Histochemical Analysis Reveals Organ-Specific Quantitative Trait Loci for Enzyme Activities in Arabidopsis

Abstract

To identify genetic loci involved in the regulation of organ-specific enzyme activities, a specific histochemical staining protocol was used in combination with quantitative trait locus (QTL) analysis. Using phosphoglucomutase (PGM) as an example, it is shown that enzyme activity can specifically, and with high resolution, be visualized in non-sectioned seedlings of Arabidopsis. The intensities of staining were converted to quantitative data and used as trait for QTL analysis using Landsberg erecta ϫ Cape Verde Islands recombinant inbred lines. Independently, PGM activities were quantified in whole-seedling extracts, and these data were also used for QTL analysis. On the basis of extract data, six significant (P Ͻ 0.05) loci affecting PGM activity were found. From the histochemical data, one or more specific QTLs were found for each organ analyzed (cotyledons, shoot apex, hypocotyl, root, root neck, root tip, and root hairs). Loci detected for PGM activity in extracts colocated with loci for histochemical staining. QTLs were found coinciding with positions of (putative) PGM genes but also at other positions, the latter ones supposedly pointing toward regulatory genes. Some of this type of loci were also organ specific. It is concluded that QTL analysis based on histochemical data is feasible and may reveal organ-specific loci involved in the regulation of metabolic pathways.Carbohydrates constitute the major part of plant biomass, and carbohydrate metabolism is one of the central biochemical pathways in plant cells. Hence understanding carbohydrate metabolism and its regulation is of crucial importance. Over the last few decades, most of the genes encoding the enzymes catalyzing the various steps of carbohydrate metabolic routes, have been unraveled. However, this does not automatically imply that the regulatory mechanisms of these routes have become clear as well. Carbohydrate metabolism as a whole, but also the individual steps of the various pathways, are likely to be controlled by a plethora of genes, encoding the enzymes and regulators at different levels.Quantitative trait locus (QTL) analysis is a powerful approach to identify genes involved in processes controlled by many genes when genetic variation for these genes is present. This has been shown in Arabidopsis for traits such as flowering time (Koornneef et al., 1998; Ungerer et al., 2002), nitrogen use efficiency (Loudet et al., 2003), and salt tolerance (Quesada et al., 2002). A few papers have shown the potential of this approach in genetic mapping of enzyme activities. Suggestions for possible candidate genes have often been inferred from a similar map position of these QTLs with genes known to be involved in the process under study (Arabidopsis [Mitchell-Olds and Pedersen, 1998]; maize [Prioul et al., 1999]; tomato [Fridman et al., 2002]; rice [Hu et al., 2001]).Now that the genome of Arabidopsis has been sequenced (Arabidopsis Genome Initiative, 2000), the map position of most genes encoding the enzymes involved in primary metabolism are know...

Vacuolar invertase regulates elongation of Arabidopsis thaliana roots as revealed by QTL and mutant analysis

“…The seedlings were studied for staining with a binocular (Leica, Wetzlar, Germany); the intensities of staining patterns were (semi-) quantified on an arbitrary scale, ranging from 0 to 5, indicating no and very intense staining, respectively (17). Details are listed in Supporting Materials and Methods.…”

“…Seedlings were grown in light (Philips TLD 50W͞830 fluorescent tubes, 13 W͞m 2 for 7 days as described in ref. 17). Samples were taken 4 to 6 h after the start of the light period and immediately frozen in liquid nitrogen (for enzyme extraction) or fixed (for histochemical staining and for length measurements).…”

“…To test whether activities in specific organs might be related to elongation, the activities of Inv and Susy, as revealed by histochemical staining, were semiquantified as described by Sergeeva et al (17). It should be noted that this method does not discriminate between Vac-Inv and CW-Inv.…”

“…QTL analysis uses naturally occurring within-species allelic variation and is especially suited to study polygenic traits (14). It has been used to find loci involved in regulation of enzyme activities: Inv, phosphoglucomutase, and other enzymes in Arabidopsis and maize (15)(16)(17).QTLs for Inv have been studied in tomato (18,19) and potato (20), where genetic colocation or heterologous complementation indicate that Inv polymorphisms are responsible for sugar content͞ composition.As a model system, Arabidopsis seedlings were chosen. In seedlings, hypocotyl, root, and shoot apex are the main sinks, assimilates being supplied by the cotyledons.…”

“…QTL analysis uses naturally occurring within-species allelic variation and is especially suited to study polygenic traits (14). It has been used to find loci involved in regulation of enzyme activities: Inv, phosphoglucomutase, and other enzymes in Arabidopsis and maize (15)(16)(17).…”