Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes

In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays.The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively).Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.     

The influence of the genes An1, An2, and An4 on the activity of the enzyme UDP-glucose:Flavonoid 3-O-glucosyltransferase in flowers of Petunia hybrida

A relation between gene dosage and UDP-glucose:flavonoid 3-O-glucosyl-transferase (UFGT) activity was found in homozygous dominant and recessive parental lines and their F₁ progeny for both of the genes An1 and An2. In both F₂ crosses, progeny plants could be classified as belonging to groups showing either a low or a medium to high UFGT activity. Test crosses showed that heterozygous and homozygous dominant plants were present throughout the medium- to high-active group. The dosage relation in F₂ plants is most probably confounded by the segregation of modifiers. Thermal inactivation experiments indicated that structurally different UFGT enzymes are formed in homozygous dominant lines as well as in lines homozygous recessive for either An1 or An2. Lines homozygous recessive for the gene An4 contain a UFGT with a half-life time at 55° C of less than 8 min, whereas UFGTs from lines homozygous dominant for An4 show a half-life time of 25 min or above, with one exception. This relation was confirmed in the F₂ progeny; heterozygotes for An4 showed an intermediate half-life time. It is concluded that An4 might be the structural gene for the enzyme; An1 and An2 are both regulatory genes. UFGT activity in flowerbuds of An4/An4 plants seems to be lower than in an4/an4 plants. Anthers of flowers of an4/an4 lines, however, are virtually devoid of UFGT activity.     

Involvement of two differenturf-s related mitochondrial sequences in the molecular evolution of the CMS-specificS-Pcf locus in petunia

In petunia, a mitochondrial (mt) locus,S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). TheS-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF,Pcf, contains parts of theatp9 andcoxII genes and an unidentified reading frame,urf-s. The second and third ORFs contain NADH dehydrogenase subunit 3 (nad3) and ribosomal protein S12 (rps12) sequences, respectively. Thenad3 andrps12 sequences included in theS-Pcf locus are identical to the corresponding sequences on the mt genome of fertile petunia. In both CMS and fertile petunia, only a single copy ofnad3 andrps12 has been detected on the physical map of the main mt genome. The origin of theurf-s sequence and the molecular events leading to the formation of the chimericS-Pcf locus are not known. This paper presents evidence indicating that two different mt sequences, related tourf-s and found in fertile petunia lines (orf-h and Rf-1), might have been involved in the molecular evolution of theS-Pcf locus. Southern analysis of mtDNA derived from both fertile and sterile petunia plants suggests that one of theseurf-s related sequences (showing 100% homology tourf-s and termedorf-h) is located on a sublimon. An additional, low-homologyurf-s related sequence (Rf-1) is shown to be located on the main mt genome 5′ to thenad3 gene. It is, thus, suggested that the sequence of events leading to the generation of theS-Pcf locus might have involved introduction of theorf-h sequence, via homologous recombination, into the main mt genome 5′ tonad3 at the region where the Rf-1 sequence is located. Contribution [No. 1581-E (1995 series)] from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel 50 250     

Cloning of Petunia hybrida chloroplast DNA sequences capable of autonomous replication in yeast

Summary Sequences from Petunia hybrida chloroplast DNA which have the property to promote autonomous replication in Saccharomyces cerevisiae were cloned in vector YIp5. Seven cloned chloroplast DNA fragments are localized at one of two different sites on the chloroplast genome. One site, arsA was mapped on a 1.8 Kb fragment at position 27.0–28.8 Kb on the P. hybrida chloroplast genome. The plasmids containing this arsA are stable both in yeast and E. coli. The other site, arsB, was shown to be very unstable and is located either in the small single copy region close to the inverted repeat or just in the inverted repeat. The functioning of these sequences as a possible origin of replication in vivo is discussed.     

非洲菊亲环素家族基因GCyP的克隆与生物信息学分析

亲环素(Cyclophilins,CyPs)具有肽脯氨酰顺反异构酶活性,催化蛋白质折叠过程,并且起着分子伴侣的作用,同时参与mRNA加工和信号转导等生物学过程.本研究在抑制性消减杂交获得表达序列标签(EST)的基础上,通过生物信息学方法获得了非洲菊亲环素家族基因GCyP的cDNA.进而设计特异引物,以非洲菊舌状花为材料,通过RT-PCR扩增并经T-A克隆后测序,获得一条长度为732 bp的序列.该序列经生物信息学分析确认其为GCyP,编码的GCyP含172个氨基酸残基,属于单结构域胞质型亲环素.GCyP序列提交GenBank后接收,登录号为EU1269167.     

Acid-stable α-Amylase of Black Aspergilli

Some general properties of the acid-stable dextrinizing amylase of black Aspergillus were investigated comparing with those of Taka-amylase A. The mode of action on starch of this amylase was quite similar to that of Taka-amylase A. Saccharifying degree at red point in starch-iodine color reaction was 5.1% and the limit of starch saccharification was a little over 40 per cent calculated as glucose with both amylases. Maltase activity was absent. Degradation products in the course of starch hydrolysis were also quite similar and they mutarotated downward. So this amylase was decided to be α-type. Thermal stability of the acid-stable α-amylase was higher than that of Taka-amylase A. Its acid stability was much higher than that of Taka-amylase A. Taka-amylase A was inactivated completely at pH 2.2, 37°C, for 30 min, but the acid-stable α-amylase retained 87% of its original activity.

From the amylase preparation of black Aspergillus acid-stable α-amylase and acidunstable α-amylase were separated by gel filtration on sephadex G-100 column. From the acid-unstable α-amylase fraction this enzyme was purified by fractionations with rivanol and acetone, and finally obtained as a homogeneous protein after gel filtration with sephadex G-50. Comparison of some general properties between the two α-amylases was carried out. Catalytic action was quite similar with both enzymes, but dextrinizing unit per mg enzyme protein of the acid-unstable α-amylase was about 5.6 times as large as that of the acid-stable α-amylase. The acid-unstable α-amylase was less heat-stable than the acid-stable α-amylase. Acid stability and pH-activity curve were compared with both α-amylases. High stability of the acid-stable α-amylase in acidic condition was observed, but, in alkaline range, it was more sensitive than the acid-unstable α-amylase.     

Increasing the variability of Lycopersicon peruvianum Mill. by protoplast fusion with Petunia hybrida L.

Somatic hybridization of Lycopersicon peruvianum and Petunia hybrida was carried out to transfer cytoplasmic male sterility from Petunia to Lycopersicon. Cytological, morphological and biochemical analyses were performed to characterize the regenerated plants. Two regenerated plants, R₃ and R₆, were male sterile. R₃ possessed chromosomes morphologically similar to those of both parental types. Leaf morphologies of these two plants and a third plant, R₇, were intermediate between the two parents. The stability of RUBPCase was verified during parental plant development and after in vitro culture. Plant R₇ presented a new form of the large subunit of RUBPCase.     

Exogenous ethylene influences flower opening of cut roses (<Emphasis Type="Italic">Rosa hybrida</Emphasis>) by regulating the genes encoding ethylene biosynthesis enzymes

The purpose of this paper is to investigate the differential responses of flower opening to ethylene in two cut rose cultivars, ‘Samantha’, whose opening process is promoted, and ‘Kardinal’, whose opening process is inhibited by ethylene. Ethylene production and 1-aminocyclopropane-1-carboxylate (ACC) synthase and oxidase activities were determined first. After ethylene treatment, ethylene production, ACC synthase (ACS) and ACC oxidase (ACO) activities in petals increased and peaked at the earlier stage (stage 3) in ‘Samantha’, and they were much more dramatically enhanced and peaked at the later stage (stage 4) in ‘Kardinal’ than control during vasing. cDNA fragments of three Rh-ACSs and one Rh-ACO genes were cloned and designated as Rh-ACS1, Rh-ACS2, Rh-ACS3 and Rh-ACO1 respectively. Northern blotting analysis revealed that, among three genes of ACS, ethylene-induced expression patterns of Rh-ACS3 gene corresponded to ACS activity and ethylene production in both cultivars. A more dramatic accumulation of Rh-ACS3 mRNA was induced by ethylene in ‘Kardinal’ than that of ‘Samantha’. As an ethylene action inhibitor, STS at concentration of 0.2 mmol/L generally inhibited the expression of Rh-ACSs and Rh-ACO in both cultivars, although it induced the expression of Rh-ACS3 transiently in ‘Kardinal’. Our results suggests that ‘Kardinal’ is more sensitive to ethylene than ‘Samantha’; and the changes of Rh-ACS3 expression caused by ethylene might be related to the acceleration of flower opening in ‘Samantha’ and the inhibition in ‘Kardinal’. Additional results indicated that three Rh-ACSs genes were differentially associated with flower opening and senescence as well as wounding.     

Genetics of the peroxidase isoenzymes in Petunia

Summary Three electrophoretic variants of the peroxidase b isoenzymes in Petunia have been found. The encoding gene prxB is shown to be located on chromosome I by its linkage with the gene Hfl. Analysis of prxB heterozygotes showed a gradual increase of the electrophoretic mobility of all three PRXb allozymes during development and differential expression in enzyme activity of three prxB alleles. The location of prxB on chromosome I was confirmed by an allelic dosage effect in trisomies I, trisomie segregation and the construction of trisomies I with triple-banded PRXb phenotype. From telotrisomic analysis it was concluded that prxB and Hfl are located on the same arm of chromosome I. The unexpected linkage of prxB and Hfl with the gene Fl in one of the crosses was suggested to be caused by a translocation in line SI, involving the gene Fl.     

Sporophyte-gametophyte interactions between anther and male gametophyte in petunia

Sporophyte-gametophyte interactions between anther and male gametophyte were investigated in two (fertile and sterile) clones of petunia (Petunia hybrida L.) with different reproductive strategies. Structural and functional reorganization of sporophyte tissues in the developing anther of fertile clone is closely coordinated with each of the successive stages of male gametophyte development (from meiosis to the formation of binuclear pollen) and comprises not only destruction of tapetum and three middle layers of the wall but also an activation of gas exchange and a rise in the content of sugars (sucrose, fructose, and glucose). In sterile clone, degradation of tapetum and anomalies in the development of sporogenic tissue were simultaneously observed in the prophase of meiosis. The death of microsporocytes and degeneration of tapetum were accompanied by a decrease in the level of sucrose delivered to the anther tissues and changes in the ratio between sucrose and hexoses in favor of glucose.