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 x 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 < 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.

Figure 1.

Frequency distribution of PGM activities in extracts of seedlings of 142 lines of the Cvi × Ler recombinant inbred population. Arrows indicate activities of the parental lines.

Figure 2.

Examples of staining for PGM activities in 7-d-old seedlings of Ler (A), Cvi (B), Col (C), PGMp mutant (D), and control (Col; E). In the control, Glc1P was omitted from the incubation. F, A detail of the hypocotyl-root transition, including staining of root hairs (Ler).

Figure 3.

Correlation between PGM activities as quantified in whole-seedling extracts (y axis) and total PGM activity, calculated based on scoring of staining of separate organs (x axis), in the Cvi × Ler recombinant inbred population. PGM activities in extracts are given in nanomoles per second per gram dry weight. For each line, staining figures for the whole seedlings were calculated as a weighed sum of data of each organ, assuming relative proportions per organ to be 40%, 5%, 15%, 25%, 5%, 5%, and 5% for cotyledons, apex, hypocotyl, root, root neck, root tip, and root hairs, respectively. Lines were divided into eight classes based on weighed means of staining and pooled. Averages for pooled lines were calculated and plotted ± se.

Figure 4.

Ler/Cvi linkage map showing the genetic locations of QTLs affecting PGM activities, determined in whole-seedlings extracts, and in various organs of intact seedlings. The lengths of the arrows indicate the 2-LOD support intervals. The directions of the arrows indicate the Ler allelic phenotype (upward increasing, downward decreasing). The locations of (putative) PGM genes on chromosomes 1 and 5 is indicated: 1, At1g23190; 2, At1g70820; 3, At1g70730; 4, At5g17530; 5, At5g51820.

Figure 5.

QTL likelihood maps for the activities of PGM in root hairs at different positions along the root as quantified from histochemical stainings. Only data for chromosome 1 are presented. LODs above 2.6 (horizontal dotted line) are significant at P = 0.05.