Bruce A. Stermer

HMG-CoA reductase gene families that differentially accumulate transcripts in potato tubers are developmentally expressed in floral tissues

We isolated two full-length cDNA clones encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) from potato (Solanum tuberosum) L. tubers. The clones, designated hmg2.2 and hmg3.3, are members of previously described gene subfamilies. In addition to being induced by arachidonic acid in tubers, hmg2.2 transcript accumulates developmentally in young flowers, and in mature sepals and ovaries, whereas transcript for hmg3.3 accumulates in mature petals and anthers. Our data suggest that members of specific HMGR-encoding gene sub-families might be involved in both defense responses and flower development. Accumulation of different HMGR transcripts could provide some control of isoprenoid biosynthesis by producing isoforms specific for classes of end-products produced in particular tissues.

Features of the hmg 1 subfamily of genes encoding HMG-CoA reductase in potato

Abstract3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the β-glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R2 pollen of R1 progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.

Analysis of elicitor-inducible transcripts encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in potato

Abstract 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme of sesquiterpenoid phytoalexin biosynthesis in potato (Solanum tuberosum L.). HMGR cDNAs were selectively amplified by the polymerase chain reaction (PCR) from a first strand cDNA synthesized from the total RNA of tuber tissue exposed to the elicitor arachidonic acid. The identity of the cDNAs encoding HMGR was supported by a 69–73% nucleic acid similarity with Arabidopsis HMG1. DNA sequence analysis indicated PCR amplification of cDNAs representing transcripts from two different HMGR genes. The use of the potato HMGR cDNAs to probe Southern blots demonstrated the presence of a small HMGR gene family in potato, at least one member of which was activated to produce approx. 2·6 kilobase transcripts in response to arachidonic acid. We discuss these results in relation to the properties of potato HMGR and the induction kinetics of its activity in elicited tuber tissue.

Rapid changes in protein synthesis after application of arachidonic acid to potato tuber tissue

Abstract Early changes in protein synthesis were investigated in potato ( Solanum tuberosum L.) cv. Kennebec alter application of arachidonic acid (AA), an elicitor of sesquiterpenoid phytoalexin accumulation and other responses associated with hypersensitive resistance in potato. In vivo labelling of disks from potato tubers with [ 3 H]leucine showed that the rate of net protein synthesis in underlying tissues nearly doubled within 6 h of the application of AA. This is one of the earliest active responses to the elicitor known. Uptake of leucine was inhibited in the surface tissue during the first 9 h after treatment with AA but removal of the upper 1 mm of treated tissue immediately before application of leucine avoided the interference. Application of other polyunsaturated fatty acids to tuber disks also caused transient increases in protein synthesis of subsurface tissues. However, AA always induced the largest and most prolonged increases. These results clearly show that AA has marked effects on the protein metabolism of potato tubers soon after its application.

Induction of early mevalonate pathway enzymes and biosynthesis of end products in potato (Solanum tuberosum) tubers by wounding and elicitation

Abstract In plants, several important classes of terpenoid compounds are synthesized via the mevalonate pathway. In addition to essential constitutive metabolites, potato (Solanum tuberosum L.) tubers synthesize antifungal sesquiterpenoid phytoalexins in response to fungal infection or arachidonic acid elicitation, and toxic steroid glycoalkaloids in response to wounding. The activity of the early pathway enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has previously been shown to increase rapidly and then decrease in response to these stimuli. During an investigation of the possible post-translational control of this enzyme, it was found that the inclusion of the cysteine protease inhibitors leupeptin and E-64 { N-[N-( l -3-trans- carboxyran-2-carbonyl)- l -leucyl]agmatine } in the enzyme extraction buffer increased nine-fold the total HMGR activity recovered in the microsomal fraction and greatly increased the ratio of microsomal to soluble activity. Incubation of microsomal HMGR preparations with soluble protein extracts, Mg2+ and ATP caused an apparent inhibition of HMGR, consistent with published reports of post-translational inactivation of HMGR by phosphorylation. The apparent inhibition was completely reversed, however, by 5 mM mevalonate and was found to be an artefact caused by the presence of mevalonate kinase, the next enzyme in the pathway, in the soluble fraction. HPLC assays for mevalonate kinase and mevalonate phosphate kinase were developed and used to measure the activities of these enzymes following wounding and elicitation. While HMGR levels increased 30-fold following arachidonic acid treatment and 15-fold following wounding, mevalonate kinase and mevalonate phosphate kinase only increased two- to four-fold following these treatments, and the levels in arachidonic acid treated tubers were only 20–40% higher than in wounded tubers. While HMGR levels are extremely low in untreated tissues, the activities of the two kinases are ralatively high, suggesting that they do not serve as control points for the synthesis of terpenoids.

Transcriptional Regulation of Phytoalexin Biosynthetic Genes

In legumes, isoflavonoid derivatives function as antimicrobial phytoalexins, whereas phytoalexins of solanaceous species are of terpenoid origin. The phenylpropanoid and isoprenoid pathways leading to these phytoalexins are involved in the synthesis of a wide range of secondary metabolites with important functions in plant growth, development and responses to the environment. Elicitation of phytoalexin biosynthesis involves transcriptional activation of the genes encoding enzymes of general phenylpropanoid/terpenoid biosynthesis, and of the genes for the specific branch pathways leading to antimicrobial compounds. In order to understand the molecular controls determining the developmental and environmental regulation of the general and specific enzymes of phytoalexin synthesis, we are studying the promoter regions of three elicitor inducible genes, chalcone synthase (chs, isoflavonoid pathway, general), isoflavone reductase (ifr,isoflavonoid pathway, specific) and 3-hydroxy-3-methylglutaryl CoA reductase (hmgr,terpenoid pathway, general). We describe cis-elements and trans-factors involved in the expression of these genes in relation to their tissue specific expression and response to biotic stress. Two elements, the G-box and H-box, located within 50 bp of the TATA box, are important for regulation of expression of chs and probably hmgr, but are not present in the alfalfa ifr promoter.

Elicitors and Defense Gene Activation in Cultured Cells

Active defense of plants against fungal or bacterial pathogens often involves the rapid death of cells in intimate contact with the invading microorganism. Accompanying this so-called hypersensitive response (HR) are rapid localized changes in host metabolism which lead to the synthesis of potential defensive barriers; these include the accumulation of low Mr antimicrobial compounds termed phytoalexins, the deposition of phenolic material and hydroxyproline-rich glycoproteins (HRGPs) in the host cell wall, and the synthesis of hydrolytic enzymes. The exact relationship between the HR and phytoalexin accumulation is still somewhat vague; in some systems, hypersensitive cell death appears to be a pre-requisite for induction and accumulation of phytoalexins in neighboring healthy cells (Bailey 1982), whereas phytoalexin synthesis can be initiated in suspension cultured cells in the absence of serious effects on cell viability (Hamdan and Dixon 1986). It is, however, well documented that molecules from plant pathogenic fungi have the ability to induce both hypersensitive-type cell necrosis and/or phytoalexin accumulation in plant cells. These so-called elicitors often originate from the fungal cell wall.