Hanan Itzhaki

Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene

Anthocyanins are the major pigments contributing to carnation flowercoloration. Most carnation varieties are sterile and hence molecular breedingis an attractive approach to creating novel colors in this commercially importantcrop. Characterization of anthocyanins in the flowers of the modern carnationcv. Eilat revealed that only the orange pelargonidin accumulates, due to a lackof both flavonoid 3′,5′-hydroxylase and flavonoid3′-hydroxylase activities. To modify flower color in cv. Eilat, we usedantisense suppression to block the expression of a gene encoding flavanone3-hydroxylase, a key step in the anthocyanin pathway. The transgenic plantsexhibited flower color modifications ranging from attenuation to complete lossof their original orange/reddish color. In the latter, only traces ofpelargonidin were detected. Dramatic suppression of flavanone 3-hydroxylaselevel/activity in these transgenes was confirmed by northern blot, RT-PCR andenzymatic assays. The new phenotype has been stable for over 4 years ofvegetative propagation. Moreover, transgenic plants with severe colormodification were more fragrant than control plants. GC-MS headspace analysesrevealed that transgenic anti-f3h flowers emit higherlevels of methyl benzoate. The possible interrelation between pathways leadingto anthocyanin and fragrance production is discussed.

Plant mitochondria contain proteolytic and regulatory subunits of the ATP-dependent Clp protease

The proteolytic machinery of plant organelles is largely unknown, although indications so far point to several proteases of bacterial origin. In this study an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX, a molecular chaperone and regulatory subunit of the ATP-dependent, serine-type Clp protease. Computer analysis of the predicted plant ClpX revealed a putative mitochondrial transit peptide at the N-terminus, as well as overall sequence similarity to other eukaryotic ClpX homologues. Specific polyclonal antibodies were made to the Arabidopsis ClpX protein and used to confirm its localization in plant mitochondria. In addition to ClpX, a ClpP protein located in mitochondria was also identified from the numerous ClpP isomers in Arabidopsis. Localization of this nuclear-encoded protein, termed ClpP2, was determined first by its close sequence similarity to mitochondrial ClpP human, and later experimentally using ClpP2-specific antibodies with isolated plant organellar fractions. In Arabidopsis, transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions, with no significant variation in mRNA level (i.e. 2-fold) for each gene between samples. Using β-casein as a substrate, plant mitochondria were found to possess an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease. The recent discovery of homologous mitochondrial ClpX and ClpP proteins in mammals suggests that this type of protease may be common to multicellular eukaryotes.

RAPD and RFLP markers tightly linked to the locus controlling carnation (Dianthus caryophyllus) flower type

Abstract Flower doubleness as a breeding characteristic is of major importance in carnation (Dianthus caryophyllus), one of the major cut-flowers sold worldwide, since flower architecture is of the utmost value in ornamentals. Based on the number of petals per flower, carnations are grouped into “single”, “semi-double” and “double” flower types. The first have five petals and are easily distinguishable, but of no economic value to the carnation industry. Flowers of standard and spray varieties, which constitute the largest market share, are usually of the double and semi-double type, respectively. These flower types are not easily distinguishable due to phenotypic overlaps caused by environmental conditions. To study the inheritance of this trait, several progeny segregating for flower type were prepared. Based on the number of single-flower type fullsibs among the offspring, we found that this phenotype is expressed only in plants homozygous for the recessive allele and that a dominant mutation in this allele causes an increase in petal number. Using random decamer primers, we identified a random amplified polymorphic DNA (RAPD) marker which is tightly linked to this recessive allele. The RAPD marker was cloned and used to generate a restriction fragment length polymorphic (RFLP) marker. This RFLP marker could discriminate with 100% accuracy between the semi-double and double- flower phenotypes in carnations of both Mediterranean and American groups. The advantages of RFLP over RAPD markers and their applicability to markerassisted selection in carnation are discussed.

CHRC, Encoding a Chromoplast-specific Carotenoid-associated Protein, Is an Early Gibberellic Acid-responsive Gene

CHRC, a corolla-specific carotenoid-associated protein, is a major component of carotenoid-lipoprotein complexes inCucumis sativus chromoplasts. Using an in vitroflower bud culture system that mimics in vivo flower development, CHRC mRNA levels in corollas were shown to be specifically up-regulated by gibberellic acid. The response to gibberellic acid was very rapid (within 20 min) and insensitive to protein synthesis inhibition by cycloheximide. Abscisic acid, known to antagonize gibberellin in many developmental systems, strongly down-regulated CHRC mRNA levels. The gibberellin synthesis inhibitor paclobutrazol exhibited a similar negative effect on CHRC expression. Inclusion of exogenous gibberellic acid into the in vitro bud culture system with the paclobutrazol not only prevented the CHRC mRNA down-regulation, it up-regulated transcript accumulation to the level of gibberellic acid-treated corollas. CHRC mRNA accumulation in response to gibberellic acid displayed a dose-dependent increase up to 10−4 m gibberellic acid. The up-regulation could be detected with as little as 10−7 m gibberellic acid. Based on these data, we suggest thatCHRC is the first structural gene identified to date whose expression is regulated by gibberellic acid in a primary fashion. The critical role of the rapid response of CHRC to gibberellic acid in aiding carotenoid sequestration while preserving chromoplast structural organization is discussed.

Generation of Transgenic Carnation Plants with Novel Characteristics by Combining Microprojectile Bombardment with Agrobacterium Tumefaciens Transformation

As one of the major contributors to the cut-flower market and a commercial leader in terms of number of stems sold worldwide (Jensen, Malter 1995), carnation (Dianthus caryophyllus L.) has been an important target for the breeding of new varieties with novel characteristics. Although new carnation varieties are continuously being produced through classical breeding, their high heterozygosity and limited gene pool, and a lack of knowledge regarding their genetic makeup, severely restrict such breeding programs (Woodson 1991). Thus, the possibility of genetically transforming carnation, as well as other cut-flower species, via direct gene transfer is quite attractive (reviewed by Zuker et al. 1998).

Phosphatidylcholine turnover during senescence of rose petals

Abstract Senescence of rose ( Rosa hyb ., cv. Mercedes) petals is accompanied by intensive modifications in membrane properties, where a decline in phospholipid content is a major change. Pulse-chase experiments with [ 14 C]choline chloride revealed that the decline in phosphatidylcholine (PC) with age is a result of a lower biosynthetic capacity of older petals and not of an increased degradation. The enzyme cytidine-diphospho-choline diacylglycerol phosphorylcholine phosphotransferase (CDP-cholinephosphotransferase, EC 2.7.8.2) was therefore studied in detail. This enzyme is involved in PC synthesis and its activity was demonstrated in microsomal fractions and found to be similar to that reported for other plant systems. The enzymes V max was higher in microsomes of young petals than of old ones. This, coupled with the reduction in protein content, resulted consequently in a decline in the total activity of the enzyme during the petal senescence. Modifications in the thiol groups of the membrane proteins resulted in changes in the enzyme activity. The activity of the enzyme was enhanced by the addition of egg lecithin and partially inhibited by cholesterol. In addition, it was altered by detergents known to influence the enzymes membranous microenvironment. Senescence of petals has previously been shown to be accompanied by a decrease in membrane lipid fluidity and protein thiol content. Therefore it is possible that the observed age-related changes in enzyme activity, which lead in turn to the overall reduction in petal phospholipid content, are a result of these membrane changes.

Substrate stimulation of an enzyme converting 1-aminocyclopropane-1-carboxylic acid to ethylene

Abstract The rate of conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, catalyzed by a solubilized membrane fraction, gradually increases with time up to a factor of 5 at 120 min. The effects of the substrate and the products of this reaction on the reaction rate were tested. Preincubation of the solubilized enzyme with ethylene had no effect on the reaction rate, while CO 2 increased it by 40% and CN − inhibited it by 65%. Pre-incubation of the enzyme with ACC significantly increased the reaction rate, in a manner resembling the observed time-dependent increase. Examination of the substrate-dependent activity over a wide range of concentrations reveals a sigmoidal curve. Analysis of the data by means of a Hill plot suggests that the enzyme converting ACC to ethylene has a K m of 4.1 mM for ACC, and possesses at least two catalytic subunits.

The Proteolytic Machinery of Chloroplasts: Homologues of Bacterial Proteases

In the past 10–15 years, many examples of non-specific and specific degradation of proteins in the chloroplast have accumulated [for review, see (1)]. These include degradation of photo- and oxidatively damaged proteins, proteins whose levels are regulated by different environmental conditions, unassembled proteins, and proteins lacking their prosthetic groups. However, the proteases involved in their degradation were largely unknown. As a first step toward identification of the proteases involved in these specific proteolytic processes, we identified and characterized several chloroplast proteases, all of which are homologues of bacterial proteases. In this paper, we provide an overview of these proteases, present recent related data, and discuss the implications of these on our understanding of proteolytic processes in chloroplasts.

Molecular Cloning and Characterization of ClpX, a Potential Regulator of Chloroplastic Clp Protease

Bacterial Clp protease is an ATP-dependent serine protease. It has a barrel-like structure, composed of two rings of 7 subunits of the proteolytic subunit ClpP, nested between two rings of hexamers of regulatory subunits, either ClpA or ClpX. The association between the proteolytic and the regulatory ATPase subunits requires binding of ATP, whereas degradation of proteins by this complex is dependent on ATP hydrolysis. The chloroplastic proteolytic subunit ClpP is encoded in the chloroplast genome (1, 2), but apparently it has also a nuclear-encoded, chloroplast-targeted homologue (3). The chloroplastic structural and functional homologue of the regulatory subunit ClpA, designated ClpC, is encoded in the nucleus and imported into the chloroplast (4, 5). ClpP and ClpC are localized to the stroma and expressed constitutively (6–8) Similar to the bacterial protease, the association between the two is dependent on ATP. When the regulatory subunit ClpC is immuno-precipitated with a specific antibody, the proteolytic subunit ClpP is also precipitated, but only in the presence of Mg·ATP or its non-hydrolyzable analog ATP-γ-S (9). Coimmunoprecipitated ClpC-P is proteolytically active, and can degrade mature 0E33 (9), which we previously demonstrated to be degraded when mistargeted to the stroma, by a soluble ATP-dependent serine protease (7).

Characterization of an Endogenous Inhibitor of Ethylene Biosynthesis in Carnation Petals

A membrane-bound enzymatic conversion of 1 — aminocyclopropane — 1 — carboxylic acid (ACC) to ethylene was recently demonstrated in carnation petals by Mayak et al. (1). They also reported the presence of a cytoplasmic inhibitor of this reaction. The aim of this work was to characterize this inhibitor.