Isocitrate Lyase and Malate Synthase Genes from Brassica napus L. Are Active in Pollen

Abstract

To investigate if pollen possesses glyoxysomal function, we analyzed the activities of isocitrate lyase and malate synthase genes. Because the activities of these enzymes were exceedingly low in pollen extracts, we constructed fusion genes encoding j3-glucuronidase (CUS) that are regulated by isocitrate lyase or malate synthase promoters from Brassica napus L. to increase the sensitivity of our assays. Expression of the fusion genes in transgenic tobacco was qualitatively similar to that of the endogenous genes; CUS activity was low in dry seeds, maximal in seedlings, and very low or undetectable in leaves, indicating that the promoters are regulated correctly. We showed that isocitrate lyase and malate synthase genes are active at specific stages of pollen development and that their activities are not enhanced during pollen germination in transgenic tobacco. We also confirmed that the endogenous genes are active by showing that the corresponding mRNAs could be detected in pollen at specific stages of development. The activation of the isocitrate lyase and malate synthase genes suggests that glyoxysomal function is induced during pollen development.Glyoxysomes are specialized peroxisomes that play a pivotal role in the postgerminative growth of oilseed plants (Beevers, 1979;Huang et al., 1983;Trelease and Doman, 1984). These organelles contain the glyoxylate cycle and /3-oxidation enzymes that catalyze the net conversion of fatty acids into succinate and, thus, account for the ability of plants to utilize lipids as a carbon source for carbohydrate synthesis. The carbohydrates generated as a result of these reactions serve as nutrients for seedlings until they become photosynthetically active.Glyoxysomal function is induced at several stages of the plant life cycle when peroxisomes with different metabolic roles are converted into glyoxysomes. The induction of glyoxysomal function is generally monitored using two key glyoxylate cycle enzymes, isocitrate lyase and malate synthase, which are required for glyoxysomal function and are associated exclusively with glyoxysomes (reviewed by Olsen and

Dynamic Adaption of Metabolic Pathways during Germination and Growth of Lily Pollen Tubes after Inhibition of the Electron Transport Chain

“…Details of identified enzymes of specific metabolic pathways, e.g. isocitrate lyase for the glyoxylate cycle (Zhang et al, 1994) or an alcohol dehydrogenase active in ethanolic fermentation (Mellema et al, 2002), are given in Supplemental Table S2, with the corresponding sequences accessible via the Sequence Read Archive of the European Nucleotide Archive (http://www.ebi.ac.uk/ena/data/view/ ERP002303; accession no. ERP002303).…”

“…So far, one may postulate that the metabolism of pollen is similar to that of sporophytic tissue if marker enzymes of specific metabolic pathways were identified, e.g. isocitrate lyase and malate synthetase for glyoxysomal function (Zhang et al, 1994). But detailed studies may reveal that at least parts of the pollen metabolism might be different due to its specific physiological role as suggested by Colaço et al (2012).…”

The COMATOSE ATP-Binding Cassette Transporter Is Required for Full Fertility in Arabidopsis

“…There is some disagreement in the literature as to whether the glyoxylate cycle operates in growing pollen tubes, dependent on the species examined (Zhang et al, 1994;Mellema et al, 2002). However, examination of microarray data indicates that transcripts encoding the glyoxylate cycle enzymes isocitrate lyase and malate synthase are virtually absent from pollen and indeed also from other floral tissues in Arabidopsis (Zimmermann et al, 2004).…”

DNA Sequences That Activate Isocitrate Lyase Gene Expression during Late Embryogenesis and during Postgerminative Growth

“…Although there are several isocitrate lyase genes in Brassica napus L., we showed that at least one gene is active during seed development, postgerminative growth, and pollen maturation (Zhang et al, 1993(Zhang et al, , 1994. We also demonstrated that the 3.5 kb of DNA upstream of this gene contains sufficient information to specify its expression at each of these diverse developmental stages (Zhang et al, 1993(Zhang et al, , 1994. Thus, a single isocitrate lyase gene can be expressed at several stages of the life cycle.…”

“…One hundred microliters of 0.5 mM luciferin (Sigma, diluted from a 10 mM stock in 1 mM Tricine, pH 7.8) was injected into the reaction mix, and the amount of light emitted within the first 10 s after luciferin injection was recorded using a Moonlight 2010 luminometer (Analytical Luminescence, San Diego, CA). GUS assays were performed as described by Zhang et al (1994). …”