Different myrosinase and idioblast distribution in Arabidopsis and Brassica napus

Abstract

Myrosinase (EC 3.2.3.1) is a glucosinolate-degrading enzyme mainly found in special idioblasts, myrosin cells, in Brassicaceae. This two-component system of secondary products and degradative enzymes is important in plant-insect interactions. Immunocytochemical analysis of Arabidopsis localized myrosinase exclusively to myrosin cells in the phloem parenchyma, whereas no myrosin cells were detected in the ground tissue. In Brassica napus, myrosinase could be detected in myrosin cells both in the phloem parenchyma and in the ground tissue. The myrosin cells were similar in Arabidopsis and B. napus and were found to be different from the companion cells and the glucosinolate-containing S-cells present in Arabidopsis. Confocal laser scanning immunomicroscopy analysis of myrosin cells in B. napus embryos showed that the myrosin grains constitute a continuous reticular system in the cell. These findings indicate that in the two species studied, initial cells creating the ground tissue have different potential for making idioblasts and suggest that the myrosinase-glucosinolate system has at least partly different functions. Several myrosinases in B. napus extracts are recovered in complex together with myrosinase-binding protein (MBP), and the localization of MBP was therefore studied in situ. The expression of MBP was highest in germinating seedlings of B. napus and was found in every cell except the myrosin cells of the ground tissue. Rapid disappearance of the MBP from the non-myrosin cells and emergence of MBP in the myrosin cells resulted in an apparent colocalization of MBP and myrosinase in 7-d-old seedlings.

Figure 1

General structure of glucosinolates and their possible products after myrosinase cleavage. R denotes amino acid-derived side chains. Epithiospecifier protein (ESP) together with the pH and other factors are critical parameters determining which product is formed from the aglucone.

Figure 2

Western-blot analysis (a) and myrosinase activity measurements (b) of protein extracts from Arabidopsis and B. napus. The monoclonal antibody 3D7 was used to detect myrosinase in leaves (a, lane 1) and seeds (lane 2) from Arabidopsis and seeds from B. napus (lane 3). The monoclonal antibody 34:14 was used to detect MBP from Arabidopsis leaves (lane 4) and B. napus seeds (lane 5). b, Specific myrosinase activity in leaves (column 1) and seeds (column 2) from Arabidopsis, and seeds from B. napus (column 3).

Figure 3

Immunohistochemical analysis of myrosinase in Arabidopsis using the monoclonal antibody 3D7. In fully expanded leaves from 25-d-old plants, myrosinase were found in often pair-wise-occurring idioblastic cells of the phloem parenchyma (a and b). b, Larger magnification of a. In flower buds, some cells contained myrosinase (c and f), even in the developing petal and sepal (c). In 10-d-old siliques, the myrosinase containing idioblastic phloem parenchyma cells were larger and the staining was more granulated (d and g). d, Arrows indicate long myrosinase-expressing cells. e, The yellowish staining outside the endosperm is retained unoxidized substrate and is therefore regarded as background. In 5-d-old seedlings, developing myrosinase-containing phloem cells were only found in the axis. Xylem is marked with + and the size bars correspond to 10 μm.

Figure 4

LM studies of Arabidopsis myrosin cells in young rosette leaf (a–d) and in pedicel (e). TEM study of S-cells in pedicel (f). a, ABB staining of paraffin-embedded material that included a myrosin cell (arrow) peripherally in the phloem (P). b, Immunohistochemical analysis of myrosinase on a section consecutive to the one shown in a. The antimyrosinase antibody 3D7 indicated myrosinase expression in the same myrosin cell (arrow) as in a. The leaf blade in c was embedded in glycol methacrylate, sectioned paradermally, and stained with ABB. It shows the phloem (P), including two long and relatively broad myrosin cells (M), and mesophyll cells. A paradermal section of a leaf blade (d) embedded in epoxy resin and stained with toluidine blue, including a vascular strand cut slightly obliquely longitudinally, showed phloem (P) and two adjacent myrosin cells (M1 and M2) located in the phloem parenchyma. e, Transverse section treated as d of a pedicel, with xylem (X), phloem (P), S-cells (S-C), and myrosin cells (M). The S-cells are located between the phloem and the cells (asterisks) of the starch sheath. The myrosin cell to the right is in direct contact with an S-cell. f, Two S-cells (S-C) and the outer part of the phloem with mature sieve-tube members (ST), immature members (iST), companion cells (CC), and phloem parenchyma cell (PPC) are shown in transverse section. The S-cells had large, empty vacuoles and a thin cytoplasmic layer in which a dilated cisternae (DC) of the endoplasmic reticulum (ER) was located. Size bars in a through e correspond to 10 μm and in f to 1 μm.

Figure 5

TEM analysis of Arabidopsis phloem of rosette leaf (a–c) and pedicel (d). a, Immunogold labeling with the 3D7 antibody in a peripheral vacuole (V) surrounded by cytoplasm in a myrosin cell. Abundant ribosomes in the cytoplasm were sometimes organized as polysomes (R). To the left, two neighboring cells and the common cell wall (CW) are shown. b, The myrosin cells M1 and M2, also shown in Figure 4d, both exhibited the characteristic high protein content of vacuoles (V) in myrosin cells. The cytoplasm had abundant distended rER and mitochondria. Next to M1, two bundle sheath cells with cell sap in the vacuoles were located. c, Higher magnification of a myrosin cell with cell wall (CW), cytoplasm with mitochondria (Mi), distended rER (ER), and the vacuole (V) with electron-dense homogeneous, granular material. The distended ER had abundant ribosomes on the membrane, and the moderately electron-dense material inside the ER resembled the most peripheral part of the vacuolar material. d, The phloem with sieve tube members (ST), companion cell (CC), and phloem parenchyma cell (PPC). The sieve tube members were connected by a sieve plate (SP), and the upper member showed three sieve element plastids and P protein. The companion cells had a fairly electron-dense cytoplasm and long, narrow vacuoles. In the ordinary phloem parenchyma cell, the vacuole filled most of the cell lumen and in the cytoplasm was a small DC of the ER with granular content and ribosomes on the membrane. Size bars correspond to 1 μm.

Figure 6

Immunohistochemical analysis of myrosinase in B. napus by use of the monoclonal antibody 3D7. In undeveloped flower buds, no staining could be detected, but in the pedicel, phloem-specific expression could be found (b). In the shoot apex (a), old flower buds (d), siliques (g), and leaves (h and i), myrosinase could be detected in myrosin cells in the ground tissue and the phloem. In mature petals (e) and stems (c and f), only phloem-specific expression was found. c, Transverse section of a stem; f, Longitudinal section with the outside uppermost. In root, ground tissue idioblasts containing myrosinase were visible (j). Xylem is marked with + and the size bars correspond to 10 μm.

Figure 7

Confocal microscopy analysis of a myrosin cell in B. napus. Myrosinase was detected by use of the 3D7 monoclonal antibody. A, Myrosin cell at high magnification showing the distribution of myrosinase in a reticular system rather than separate myrosin grains. This part is presented as a stereo anaglyph achieved as surface-extracted maximum intensity projection of a series of confocal images. B, Single confocal image presenting an overview of part of a cotyledon from a mature embryo. The arrow indicates the single myrosin cell shown in a. Size bars correspond to 5 μm (A) and 50 μm (B).

Figure 8

Immunohistochemical analysis of myrosinase and MBP expression in B. napus seedlings using the monoclonal antibodies 3D7 and S4C6, respectively. Myrosinase was detected in a, b, and e and MBP in consecutive sections in c, d, and f. In 2-d-old seedlings, myrosinase was found in myrosin cells (a and b) and MBP was found in all cells of the ground tissue except for the myrosin cells (c and d). In 7-d-old seedlings, myrosinase and MBP were colocalized (e and f). Size bars correspond to 10 μm. Arrows indicate the same myrosin cell in consecutive sections.