Class I beta-1,3-glucanase and chitinase are expressed in the micropylar endosperm of tomato seeds prior to radicle emergence

Abstract

beta-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. beta-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. beta-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of beta-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 microM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both beta-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed.

Figure 1

β-1,3-Glucanase and chitinase activity of the micropylar region (endosperm cap and radicle tip) and the rest of tomato seeds (lateral endosperm and embryo) during imbibition and germination. A, Tomato cv Moneymaker seeds were imbibed in water at 25°C and β-1,3-glucanase enzyme activity was assayed radiometrically using reduced [³H]laminarin as substrate. B, Chitinase enzyme activity was assayed in the same seeds using reduced [³H]chitin as substrate. C, The time course of germination (radicle emergence) in water at 25°C. Both germinated and ungerminated seeds were included in enzyme extractions at each time point. Error bars indicate ±se.

Figure 2

Gel-blot hybridization assays of RNA from dissected tissues of tomato cv Moneymaker seeds using riboprobes from cDNAs of different classes of tomato β-1,3-glucanases or chitinase: GluB (class I basic intracellular β-1, 3-glucanase); GluA (class II acidic extracellular β-1, 3-glucanase); and Chi9 (class I basic intracellular chitinase). G46 is a cDNA for a ribosomal protein used to demonstrate equal loading of the lanes (Cooley et al., 1999). Seeds were imbibed for the indicated times in water prior to dissection and extraction of RNA. Total RNA (10 μg) was loaded in each lane. The RNA sample of dry (0 h) micropylar region tissues includes endosperm caps and radicle tips. In addition, no signal was detected using probes for Tom PR-Q'a (class III acidic β-1, 3-glucanase), Tom PR-Q'b (class III basic β-1, 3-glucanase), Chi3 and Chi17 (class II acidic extracellular chitinases), or Chi2;1 (Chi14; class II basic chitinase) (data not shown).

Figure 3

Localization of tomato class I β-1,3-glucanase and chitinase mRNAs in individual tomato cv Moneymaker seeds by tissue printing. After 48 h of imbibition in water at 25°C, individual ungerminated tomato seeds were bisected and the cut surface of each half was printed directly on separate nylon membranes. The mirror-image tissue prints were then hybridized with antisense (A and C) or sense (B and D) riboprobes for class I tomato β-1,3-glucanase (GluB; A and B) or class I chitinase (Chi9; C and D).

Figure 4

β-1,3-Glucanase and chitinase protein accumulation in the micropylar region and the rest of tomato seeds during imbibition and germination. Tomato cv Moneymaker seeds were imbibed in water at 25°C for the times indicated. Western blots of tomato seed proteins are shown using antiserum to tobacco class I β-1,3-glucanase (A) and to tobacco class I chitinase (C), respectively. The left-most lane in A contains authentic tobacco 33-kD class I β-1,3-glucanase (Gln I) and in C contains tobacco class I chitinase A (34 kD) and chitinase B (32 kD; Chn I). The bacterially expressed fusion proteins of maltose-binding protein and tomato β-1,3-glucanase (pMAL-GluB; B) and of maltose-binding protein and chitinase (pMAL-Chi9; D) were immunoblotted with the antisera used in A and C, respectively. The left lanes in B and D were loaded with the purified fusion proteins (approximately 77 kD), whereas the right lanes were loaded with the same samples after protease cleavage of the fusion protein to separate the maltose-binding protein from the tomato proteins.

Figure 5

Enzyme activities and immunoblot assays of β-1,3-glucanase (A) and chitinase (B) of gib-1 tomato seeds after different imbibition times in water or in 100 μm GA₄₊₇ at 25°C. The inset in B shows the germination time courses for seeds in either water or GA. Error bars indicate ±se.

Figure 6

Gel blot analyses of tomato class I β-1,3-glucanase (A) and class I chitinase (B) mRNA expression in response to 100 μm ABA treatment during tomato cv Moneymaker seed imbibition and germination. Seeds were imbibed for the indicated times in water or 100 μm ABA prior to dissection and extraction of RNA. The membranes were hybridized to riboprobes for either tomato class I β-1,3-glucanase (GluB; A) or class I chitinase (Chi9; B). G46 is a cDNA for a ribosomal protein to demonstrate equal loading of lanes with 10 μg of total RNA. The RNA sample of dry micropylar region tissues (0 h) includes both endosperm caps and radicle tips.

Figure 7

Enzyme assays and protein gel-blot analyses of tomato class I β-1,3-glucanase (A) and class I chitinase (B) in response to 100 μm ABA treatment during tomato cv Moneymaker seed imbibition and germination. The inset in B shows the germination time courses for seeds in either water or ABA. Error bars indicate ±se.

Figure 8

Time course of the decrease in endosperm cap puncture force of tomato cv Moneymaker seeds incubated in water or 100 μm ABA at 25°C. The endosperm caps were dissected from ungerminated seeds imbibed for the indicated times.