The gibberellin-induced, cysteine-rich protein GIP2 from Petunia hybrida exhibits in planta antioxidant activity

Numerous GAST-like genes have been identified in various plant species. All code for small proteins with a conserved C-terminal region in which 12 cysteines are located in exactly the same positions. We have previously identified five gibberellin (GA)-induced GAST1-like genes in petunia, GIP1-5. GIP2 is expressed in elongating zones, and its suppression in transgenic petunia plants inhibits stem elongation, suggesting a role for the protein in GA-induced cell growth. However, nothing is known about the biochemical activity of GIP2 or any other GAST-like protein. As all contain putative catalytic disulfide bonds (putative redox-active cysteines), we speculated that they might be involved in redox regulation. Expression analysis of GIP2, GIP4 and GIP5 revealed that they are induced by H(2)O(2). To study whether GIP2 modulates H(2)O(2) levels, we generated transgenic petunia plants expressing GIP2 under the regulation of the ubiquitous CaMV 35S promoter. The transgene reduced H(2)O(2) levels in leaves following wounding. It also reduced the levels of H(2)O(2) in guard cells following osmotic stress and ABA treatments, leading to the suppression of stomatal closure. In addition, the transgene promoted stem and corolla elongation. As reactive oxygen species (ROS) are involved in cell elongation, we suggest that GIP2 affects growth by regulating the levels of ROS. As all known GAST-like proteins contain putative redox-active cysteines, they may all act as antioxidants.     

矮牵牛花期一些生理指标的变化

选择了 3种颜色的矮牵牛 (PetuniahybridaVilm) :粉红色、杂色和红色 ,将其开花过程分为 4个时期 :未出现花芽、花芽期、花蕾期和开花期 ,测定各时期MDA、可溶性糖、激素水平和多胺含量等指标的变化。结果表明 ,从无花芽期到开花期MDA含量有所升高 ;可溶性糖含量呈现降低的趋势。在粉红色的矮牵牛叶片中 ,IAA含量在开花期升高 ;GA含量在无花芽期和花芽期时较高 ;而ZRs则在花蕾期较低 ,在开花期时含量上升。 3种多胺含量的变化不同 ,腐胺在整个花期略有上升 ,精胺和亚精胺则略有下降     

PhEXPA1, a Petunia hybrida expansin,is involved in cell wall metabolism and in plant architecture specification

Expansins are wall-loosening proteins that induce wall stress relaxation and irreversible wall extension in a pH-dependent manner. Despite a substantial body of work has been performed on the characterization of many expansins genes in different plant species, the knowledge about their precise biological roles during plant development remains scarce. To yield insights into the expansion process in Petunia hybrida, PhEXPA1, an expansin gene preferentially expressed in petal limb, has been characterized. The constitutive overexpression of PhEXPA1 significantly increased expansin activity, cells size and organ dimensions. Moreover, 35S::PhEXPA1 transgenic plants exhibited an altered cell wall polymer composition and a precocious timing of axillary meristem development compared with wild-type plants. These findings supported a previous hypothesis that expansins are not merely structural proteins involved in plant cell wall metabolism but they also take part in many plant development processes. Here, to support this expansins dual role, we discuss about differential cell wall-related genes expressed in PhEXPA1 expression mutants and gradients of altered petunia branching pattern.     

The role of SPY and its TPR domain in the regulation of gibberellin action throughout the life cycle of Petunia hybrida plants

SPY acts as a negative regulator of gibberellin (GA) action in Arabidopsis, but its mode of action and regulation are still unknown. SPY over-expression in transgenic petunia plants affected various GA-regulated processes, including seed germination, shoot elongation, flower initiation, flower development and the expression of a GA-induced gene, GIP. A similar phenotype was obtained when wild-type petunia plants were treated with the GA-biosynthesis inhibitor, paclobutrazol. The N-terminus of SPY contains tetratricopeptide repeats (TPR). TPR motifs participate in protein-protein interactions, suggesting that SPY is part of a multiprotein complex. To test this hypothesis, we over-expressed the SPY's TPR region without the catalytic domain in transgenic petunia and generated a dominant-negative SPY mutant. The transgenic seeds were able to germinate on paclobutrazol, suggesting an enhanced GA signal. We cloned the petunia SPY homologue, PhSPY, and showed that its mRNA level is not affected by GA or ABA. The results of this study support the role of SPY as a negative regulator of GA action, suggest that the TPR domain is required for the interaction with other proteins to form an active complex and indicate that different plants use similar mechanisms to transduce the GA signal.     

矮牵牛叶肉原生质体再生壁多糖的形成及转化

用~3H—葡萄糖饲喂矮牵牛叶肉原生质体,发现放射性集中在半纤维素部分,占整个再生壁的83％～90％,而纤维素中约占9％。纸层析分析证明,葡萄糖占半纤维素部分中性糖总量的70％,再生壁主要是由非纤维素葡聚糖组成。原生质体在含有~3H—葡萄糖的培养基中培养4d后,24h追踪表明,部分半纤维素转化成纤维素。加入香豆素,至少3d之内抑制半纤维素向纤维素转化;除去抑制剂,半纤维素含量迅速降低而纤维素增加。所以认为矮牵牛叶向原生质体壁再生过程中半纤维素是合成纤维素的前体。     

矮牵牛育种研究进展

从常规育种、基因工程育种、体细胞育种和单倍体育种4个方面评述了矮牵牛(Petunia hybrida Vilm.)育种研究进展.国外对矮牵牛育种研究较多,新品种面世推陈出新,国内对其研究则较为薄弱.其常规育种最为成功,不断有新品种推出,基因工程育种也取得了一定成就,已有新品种面世,而体细胞育种及单倍体育种尚无新品种产生,但对这两种方法在育种上应用的可能性和前景作出了一定探索.     

S-alleles are retained and expressed in a self-compatible cultivar of Petunia hybrida.

Summary We identified two S-allele-associated proteins (S-proteins) in a self-compatible cultivar of Petunia hybrida based on their segregation in F₁ hybrids between P. hybrida and its self-incompatible relative, Petunia inflata (with S₂S₂ genotype), and in selfed progeny of P. hybrida. These two S-proteins, designated S_x-protein (24 kDa) and S_o- protein (31 kDa), are pistil specific, and their expression follows a temporal and spatial pattern similar to that of S-proteins characterized in self-incompatible solanaceous species. Their amino-terminal sequences also share a high degree of similarity with those of solanaceous S-proteins. Selfing of P. hybrida yielded plants with S_oS_o, SxSo, and S_xS_x genotypes in an approximately 1:2:1 ratio, indicating that the S_x- and S_o-alleles, though expressed in the pistil, failed to elicit a self-incompatibility response. The S₂-allele of P. inflata is expressed in all the F₁ hybrids, rendering them capable of rejecting pollen bearing the S₂-allele. The S_o-allele is not functional in the F1 hybrids, because all the F₁ progeny with S₂S_o genotype are self-compatible. However, in F₁ hybrids with S₂S_x genotype, approximately half are self-incompatible and half are self-compatible, indicating that the function of the S_x-allele depends on the genetic background. These results strongly suggest that the presence of functional S-alleles alone is not sufficient for expression of a self-incompatibility phenotype, and reaffirm the multigenic nature of gametophytic self-incompatibility suggested by earlier genetic studies.     

NEC1, a novel gene, highly expressed in nectary tissue of Petunia hybrida

To study the molecular regulation of nectary development, we cloned NEC1, a gene predominantly expressed in the nectaries of Petunia hybrida, by using the differential display RT-PCR technique. The secondary structure of the putative NEC1 protein is reminiscent of a transmembrane protein, indicating that the protein is incorporated into the cell membrane or the cytoplast membrane. Immunolocalization revealed that NEC1 protein is present in the nectaries. Northern blot analyses showed that NEC1 is highly expressed in nectary tissue and weakly in the stamen. GUS expression driven by the NEC1 promoter revealed GUS activity in the outer nectary parenchyma cells, the upper part of the filament and the anther stomium. The same expression pattern was observed in Brassica napus. GUS expression was observed as blue spots on the surface of very young nectaries that do not secrete nectar and do accumulate starch. GUS expression was highest in open flowers in which active secretion of nectar and starch hydrolysis had taken place. Ectopic expression of NEC1 resulted in transgenic plants that displayed a phenotype with leaves having 3-4 times more phloem bundles in mid-veins than the wild-type Petunia. The possible role of NEC1 gene in sugar metabolism and nectar secretion is discussed.     

Petunia flower longevity: the role of sensitivity to ethylene

Two petunia ( Petunia hybrida L.) lines, differing in their flower longevity, were studied, Similar tendencies were found in the changes of corolla fresh weight, electrolyte leakage and membrane microviscosity over the life spans of the two lines. Ethylene production by flowers of the two lines showed a similar pattern, peaking at 3 nl flower⁻¹ h⁻¹. However, in flowers of the short-lived line, ethylene production peaked at 6 days of age, but in the long-lived line, the peak appeared at 10 days of age. A large difference was found in the responsiveness of the flower to ethylene, Flowers of the short-lived line responded to a similar ethylene by immediate wilting, while those of the long-lived line responded to a similar ethylene treatment only after two days. Differences in sensitivity to ethylene were also, observed when the flowers were treated continuosly with (aminooxy)acetic acid, which blocks ethylene synthesis. Flowers of both lines responded to ethylene treatment by increased ethylene production to a similar rate. Differential sensitivity to ethylene, independent of ethylene production, seemingly governs flower longevity in the two petunia lines studied.     

增强UV-B对矮牵牛花瓣中生理生化物质变化的影响

比较研究了普通光照(CK)和增强UV-B处理条件下,粉红色矮牵牛(Petunia hybrida)花瓣中花色素苷、辅色素、丙二醛和还原糖的含量以及苯丙氨酸解氨酶(PAL)活性的变化.结果表明,UV-B照射处理组花瓣中花色素苷、辅色素和还原糖含量均高于对照(CK),PAL活性增强,叶面积、叶宽和周长均小于对照组,MDA含量则低于对照组.综合分析表明,粉红色矮牵牛承受增强UV-B处理有一个阈值,大致为960.5 kJ?m-2;并且花色素苷和辅色素的作用不同,花色素苷以抗氧化为主,而辅色素以对UV-B的屏蔽作用为主.     

The regulation of respiration in cell suspensions of Petunia hybrida, studied by inhibiting mitochondrial protein synthesis with chloramphenicol

When Petunia hybrida L. cv. Rosy Morn Fertile suspension cells were inoculated in fresh medium with chloramphenicol (CAP), the activity of cytochrome C oxidase (EC 1. 9. 3. 1), and the respiration via the cytochrome pathway of isolated mitochondria decreased, while in untreated cells these parameters more than doubled in 2–3 days. However, the in vivo respiratory activity of the cytochrome pathway of CAP-treated cells showed a similar course in time as that of untreated cells, even in the presence of an uncoupler, a large rise during the first 2–3 days followed by a decline. This leads to the conclusion that the respiration via the cytochrome pathway, even when measured in the presence of an uncoupler, is not the capacity of this pathway. Furthermore, the results suggest that, although new-synthesis of proteins occurs directly after in-oculation, a large overcapacity must be present of cytochrome pathway elements (at least of those that are mitochondrial encoded). CAP had little effect on the uninhibited respiration and the cyanide-resistant, alternative pathway of the Petunia cells. However, the engagement of the alternative pathway (in the presence or absence of uncoupler) was increased in CAP-treated cells, especially after day 3 of the batch cycle, possibly as an effect of higher sugar degradation in combination with substrate phosphorylation to compensate the loss of ATP-synthesizing ability of the cytochrome pathway. It will be discussed that in general one should be careful using the term 'capacity' for the respiratory pathways.     

Immunocytochemical evidence of calreticulin-like protein in pollen tubes and styles of Petunia hybrida Hort.

With a polyclonal antibody raised against calreticulin (CRT) the locations where the protein occurs in unpollinated and pollinated styles of Petunia hybrida were localized. The epitopes binding the CRT antibody were immunolocalized preferentially in pollen tubes. In transmitting tract cells, both before and after pollination, the level of CRT was low. The protein was mainly localized in the cytosol and around dictyosomes of transmitting-tract cells. In pollen tubes, a high level of CRT was found at their tips rich in endoplasmatic reticulum, cisternae piles of reticular and/or dictyosomal origin, and vesicles. Binding sites of the CRT antibody were also found in the internal callosic cell wall of the pollen tube. These results indicate a role of CRT in cells directly participating in pollen-pistil interaction.     

Ga4/7 promotes stem growth and flowering in a genetic line of Aquilegia x hybrida Sims

Rose-White is a unique genetic line of Aquilegia x hybrida Sims that will flower without a cold period, does not have an extended juvenile growth phase, and is responsive to gibberellin treatment. Plants treated at the 7-leaf stage with GA_4/7 bolted and flowered 20 days earlier than untreated plants. Gibberellin treatment did not increase the growth rate (leaves/day), but induced flowering after the production of fewer leaves. Plant height and the number of flowers per plant increased significantly. GA_4/7 treatment delayed flower senescence and abscission. Flower longevity was extended 3-fold when compared to untreated plants. Treatment response was more pronounced at lower temperatures (19°C day/12°C night) than at higher growing temperatures (24°C day/19°C night). GA₃ was much less effective than GA_4/7 in promoting flowering, and there was no response to benzyladenine.     

Identification of a S-ribonuclease-binding protein in Petunia hybrida

To investigate protein-protein interactions in gametophytic self-incompatibility, we used a yeast two-hybrid assay to identify proteins that could interact with the S-ribonuclease protein. These assays identified a pollen-expressed protein, which we have named PhSBP1, that appears to bind with a high degree of specificity to the Petunia hybrida S-ribonuclease. Although PhSBP1 activates reporter gene expression only when expressed in tandem with a S-RNAse bait protein, binding is not allele-specific. Sequence analysis demonstrated that PhSBP1 contained a C-terminal cysteine-rich region that includes a RING-HC domain. Because many RING-finger domain proteins appear to function as E3 ubiquitin ligases, our results suggest that ubiquitination and protein degradation may play a role in regulating self-incompatibility interactions. Together, these results suggest that PhSBP1 may be a candidate for the recently proposed general inhibitor (RI) of self-incompatibility ribonucleases.     

Quantitative analysis of protein changes during meristem intiation and bud development in protoplast-derived Petunia hybrida callus

Small calluses from Petunia hybrida leaf protoplasts differentiated into meristems and then vegetative buds in the presence of high cytokinin concentrations. Total proteins from calluses grown at two cytokinin concentrations, of which only the highest induced bud formation, were analysed for molecular markers of cytokinin action by two-dimensional electrophoresis. The protein patterns were also compared to those of differentiated shoots and roots and to those of calluses grown under various hormonal conditions. Five percent of the total proteins detected by this method varied significantly in their level of expression according to the treatment. Quantification by image analysis demonstrated the occurrence of five groups of polypeptides with different expression patterns. Polypeptides from group 1 marked the development of buds and were abundant in young photosynthetic organs from whole plants. Polypeptides from group 5 increased only in calluses grown on control medium, i.e. in the presence of the lowest cytokinin concentration. No protein increased specifically during meristem formation. The possible action of cytokinin on regeneration is discussed in relation to these results.     

Effects of uniconazole and GA3 on cold-induced stem elongation and flowering of Raphanus sativus L.

The effects of gibberellin (GA) on cold-induced stem elongation and flowering of Japanese radish (Raphanus sativus L.) were investigated using application of GA3 and a GA-biosynthesis inhibitor, uniconazole (UCZ). UCZ very strongly inhibited stem elongation and delayed flowering, and the inhibition and delay were completely reversed by GA3 application. These results suggest that GA is necessary not only in the stem elongation but also in the flowering. When cold treatment (CT) was conducted on the plants whose GA level was lowered by UCZ, GA3 applied after CT completely reversed the delay of flowering. Thus low GA level probably did not retard cold induction. Microscopic observation of apical meristem showed that UCZ delayed flowering by delaying the shift from vegetative to dome-shaped meristem. This result suggested that low GA level delayed floral evocation. Consequently it was suggested that low GA level retarded physiological process involved in long day induction or in floral evocation, resulting in delay of floral evocation.     

Fertile somatic hybrids between Petunia hybrida and a wild species,Petunia variabilis

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F₂ and BC₁ seedlings were obtained.     

镉胁迫对矮牵牛种子萌发、幼苗生长及抗氧化酶活性的影响

采用营养液培养的方法研究了不同浓度镉Cd（0、1、2和4 mmol·L^-1）处理对矮牵牛（Petunia hybrida）种子萌发、幼苗生长及生理特性的影响。结果发现：与对照相比,较低浓度（1 mmol·L^-1）Cd处理下矮牵牛种子萌发和幼苗生长均显著被抑制,当Cd浓度增至4 mmol·L^-1时完全抑制了矮牵牛种子的萌发。Cd胁迫在低浓度时激活矮牵牛幼苗叶片过氧化物酶（POD）、过氧化氢酶（CAT）活性,而高浓度产生抑制效应;抗坏血酸过氧化物酶（APX）和超氧化物歧化酶（SOD）活性与对照相比均显著升高,丙二醛（MDA）含量无显著变化。不同浓度的乙二胺四乙酸(EDTA)的加入使4 mmol·L^-1 Cd胁迫下矮牵牛幼苗叶片POD和APX活性增加,但不影响SOD和CAT活性;此外,6 mmol·L^-1 EDTA使Cd胁迫下矮牵牛幼苗叶片中MDA含量增加。结果表明,Cd胁迫抑制矮牵牛种子萌发和幼苗生长,却使幼苗叶片POD、CAT和APX活性增加,EDTA的加入能够进一步提高Cd胁迫幼苗POD和APX活性。因此,矮牵牛幼苗在Cd胁迫下具有较强的抗氧化损伤的能力。     

Self-incompatibility alleles control a low molecular weight,basic protein in pistils of Petunia hybrida

Summary Proteins extracted from the pistils of several clones of Petunia hybrida carrying differing pairs of S alleles were examined by gel electrophoresis. The major protein of pistils, a basic glycoprotein of relatively low molecular weight, showed properties which varied in a simple manner with the S genotype. For each S allele we were able to assign a specific molecular weight (ranging from 27,000 to 33,000) and isoelectric point (in the range 8.3 to 8.7) for this putative S protein. Pistils homozygous at the S locus showed only one major protein on two-dimensional gel electrophoresis, while pistils from plants heterozygous at the S locus showed two. No evidence was obtained for the presence of this putative S protein in pollen extracts.