive hybrids between the tetraploid Viola riviniana and the diploid V. reichenbachiana invaded pine forests of the Dübener Heide (central Germany), an area affected by calcareous pollutants. The spread of these violet populations was correlated with the impact of pollution on habitat conditions. We compared morphology, cytology and random amplified polymorphic DNA (RAPD) bands among six Viola populations from the Dübener Heide and three populations of each pure species. RAPD analysis using 12 primers revealed 141 scorable bands. We considered bands as species specific if they occurred in at least 75% of the plants in one pure violet species but in none of the other. Seven bands were specific to V. riviniana and 11 bands were specific to V. reichenbachiana . Two plants of a V. reichenbachiana population were identified as hybrids. Of the Viola populations from the Dübener Heide, one was diploid and could be classified as V. reichenbachiana by morphology and RAPD markers. However, the majority of the Dübener Heide populations were tetraploid, and of a more variable morphology than V. riviniana and V. reichenbachiana , showing different combinations of intermediate characters, characters of the pure species and extreme characters. Despite their overall genetic similarity to V. riviniana , these plants could be identified as introgressive hybrids between V. riviniana and V. reichenbachiana by species-specific RAPD bands. Therefore, we propose that recurrent hybridization and backcrossing resulted in novel genotypes adapted to the changed environment of polluted pine forests.     

三色堇与角堇种间遗传连锁图谱构建

该研究以二倍体三色堇和角堇为亲本杂交产生的66株F2代分离群体为作图群体,采用SRAP标记技术进行基因分型,利用JoinMap4.0软件构建了首张三色堇与角堇的种间遗传连锁图谱。结果表明:(1)从256对SRAP引物组合中筛选获得50对多态性好、标记位点清晰且稳定的引物组合。(2)通过对三色堇F2代群体的PCR扩增,共获得118个SRAP多态性标记位点,其中偏分离标记率为24.6%,符合遗传作图需要。(3)成功构建了三色堇和角堇的种间分子遗传连锁图谱,该图谱有15个连锁群,67个SRAP标记,连锁群长度范围1.6~52.2 cM,覆盖基因组总长度327.9 cM,标记间平均图距为4.9 cM。研究结果为三色堇和角堇高密度遗传图谱构建和重要性状的基因定位及分子标记辅助选择育种奠定了基础。