Characterization of proteolytic activity during senescence in daylilies

From 12 to 24 h after the opening of daylily flowers ( Hemerocallis hybrid cv. Stella d'Oro), the petals begin to degrade and the protein levels of soluble, microsomal‐ and plastid‐enriched fractions decrease by 50%, on a per petal basis. To help determine some of the components for the cell death program in daylily petals, we studied the mechanisms that regulate this loss of protein. Enzyme activities capable of digesting native daylily protein, gelatin, and azocasein markedly increase after flower opening, and their appearance is inhibited by the translation inhibitor, cycloheximide. Protein hydrolysis in vitro is prevented by inhibitors of cysteine, serine and metalloproteinases. Immunoblots using antibodies to ubiquitin pathway enzymes indicate that the ubiquitin system is not senescence specific. However, ion leakage is delayed by two inhibitors of the 26S proteasome. We propose that programmed cell death in daylily petals may involve the increase in activity of at least three classes of proteinases, and discuss the possibility that these proteinases may operate in concert with the ubiquitin pathway.     

Activities of nucleases in senescing daylily petals

The activity of nucleases during organ death was investigated using daylily petals (Hemerocallis hybrid cv. Stella d’Oro), in which the processes associated with senescence are rapid and clearly ordered. The number of nuclei with fragmented DNA as well as activities of various nucleases increase before certain other events that are related to senescence. Furthermore, DNA breakage and activities of nucleases occur earlier when senescence is accelerated by abscisic acid and occur later when senescence is retarded by cycloheximide. These results suggest that the activities of nucleases contribute to the senescence of daylily petals. Therefore, studying the regulation of nuclease gene expression may be useful for understanding components of the signal transduction system that leads to the death of these organs.     

Possible involvement of abscisic acid in senescence of daylily petals

Daylily flowers (Hemerocallis hybrid, cv. Stella d'Oro) senesce and die autonomously over a 24 h period after opening. Investigations were performed to determine some of the mechanisms that lead to death of the petals. The flowers are insensitive to ethylene, but exogenous ABA prematurely upregulates events that occur during natural senescence, such as loss or differential membrane permeability, increases in lipid peroxidation and the induction of proteinase and RNase activities. Furthermore, the same patterns of proteinase and RNase activities appearing on activity gels during natural senescence are induced prematurely by ABA. The mRNA profile from ABA-treated, prematurely senescing petals visualized by differential display shows a high degree of similarity to the mRNA profile of naturally senescing petals 18 h later. In addition, endogenous ABA increases before flower opening and continues to increase during petal senescence. An osmotic stress by sorbitol increases endogenous levels of ABA and upregulates the same parameters of senescence as those occurring during natural senescence and after application of ABA. The mRNA profile from sorbitol-treated, prematurely senescing petals, but somewhat less similarity to mRNA from ABA-treated petals. The possibility is discussed that ABA is a constituent of the signal transduction chain leading to programmed cell death of daylily petals.     

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

Candida albicans contains 10 putative cytochrome P450 (CYP) genes coding for enzymes that appear to play important roles in fungal survival and virulence. Here, we report the characterization of CYP52A21, a putative alkane/fatty acid hydroxylase. The recombinant CYP52A21 protein containing a 6x(His)-tag was expressed in Escherichia coli and was purified. The purified protein, reconstituted with rat NADPH-cytochrome P450 reductase, omega-hydroxylated dodecanoic acid to give 12-hydroxydodecanoic acid, but to a lesser extent also catalyzed (omega-1)-hydroxylation to give 11-hydroxydodecanoic acid. When 12,12,12-d(3)-dodecanoic acid was used as the substrate, there was a major shift in the oxidation from the omega- to the (omega-1)-hydroxylated product. The regioselectivity of fatty acid hydroxylation was examined with the 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids. Although all three 12-halododecanoic acids bound to CYP52A21 with similar affinities, the production of 12-oxododecanoic acid decreased as the size of the terminal halide increased. The regioselectivity of CYP52A21 fatty acid oxidation is thus consistent with presentation of the terminal end of the fatty acid chain for oxidation via a narrow channel that limits access to other atoms of the fatty acid chain. This constricted access, in contrast to that proposed for the CYP4A family of enzymes, does not involve covalent binding of the heme to the protein.     

Development of leucine aminopeptidase activity during daylily flower growth and senescence

The possibility that exopeptidases, i.e. aminopeptidases and carboxypeptidases, in addition to the previously studied endopeptidase might also be developmentally regulated in daylily petals was examined. The level of leucine aminopeptidase and endopeptidase activities changed after the flower was fully open while that of carboxypeptidase activity remained relatively unchanged throughout senescence. Leucine aminopeptidase activity seemed to increase after the flower was fully open and peaked several hours earlier than endopeptidase did. Taken together, it is postulated that leucine aminopeptidase might play a role in protein turnover during flower opening and in the initiation of protein hydrolysis associated with petal senescence while the endopeptidase could be responsible for the breakdown of the bulk of proteins at the later stages. The drop in leucine aminopeptidase activity associated with the onset of daylily petal senescence was effectively halted by a cycloheximide treatment of cut daylily flowers for 24 h which was previously shown to prolong the vase life of the flowers and prevent protein loss from the petals. Apart from both being developmentally regulated in daylily petals, the leucine aminopeptidase activity and the previously studied endopeptidase are different in several aspects. They appear to have different pH optima, 8 for leucine aminopeptidase and 6.2 for endopeptidase. Unlike the endopeptidase activity, no new leucine aminopeptidase isozymes appeared during petal senescence, and the leucine aminopeptidase did not appear to belong to the cysteine class of proteolytic enzymes.     

Senescence-associated mRNAs that may participate in signal transduction and protein trafficking

The differential display technique was used to generate cDNA probes in order to identify mRNAs that are up-regulated during senescence of Arabidopsis leaves. Three mRNAs were examined that had not previously been associated with senescence. The steady-state levels of these mRNAs are detectable in small amounts in mature green leaves, but increase considerably as chlorophyll levels begin to decline. This relationship to senescence occurs under natural circumstances as well as when senescence is accelerated by leaf detachment in the dark or by addition of 1-aminocyclopropane-1-carboxylic acid (ACC). Retardation of senescence by benzyladenine slows the increase of the mRNAs. One of these mRNAs appears to code for a protein (Sec 13) that may be involved in vesicle formation at the endoplasmic reticulum. Another mRNA codes for a protein with WD‐repeat motif whose function is as yet unidentified, and the third codes for a putative calcium-dependent protein kinase. A fourth cDNA has also been cloned by subtractive hybridization from senescing Arabidopsis leaves that encodes vacuolar-processing enzyme ( γ VPE). Incubation of detached leaves in darkness also caused an abrupt elevation in the steady-state levels of the γVPE , similar to that of the senescing attached leaves. The possible functions of the gene products and their involvement in cellular and biochemical processes during senescence are discussed.     

Cloning and expression of a putative cytochrome P450 gene that influences the colour of Phalaenopsis flowers

Anthocyanins are responsible for reds through blues in flowers. Blue and violet flowers generally contain derivatives of delphinidin, whereas red and pink flowers contain derivatives of cyanidin or pelargonidin. Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes. Flavonoid-3',5'-hydroxylase, a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3',5'-hydroxylated anthocyanins, generally required for blue or purple flowers. Here we report on the isolation of a cDNA clone of a putative flavonoid-3',5'-hydroxylase gene from Phalaenopsis that was then cloned into a plant expression vector. Transient transformation was achieved by particle bombardment of Phalaenopsis petals. The transgenic petals changed from pink to magenta, indicating that the product of the putative flavonoid-3',5'-hydroxylase gene influences anthocyanin pigment synthesis.     

Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction

Two forms of cytochrome P-450 (P-450), designated P-450MP-1 and P-450MP-2, were purified to electrophoretic homogeneity from human liver microsomes on the basis of mephenytoin 4-hydroxylase activity. Purified P-450MP-1 and P-450MP-2 contained 12-17 nmol of P-450/mg of protein and had apparent monomeric molecular weights of 48,000 and 50,000, respectively. P-450MP-1 and P-450MP-2 were found to be very similar proteins as judged by chromatographic behavior on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE- and CM-cellulose columns, spectral properties, amino acid composition, peptide mapping, double immunodiffusion analysis, immunoinhibition, and N-terminal amino acid sequences. In vitro translation of liver RNA yielded polypeptides migrating with P-450MP-1 or P-450MP-2, depending upon which form was in each sample, indicating that the two P-450s are translated from different mRNAs. When reconsituted with NADPH-cytochrome-P-450 reductase and L-alpha-dilauroyl-sn-glyceryo-3-phosphocholine, P-450MP-1 and P-450MP-2 gave apparently higher turnover numbers for mephenytoin 4-hydroxylation than did the P-450 in the microsomes. The addition of purified rat or human cytochrome b5 to the reconstituted system caused a significant increase in the hydroxylation activity; the maximum stimulation was obtained when the molar ratio of cytochrome b5 to P-450 was 3-fold. Rabbit anti-human cytochrome b5 inhibited NADH-cytochrome-c reductase and S-mephenytoin 4-hydroxylase activities in human liver microsomes. In the presence of cytochrome b5, the Km value for S-mephenytoin was 1.25 mM with all five purified cytochrome P-450s preparations, and Vmax values were 0.8-1.25 nmol of 4-hydroxy product formed per min/nmol of P-450. P-450MP is a relatively selective P-450 form that metabolizes substituted hydantoins well. Reactions catalyzed by purified P-450MP-1 and P-450MP-2 preparations and inhibited by anti-P-450MP in human liver microsomes include S-mephenytoin 4-hydroxylation, S-nirvanol 4-hydroxylation, S-mephenytoin N-demethylation, and diphenylhydantoin 4-hydroxylation. Thus, at least two very similar forms of human P-450 are involved in S-mephenytoin 4-hydroxylation, an activity which shows genetic polymorphism.     

Fatty acid omega and (omega-1)-Hydroxylation in rabbit intestinal mucosa microsomes.

The microsomes from rabbit intestinal mucosa which had been washed quickly and thoroughly with phenylmethylsulfonyl fluoride were found to catalyze the hydroxylation of fatty acids in the presence of NADPH and molecular oxygen. Myristic and palmitic acids were converted to the corresponding omega-and (omega-1)-hydroxy fatty acids, whereas lauric acid was converted only to 12-hydroxylauric acid, and capric acid, to 9-and 10-hydroxycapric acids together with an unknown polar acid.Among these fatty acids, both myristic and lauric acids appeared to be the most efficient substrates. The inhibition of the hydroxylation by SKF 525-A and carbon monoxide suggested that the activity depended upon cytochrome P-450. The specific activity of the fatty acid hydroxylation was almost constant along the small intestine, while the aminopyrine N-demethylation activity and the cytochrome P-450 content were highest at the proximal end of the intestine and progressively declined toward the caudal end. The cytochrome P-450 was solubilized from the intestinal microsomes and purified by 6-amino-n-hexyl Sepharose 4B chromatography. The partially purified cytochrome P-450 was active in fatty acid hydroxylation in combination with intestinal NADPH-cytochrome c reductase and phosphatidylcholine.     

郁金香切花瓶插期的衰老与膜脂过氧化的关系

郁金香鲜切花在瓶插过程中,叶片可溶性蛋白质含量从瓶插开始即开始下降,花瓣在瓶插初期略有上升,第３天以后随花朵的开放迅速下降。叶片、花瓣的可溶性蛋白质含量的下降与细胞膜相对透性的增加呈负相关,与ＭＤＡ含量的增加呈负相关;而细胞膜相对透性的增加与ＭＤＡ含量的增加呈正相关。同时,饱和脂肪酸所占百分比增加,不饱和脂肪酸含量下降。ＩＵＦＡ的下降与ＭＤＡ含量的增加呈负相关。在整个瓶插过程中,花瓣的可溶性蛋白质     

Ethylene-induced gene expression in carnation petals : relationship to autocatalytic ethylene production and senescence

Exposure of carnation (Dianthus caryophyllus L.) flowers to ethylene evokes the developmental program of petal senescence. The temporal relationship of several aspects of this developmental program following treatment with ethylene was investigated. Exposure of mature, presenescent flowers to 7.5 microliters per liter ethylene for at least 6 hours induced petal in-rolling and premature senescence. Autocatalytic ethylene production was induced in petals following treatment with ethylene for 12 or more hours. A number of changes in mRNA populations were noted in response to ethylene, as determined by in vitro translation of petal polyadenylated RNA. At least 6 mRNAs accumulated following ethylene exposure. The molecular weights of their in vitro translation products were 81, 58, 42, 38, 35, and 25 kilodaltons. Significant increases in abundance of most mRNAs were observed 3 hours following ethylene exposure. Ethylene exposure resulted in decreased abundance of another group of mRNAs. Treatment of flowers with competitive inhibitors of ethylene action largely prevented the induction of these ethylene responses in petals. An increase in flower age was accompanied by an increase in the capacity for ethylene to induce petal in-rolling, autocatalytic ethylene production, and changes in mRNA populations suggesting that these responses are regulated by both sensitivity to ethylene and ethylene concentration. These results indicate that changes in petal physiology resulting from exposure to ethylene may be the result of rapid changes in gene expression.     

Isolation and characterization of a novel cytochrome P-450-like pseudogene

A rabbit liver P-450-like pseudogene has been isolated from a lambda phage genomic library. Sequence analysis revealed structural homology with respect to the rat P-450b and P-450e genes as well as a similar intron-exon organization. A 5'-proximal TATA box-like sequence and two 3'-distal putative polyadenylation signals were identified, and all putative intron-exon boundaries except at the 3'-splice site of intron 2 were found to follow the GT/AG rule. With allowance for apparent deletions and insertions, the structural homology of the amino acid sequence deduced from the pseudogene with respect to rabbit P-450 isozyme 2 is lower for exons 1 through 4 (18-28%) than for exons 5 through 9 (42-65%). S1 nuclease mapping showed that mRNAs complementary to the DNA sequence of exon 9 are expressed. However, due to the alterations in the pseudogene, it appears that functional P-450 would not be produced from such mRNAs.     

Sex-dependent expression and clofibrate inducibility of cytochrome P450 4A fatty acid omega-hydroxylases. Male specificity of liver and kidney CYP4A2 mRNA and tissue-specific regulation by growth hormone and testosterone.

The induction of liver cytochrome P450 4A-catalyzed fatty acid omega-hydroxylase activity by clofibrate and other peroxisome proliferators has been proposed to be causally linked to the ensuing proliferation of peroxisomes in rat liver. Since female rats are less responsive than males to peroxisome proliferation induced by clofibrate, the influence of gender and hormonal status on the basal and clofibrate-inducible expression of the 4A P450s was examined. Northern blot analysis using gene-specific oligonucleotide probes revealed that in the liver, P450 4A1 and 4A3 mRNAs are induced to a much greater extent in male as compared to female rats following clofibrate treatment, whereas P450 4A2 mRNA is altogether absent from female rat liver. Male-specific expression of P450 4A2 mRNA was also observed in kidney. Western blot analysis indicated that a similar sex dependence characterizes both the basal expression and the clofibrate inducibility of the corresponding P450 4A proteins. This suggests that the lower responsiveness of female rats to clofibrate-induced peroxisome proliferation may reflect the lower inducibility of the P450 4A fatty acid hydroxylase enzymes in this sex. Investigation of the contribution of pituitary-dependent hormones to the male-specific expression of 4A2 revealed that this P450 mRNA is fully suppressed in liver following exposure to the continuous plasma growth hormone profile that characterizes adult female rats; in this and other regards liver P450 4A2 is regulated in a manner that is similar, but not identical to, P450 3A2, a male-specific testosterone 6 beta-hydroxylase. In contrast, kidney 4A2 expression, although also male-specific, was not suppressed by continuous growth hormone treatment, but was regulated by pathways that, in part, involve testosterone as a positive regulator. The male-specific expression of liver and kidney P450 4A2 is thus under the control of distinct pituitary-dependent hormones acting in a tissue-specific manner.     

Intracellular energy depletion triggers programmed cell death during petal senescence in tulip

Programmed cell death (PCD) in petals provides a model system to study the molecular aspects of organ senescence. In this study, the very early triggering signal for PCD during the senescence process from young green buds to 14-d-old petals of Tulipa gesneriana was determined. The opening and closing movement of petals of intact plants increased for the first 3 d and then gradually decreased. DNA degradation and cytochrome c (Cyt c) release were clearly observed in 6-d-old flowers. Oxidative stress or ethylene production can be excluded as the early signal for petal PCD. In contrast, ATP was dramatically depleted after the first day of flower opening. Sucrose supplementation to cut flowers maintained their ATP levels and the movement ability for a longer time than in those kept in water. The onset of DNA degradation, Cyt c release, and petal senescence was also delayed by sucrose supplementation to cut flowers. These results suggest that intracellular energy depletion, rather than oxidative stress or ethylene production, may be the very early signal to trigger PCD in tulip petals.