The ram1 Mutant of Arabidopsis Exhibits Severely Decreased β-Amylase Activity

Abstract

Despite extensive biochemical analyses, the biological function(s) of plant ␤-amylases remains unclear. The fact that ␤-amylases degrade starch in vitro suggests that they may play a role in starch metabolism in vivo. ␤-Amylases have also been suggested to prevent the accumulation of highly polymerized polysaccharides that might otherwise impede flux through phloem sieve pores. The identification and characterization of a mutant of Arabidopsis var. Columbia with greatly reduced levels of ␤-amylase activity is reported here. The reduced ␤-amylase 1 (ram1) mutation lies in the gene encoding the major form of ␤-amylase in Arabidopsis. Although the Arabidopsis genome contains nine known or putative ␤-amylase genes, the fact that the ram1 mutation results in almost complete loss of ␤-amylase activity in rosette leaves and inflorescences (stems) indicates that the gene affected by the ram1 mutation is responsible for most of the ␤-amylase activity present in these tissues. The leaves of ram1 plants accumulate wild-type levels of starch, soluble sugars, anthocyanin, and chlorophyll. Plants carrying the ram1 mutation also exhibit wild-type rates of phloem exudation and of overall growth. These results suggest that little to no ␤-amylase activity is required to maintain normal starch levels, rates of phloem exudation, and overall plant growth.␤-Amylases are found in vegetative tissues as well as in storage and reproductive organs, such as tubers and seeds. ␤-Amylases hydrolyze ␣-1,4-glucosidic linkages from the reducing ends of polysaccharide chains to form maltose (Beck and Ziegler, 1989). The genomes of many plant species contain several genes that encode ␤-amylases. For example, according to the database maintained by The Institute for Genomic Research (TIGR), the Arabidopsis genome includes nine genes that are identified as ␤-amylase or putative ␤-amylase genes (http://www.tigr.org/ tdb/edb2/ath1/htmls/GeneNameSearch.html). Despite the fact that they have been well characterized in vitro, the in vivo biological function(s) of ␤-amylases remains under debate (Ziegler, 1999).Several functions have been proposed for ␤-amylases in vivo. The fact that ␤-amylases hydrolyze starch in vitro (Beck and Ziegler, 1989) suggests a role for ␤-amylases in starch degradation. In addition, immunolocalization studies have shown an apparent association between ␤-amylases and starch granules (Okamoto and Akazawa, 1979;Hagenimana et al., 1992). However, these studies were conducted using polyclonal antibodies that may cross-react with starch phosphorylase. Consequently, the results of these studies should be interpreted with caution (Wang et al., 1995). In addition, starch is localized to plastids, whereas most ␤-amylases lack chloroplast transit peptides (Kreis et al., 1987;Monroe et al., 1991;Yoshida and Nakamura, 1991). Although chloroplast-localized ␤-amylases have been identified (Lao et al., 1999), the majority of ␤-amylase activity is extrachloroplastic (Beck and Ziegler, 1989;Nakamura et al., 1991). For example, 80% of the amylas...

The glabra1 Mutation Affects Cuticle Formation and Plant Responses to Microbes

“…A cursory database search identified over 200 articles in which the gl1 background has been used to study molecular, genetic, and biochemical aspects of diverse aspects of plant physiology (Liu and Zhu, 1997;Aarts et al, 1998;Bender and Fink, 1998;Cecchini et al, 1998;Xie et al, 1998;Yi and Jack, 1998;Bieza and Lois, 2001;Ellis and Turner, 2001;Laby et al, 2001;Devoto et al, 2002;He et al, 2002;Collins et al, 2003;Quiel and Bender, 2003;Barkan et al, 2006;Zhu et al, 2007;Speth et al, 2009;Jung and Niyogi, 2010;Rowe et al, 2010). Notably, many of the mutants that were isolated or crossed into the gl1 background have been studied for the defense physiologies.…”

Mutational Analysis of the Pullulanase-Type Debranching Enzyme of Maize Indicates Multiple Functions in Starch Metabolism

“…Examples from Arabidopsis are sex4 , dpe1 , and sex1 , which affect, respectively, an amylase (Zeeman et al, 1998a), a D enzyme (Critchley et al, 2001), and the R1 homolog, a dikinase that is speculated to control the rate of starch degradation (Yu et al, 2001;Ritte et al, 2002). Mutations in other starch degradative enzymes (e.g., Arabidopsis ram1 , which codes for the major form of ␤ -amylase) have no noticeable effect on starch synthesis or degradation (Laby et al, 2001). To date, a mutation affecting pullulanase-type DBE has not been identified in starch excess screens (or any other screens), so clues to its function are not available from genetic analysis.…”

Natural Variation for Carbohydrate Content in Arabidopsis. Interaction with Complex Traits Dissected by Quantitative Genetics

“…An interesting candidate gene could be a cytosolic b-amylase (At4g15210), which is expressed in shoots at the vegetative stage and is inducible by sugar. However, Ram1 mutants defective in this gene contain wild-type levels of soluble sugars and starch (Laby et al, 2001). It is remarkable that a starch phosphatase (At3g46970) is encoded in the candidate region for ST3.4.…”