Determination of Quercetin and Flavonol Synthase in Boesenbergia rotunda Rhizome.

BACKGROUND AND OBJECTIVE: Flavonols in plants are catalyzed by flavonol synthase (FLS) enzyme. FLS was reported expressed in flowers and fruits, i.e., Dianthus caryophyllus L. (Caryophyllaceae), Petunia hybrida Hort. (Solanaceae), Arabidopsis thaliana L. (Brassicaceae), Citrus unshiu Marc. (Rutaceae). However, none reported about FLS in medicinal plants, particularly those which possess anti-inflammatory activity. This study was aimed to extract and identify FLS in the rhizome of Boesenbergia rotunda (Zingiberaceae) and to determine quercetin in the ethanol extract of the rhizome. MATERIALS AND METHODS: The protein extraction of the rhizome was carried out by employing Laing and Christeller's (2004) and Wang's (2014) methods. The extracted-proteins were separated by using SDS-PAGE, followed by the measurement of FLS intensity by using Gel Analyzer. The FLS-1 of recombinant A. thaliana was employed as the standard. The determination of quercetin in the rhizome was carried out using LC-MS. RESULTS: The FLS occurred as a thick band at 38 kDa with intensity 116-158. The LC chromatogram of the extract indicated a small peak at 7.94 min similar to that of quercetin standard. The MS spectra at 7.94 min indicated that quercetin is present in the B. rotunda rhizome (m/z = 303.0549). The concentration of quercetin in the extract is 0.022% w/v. CONCLUSION: The FLS, an enzyme which plays an important role in producing quercetin, was detected in B. rotunda rhizome planted in Indonesia. As a consequence, quercetin in a small amount, was also quantified in the rhizome of this plant. This report will add a scientific insight of B. rotunda for biological sciences.

CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata.

KEY MESSAGE: Function of Petunia PiSSK1. Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S 2-haplotype and S 3-haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCFSLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1 and examined the SI behavior of a T 0 plant (S 2 S 3) with biallelic mutations in the pollen genome and two progeny plants (S 2 S 2) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise-compatible S-genotypes, but fully compatible with pistils of an S 3 S 3 transgenic plant in which production of S3-RNase was completely suppressed by an antisense S 3-RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.

UGT79B31 is responsible for the final modification step of pollen-specific flavonoid biosynthesis in Petunia hybrida.

MAIN CONCLUSION: UGT79B31 encodes flavonol 3- O -glycoside: 2″- O -glucosyltransferase, an enzyme responsible for the terminal modification of pollen-specific flavonols in Petunia hybrida. Flavonoids are known to be involved in pollen fertility in petunia (P. hybrida) and maize (Zea mays). As a first step toward elucidating the role of flavonoids in pollen, we have identified a glycosyltransferase that is responsible for the terminal modification of petunia pollen-specific flavonoids. An in silico search of the petunia transcriptome database revealed four candidate UDP-glycosyltransferase (UGT) genes. UGT79B31 was selected for further analyses based on a correlation between the accumulation pattern of flavonol glycosides in various tissues and organs and the expression profiles of the candidate genes. Arabidopsis ugt79b6 mutants that lacked kaempferol/quercetin 3-O-glucosyl(1 â†’ 2)glucosides, were complemented by transformation with UGT79B31 cDNA under the control of Arabidopsis UGT79B6 promoter, showing that UGT79B31 functions as a flavonol 3-O-glucoside: 2″-O-glucosyltransferase in planta. Recombinant UGT79B31 protein can convert kaempferol 3-O-galactoside/glucoside to kaempferol 3-O-glucosyl(1 â†’ 2)galactoside/glucoside. UGT79B31 prefers flavonol 3-O-galactosides to the 3-O-glucosides and rarely accepted the 3-O-diglycosides as sugar acceptors. UDP-glucose was the preferred sugar donor for UGT79B31. These results indicated that UGT79B31 encodes a flavonoid 3-O-glycoside: 2″-O-glucosyltransferase. Transient expression of UGT79B31 fused to green fluorescent protein (GFP) in Nicotiana benthamiana showed that UGT79B31 protein was localized in the cytosol.

Simultaneous knock-down of six ß-galactosidase genes in petunia petals prevents loss of pectic galactan but decreases petal strength.

Galactose (Gal) is incorporated into cell wall polysaccharides as flowers open, but then is lost because of ß-galactosidase activity as flowers mature and wilt. The significance of this for flower physiology resides in the role of galactan-containing polysaccharides in the cell wall, which is still largely unresolved. To investigate this, transcript accumulation of six cell wall-associated ß-galactosidases was simultaneously knocked down in 'Mitchell' petunia (Petunia axillaris x (P. axillaris x P. hybrida)) flower petals. The multi-PhBGAL RNAi construct targeted three bud- and three senescence-associated ß-galactosidase genes. The petals of the most down-regulated line (GA19) were significantly disrupted in galactose turnover during flower opening, and at the onset of senescence had retained 86% of their galactose compared with 20% in the controls. The Gal content of Na2CO3-soluble cell wall extracts and the highly insoluble polysaccharides associated with cellulose were particularly affected. Immunodetection with the antibody LM5 showed that much of the cell wall Gal in GA19 was retained as galactan, presumably the side-chains of rhamnogalacturonan-I. The flowers of GA19, despite having retained substantially more galactan, were no different from controls in their internal cell arrangement, dimensions, weight or timing of opening and senescence. However, the GA19 petals had less petal integrity (as judged by force required to cause petal fracture) after opening and showed a greater decline in this integrity with time than controls, raising the possibility that galactan loss is a mechanism for helping to maintain petal tissue cohesion after flower opening.

The acyl-activating enzyme PhAAE13 is an alternative enzymatic source of precursors for anthocyanin biosynthesis in petunia flowers.

Anthocyanins, a class of flavonoids, are responsible for the orange to blue coloration of flowers and act as visual attractors to aid pollination and seed dispersal. Malonyl-CoA is the precursor for the formation of flavonoids and anthocyanins. Previous studies have suggested that malonyl-CoA is formed almost exclusively by acetyl-CoA carboxylase, which catalyzes the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate. In the present study, the full-length cDNA of Petunia hybrida acyl-activating enzyme 13 (PhAAE13), a member of clade VII of the AAE superfamily that encodes malonyl-CoA synthetase, was isolated. The expression of PhAAE13 was highest in corollas and was down-regulated by ethylene. Virus-induced gene silencing of petunia PhAAE13 significantly reduced anthocyanin accumulation, fatty acid content, and cuticular wax components content, and increased malonic acid content in flowers. The silencing of PhAAE3 and PhAAE14, the other two genes in clade VII of the AAE superfamily, did not change the anthocyanin content in petunia flowers. This study provides strong evidence indicating that PhAAE13, among clade VII of the AAE superfamily, is specifically involved in anthocyanin biosynthesis in petunia flowers.

Transcriptome analysis reveals the same 17 S-locus F-box genes in two haplotypes of the self-incompatibility locus of Petunia inflata.

Petunia possesses self-incompatibility, by which pistils reject self-pollen but accept non-self-pollen for fertilization. Self-/non-self-recognition between pollen and pistil is regulated by the pistil-specific S-RNase gene and by multiple pollen-specific S-locus F-box (SLF) genes. To date, 10 SLF genes have been identified by various methods, and seven have been shown to be involved in pollen specificity. For a given S-haplotype, each SLF interacts with a subset of its non-self S-RNases, and an as yet unknown number of SLFs are thought to collectively mediate ubiquitination and degradation of all non-self S-RNases to allow cross-compatible pollination. To identify a complete suite of SLF genes of P. inflata, we used a de novo RNA-seq approach to analyze the pollen transcriptomes of S2-haplotype and S3-haplotype, as well as the leaf transcriptome of the S3S3 genotype. We searched for genes that fit several criteria established from the properties of the known SLF genes and identified the same seven new SLF genes in S2-haplotype and S3-haplotype, suggesting that a total of 17 SLF genes constitute pollen specificity in each S-haplotype. This finding lays the foundation for understanding how multiple SLF genes evolved and the biochemical basis for differential interactions between SLF proteins and S-RNases.

Transgenic potato plants with overexpression of dihydroflavonol reductase can serve as efficient nutrition sources.

Potato (Solanum tuberosum) is considered to be one of the most important crops cultivated in Europe and the entire world. The tubers of the potato are characterized by rich starch and protein contents and high concentrations of antioxidants, such as vitamin C and flavonoids. Notably, the presence of the phenolic antioxidants is of high importance as they have health-related properties. They are known to reduce the incidence of atherosclerosis, prevent certain kinds of cancer, and aid with many other kinds of diseases. The aim of this study was to find the most efficient way to increase the content of phenolic antioxidants in potato tubers through transgenesis. The results showed that the most efficacious way to achieve this goal was the overexpression of the dihydroflavonol reductase gene (DFR). The produced transgenic potato plants served as a nutrition source for laboratory rats; the study has confirmed their nontoxicity and nutritional benefits on the tested animals.

Production of 7-O-methyl aromadendrin, a medicinally valuable flavonoid, in Escherichia coli.

7-O-Methyl aromadendrin (7-OMA) is an aglycone moiety of one of the important flavonoid-glycosides found in several plants, such as Populus alba and Eucalyptus maculata, with various medicinal applications. To produce such valuable natural flavonoids in large quantity, an Escherichia coli cell factory has been developed to employ various plant biosynthetic pathways. Here, we report the generation of 7-OMA from its precursor, p-coumaric acid, in E. coli for the first time. Primarily, naringenin (NRN) (flavanone) synthesis was achieved by feeding p-coumaric acid and reconstructing the plant biosynthetic pathway by introducing the following structural genes: 4-coumarate-coenzyme A (CoA) ligase from Petroselinum crispum, chalcone synthase from Petunia hybrida, and chalcone isomerase from Medicago sativa. In order to increase the availability of malonyl-CoA, a critical precursor of 7-OMA, genes for the acyl-CoA carboxylase α and ß subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinica were also introduced. Thus, produced NRN was hydroxylated at position 3 by flavanone-3-hydroxylase from Arabidopsis thaliana, which was further methylated at position 7 to produce 7-OMA in the presence of 7-O-methyltransferase from Streptomyces avermitilis. Dihydrokaempferol (DHK) (aromadendrin) and sakuranetin (SKN) were produced as intermediate products. Overexpression of the genes for flavanone biosynthesis and modification pathways, along with malonyl-CoA overproduction in E. coli, produced 2.7 mg/liter (8.9 µM) 7-OMA upon supplementation with 500 µM p-coumaric acid in 24 h, whereas the strain expressing only the flavanone modification enzymes yielded 30 mg/liter (99.2 µM) 7-OMA from 500 µM NRN in 24 h.

S-RNase-based self-incompatibility in Petunia inflata.

BACKGROUND: For the Solanaceae-type self-incompatibility, also possessed by Rosaceae and Plantaginaceae, the specificity of self/non-self interactions between pollen and pistil is controlled by two polymorphic genes at the S-locus: the S-locus F-box gene (SLF or SFB) controls pollen specificity and the S-RNase gene controls pistil specificity. SCOPE: This review focuses on the work from the authors' laboratory using Petunia inflata (Solanaceae) as a model. Here, recent results on the identification and functional studies of S-RNase and SLF are summarized and a protein-degradation model is proposed to explain the biochemical mechanism for specific rejection of self-pollen tubes by the pistil. CONCLUSIONS: The protein-degradation model invokes specific degradation of non-self S-RNases in the pollen tube mediated by an SLF, and can explain compatible versus incompatible pollination and the phenomenon of competitive interaction, where SI breaks down in pollen carrying two different S-alleles. In Solanaceae, Plantaginaceae and subfamily Maloideae of Rosaceae, there also exist multiple S-locus-linked SLF/SFB-like genes that potentially function as the pollen S-gene. To date, only three such genes, all in P. inflata, have been examined, and they do not function as the pollen S-gene in the S-genotype backgrounds tested. Interestingly, subfamily Prunoideae of Rosaceae appears to possess only a single SLF/SFB gene, and competitive interaction, observed in Solanaceae, Plantaginaceae and subfamily Maloideae, has not been observed. Thus, although the cytotoxic function of S-RNase is an integral part of SI in Solanaceae, Plantaginaceae and Rosaceae, the function of SLF/SFB may have diverged. This highlights the complexity of the S-RNase-based SI mechanism. The review concludes by discussing some key experiments that will further advance our understanding of this self/non-self discrimination mechanism.

Ectopic expression of S-RNase of Petunia inflata in pollen results in its sequestration and non-cytotoxic function.

The specificity of S-RNase-based self-incompatibility (SI) is controlled by two S-locus genes, the pistil S-RNase gene and the pollen S-locus-F-box gene. S-RNase is synthesized in the transmitting cell; its signal peptide is cleaved off during secretion into the transmitting tract; and the mature "S-RNase", the subject of this study, is taken up by growing pollen tubes via an as-yet unknown mechanism. Upon uptake, S-RNase is sequestered in a vacuolar compartment in both non-self (compatible) and self (incompatible) pollen tubes, and the subsequent disruption of this compartment in incompatible pollen tubes correlates with the onset of the SI response. How the S-RNase-containing compartment is specifically disrupted in incompatible pollen tubes, however, is unknown. Here, we circumvented the uptake step of S-RNase by directly expressing S(2)-RNase, S(3)-RNase and non-glycosylated S(3)-RNase of Petunia inflata, with green fluorescent protein (GFP) fused at the C-terminus of each protein, in self (incompatible) and non-self (compatible) pollen of transgenic plants. We found that none of these ectopically expressed S-RNases affected the viability or the SI behavior of their self or non-self-pollen/pollen tubes. Based on GFP fluorescence of in vitro-germinated pollen tubes, all were sequestered in both self and non-self-pollen tubes. Moreover, the S-RNase-containing compartment was dynamic in living pollen tubes, with movement dependent on the actin-myosin-based molecular motor system. All these results suggest that glycosylation is not required for sequestration of S-RNase expressed in pollen tubes, and that the cytosol of pollen is the site of the cytotoxic action of S-RNase in SI.

A plant thiolase involved in benzoic acid biosynthesis and volatile benzenoid production.

The exact biosynthetic pathways leading to benzoic acid (BA) formation in plants are not known, but labeling experiments indicate the contribution of both beta-oxidative and non-beta-oxidative pathways. In Petunia hybrida BA is a key precursor for the production of volatile benzenoids by its flowers. Using functional genomics, we identified a 3-ketoacyl-CoA thiolase, PhKAT1, which is involved in the benzenoid biosynthetic pathway and the production of BA. PhKAT1 is localised in the peroxisomes, where it is important for the formation of benzoyl-CoA-related compounds. Silencing of PhKAT1 resulted in a major reduction in BA and benzenoid formation, leaving the production of other phenylpropanoid-related volatiles unaffected. During the night, when volatile benzenoid production is highest, it is largely the beta-oxidative pathway that contributes to the formation of BA and benzenoids. Our studies add the benzenoid biosynthetic pathway to the list of pathways in which 3-ketoacyl-CoA thiolases are involved in plants.

RNase-based gametophytic self-incompatibility evolution: Questioning the hypothesis of multiple independent recruitments of the S-pollen gene.

Multiple independent recruitments of the S-pollen component (always an F-box gene) during RNase-based gametophytic self-incompatibility evolution have recently been suggested. Therefore, different mechanisms could be used to achieve the rejection of incompatible pollen in different plant families. This hypothesis is, however, mainly based on the interpretation of phylogenetic analyses, using a small number of divergent nucleotide sequences. In this work we show, based on a large collection of F-box S-like sequences, that the inferred relationship of F-box S-pollen and F-box S-like sequences is dependent on the sequence alignment software and phylogenetic method used. Thus, at present, it is not possible to address the phylogenetic relationship of F-box S-pollen and S-like sequences from different plant families. In Petunia and Malus/Pyrus the putative S-pollen gene(s) show(s) variability patterns different than expected for an S-pollen gene, raising the question of false identification. Here we show that in Petunia, the unexpected features of the putative S-pollen gene are not incompatible with this gene's being the S-pollen gene. On the other hand, it is very unlikely that the Pyrus SFBB-gamma gene is involved in specificity determination.

Petunia phospholipase c1 is involved in pollen tube growth.

Although pollen tube growth is essential for plant fertilization and reproductive success, the regulators of the actin-related growth machinery and the cytosolic Ca2+ gradient thought to determine how these cells elongate remain poorly defined. Phospholipases, their substrates, and their phospholipid turnover products have been proposed as such regulators; however, the relevant phospholipase(s) have not been characterized. Therefore, we cloned cDNA for a pollen-expressed phosphatidylinositol 4,5-bisphosphate (PtdInsP2)-cleaving phospholipase C (PLC) from Petunia inflata, named Pet PLC1. Expressing a catalytically inactive form of Pet PLC1 in pollen tubes caused expansion of the apical Ca2+ gradient, disruption of the organization of the actin cytoskeleton, and delocalization of growth at the tube tip. These phenotypes were suppressed by depolymerizing actin with low concentrations of latrunculin B, suggesting that a critical site of action of Pet PLC1 is in regulating actin structure at the growing tip. A green fluorescent protein (GFP) fusion to Pet PLC1 caused enrichment in regions of the apical plasma membrane not undergoing rapid expansion, whereas a GFP fusion to the PtdInsP2 binding domain of mammalian PLC delta1 caused enrichment in apical regions depleted in PLC. Thus, Pet PLC1 appears to be involved in the machinery that restricts growth to the very apex of the elongating pollen tube, likely through its regulatory action on PtdInsP2 distribution within the cell.

Upregulation of a tonoplast-localized cytochrome P450 during petal senescence in Petunia inflata.

BACKGROUND: Gene expression in Petunia inflata petals undergoes major changes following compatible pollination. Severe flower wilting occurs reproducibly within 36 hours, providing an excellent model for investigation of petal senescence and programmed cell death. Expression of a number of genes and various enzyme activities involved in the degradation and remobilization of macromolecules have been found to be upregulated during the early stages of petal senescence. RESULTS: By performing differential display of cDNAs during Petunia inflata petal senescence, a highly upregulated gene encoding a cytochrome P450 was identified. Analysis of the complete cDNA sequence revealed that the predicted protein is a member of the CYP74C family (CYP74C9) and is highly similar to a tomato CYP74C allene oxide synthase (AOS) that is known to be active on 9-hydroperoxides. Cloning of the petunia genomic DNA revealed an intronless gene with a promoter region that carries signals found in stress-responsive genes and potential binding sites for Myb transcription factors. Transcripts were present at detectable levels in root and stem, but were 40 times more abundant in flowers 36 hours after pollination. Ethylene and jasmonate treatment resulted in transitory increases in expression in detached flowers. A protein fusion of the CYP74C coding region to a C-terminal GFP was found to be located in the tonoplast. CONCLUSION: Though oxylipins, particularly jasmonates, are known to be involved in stress responses, the role of other products of CYP74 enzymes is less well understood. The identification of a CYP74C family member as a highly upregulated gene during petal senescence suggests that additional products of fatty acid metabolism may play important roles during programmed cell death. In contrast to the chloroplast localization of AOS proteins in the CYP74A subfamily, GFP fusion data indicates that the petunia CYP74C9 enzyme is in the tonoplast. This result suggests that the highly similar CYP74C enzymes that have been identified in two other Solanaceous plants may also be associated with the vacuole, an organelle known to have a prominent role in programmed cell death.

Modification of cytokinin levels in potato via expression of the Petunia hybrida Sho gene.

The Sho gene from Petunia hybrida encodes an enzyme for cytokinin synthesis. Here we report on the effects of Shogene expression on potato development. In contrast to transgenic potato expressing the Agrobacterium ipt gene, moderate Sho expression resulted in sufficient root development that allowed the cultivation of the Sho transformants in soil. The most pronounced effects detectable in these lines were an enhanced shoot production, delayed tuber formation, significant reduction in tuber size, and inhibition of tuber dormancy. Sho expression predominantly associated with a strong increase in 2iP glucosides, accompanied by an increase in zeatin glucosides in lines with very high Sho expression levels. The data demonstrate that it is possible to produce viable plants with enhanced cytokinin levels via constitutive Sho expression, which allows an assessment of cytokinin effects in all organs.

Pyruvate decarboxylase provides growing pollen tubes with a competitive advantage in petunia.

Rapid pollen tube growth places unique demands on energy production and biosynthetic capacity. The aim of this work is to understand how primary metabolism meets the demands of such rapid growth. Aerobically grown pollen produce ethanol in large quantities. The ethanolic fermentation pathway consists of two committed enzymes: pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). Because adh mutations do not affect male gametophyte function, the obvious question is why pollen synthesize an abundant enzyme if they could do just as well without. Using transposon tagging in Petunia hybrida, we isolated a null mutant in pollen-specific Pdc2. Growth of the mutant pollen tubes through the style is reduced, and the mutant allele shows reduced transmission through the male, when in competition with wild-type pollen. We propose that not ADH but rather PDC is the critical enzyme in a novel, pollen-specific pathway. This pathway serves to bypass pyruvate dehydrogenase enzymes and thereby maintain biosynthetic capacity and energy production under the unique conditions prevailing during pollen-pistil interaction.

Selective recruitment of Adh genes for distinct enzymatic functions in Petunia hybrida.

Alcohol dehydrogenase (ADH) activity in plants is generally associated with glycolytic fermentation, which facilitates cell survival during episodes of low-oxygen stress in water-logged roots as well as chronically hypoxic regions surrounding the vascular core. Work with tobacco and potato has implicated ADH activity in additional metabolic roles, including aerobic fermentation, acetaldehyde detoxification and carbon reutilization. Here a combination of approaches has been used to examine tissue-specific patterns of Adh gene expression in order to provide insight into the potential roles of alcohol dehydrogenases, using Petunia hybrida, a solanaceous species with well-characterized genetics. A reporter-gene study, relying on the promoters of Adh1 and Adh2 to drive expression of the gene for a green fluorescent protein derivative, mgfp5, revealed unexpectedly complex patterns of GFP fluorescence in floral tissues, particularly the stigma, style and nectary. Results of GC-MS analysis suggest the association of ADH with production of aromatic compounds in the nectary. Overall the results demonstrate selective recruitment of Adh gene family members in tissues and organs associated with diverse ADH functions.

A Rho family GTPase controls actin dynamics and tip growth via two counteracting downstream pathways in pollen tubes.

Tip growth in neuronal cells, plant cells, and fungal hyphae is known to require tip-localized Rho GTPase, calcium, and filamentous actin (F-actin), but how they interact with each other is unclear. The pollen tube is an exciting model to study spatiotemporal regulation of tip growth and F-actin dynamics. An Arabidopsis thaliana Rho family GTPase, ROP1, controls pollen tube growth by regulating apical F-actin dynamics. This paper shows that ROP1 activates two counteracting pathways involving the direct targets of tip-localized ROP1: RIC3 and RIC4. RIC4 promotes F-actin assembly, whereas RIC3 activates Ca(2+) signaling that leads to F-actin disassembly. Overproduction or depletion of either RIC4 or RIC3 causes tip growth defects that are rescued by overproduction or depletion of RIC3 or RIC4, respectively. Thus, ROP1 controls actin dynamics and tip growth through a check and balance between the two pathways. The dual and antagonistic roles of this GTPase may provide a unifying mechanism by which Rho modulates various processes dependent on actin dynamics in eukaryotic cells.

Distinct genes produce the alcohol dehydrogenases of pollen and maternal tissues in Petunia hybrida.

Analysis of cDNA clones derived from hypoxic root mRNA of Petunia hybrida has revealed the existence of a third active gene encoding alcohol dehydrogenase in petunia. A combination of RT-PCR and ADH activity gels provide evidence for the selective tissue-specific expression of these three genes in multiple floral organs and hypoxically stressed roots. Expression of adh 1 in the plant appears to be restricted to immature pollen grains; the other two genes are expressed differentially in maternal anther tissues, stigma, petals, and hypoxic root. This work underscores the utility of RT-PCR for distinguishing expression patterns of closely related genes, clarifies the expression patterns exhibited by members of this gene family, and suggests multiple functions for the adh genes of petunia.