Communication between the maternal testa and the embryo and/or endosperm affect testa attributes in tomato

Abstract

Two tomato (Lycopersicon esculentum) mutants with dark testae displaying poor germination rate and percentage on both water and 100 microM gibberellin(4 + 7) were recovered. The mutants were allelic (black seed1-1; bks1-1 and bks1-2), inherited in Mendelian fashion as a recessive gene residing on chromosome 11. They are not allelic to bs (brown seed) -1, -2, or -4, which impair seed germination and possess dark testae. The bks/bs mutants accumulated dark pigment in the cell layers of the testa above the endothelium, which itself accumulated proanthocyanidins similar to wild type. The poor germination performance of bks mutant seeds was because of impediment of the mutant testae to radicle egress. Imbibition on gibberellin(4 + 7) did not ameliorate germination percentage or rate. The toughening of the bks testa and associated poor germination were partially overcome when seeds were not dried before germination or were dried under N(2). The seeds of the bks mutant have elevated activity of at least one enzyme responsible for the detoxification of reactive oxygen species. The bks mutant is epistatic to 12 anthocyaninless mutants of tomato. Bio- and physicochemical analysis of the bks testa determined that it accumulated a melanic substance. Inheritance of bks/bs mutations contrasts with that of the anthocyaninless mutants, which are inherited according to the genotype of the maternally derived testa. This suggests that the testa manufactures components before its demise that can maximize testa strength, whereas the endosperm/embryo produces factors that are conveyed to the testa, mitigating this process.

Figure 1.

The bks and bs mutants result in a darker than usual testa. A to D, F₁ seeds from reciprocal crosses of bks1-2 and WT MT are WT in appearance. E, Pigment accumulation in bks1-2 during development. Width of the box containing the insets = 5 mm. F, Hand sections of WT and mutant seeds with dark testae. Embryo and endosperm color is unaffected relative to WT. Pieralbo (PA) is not available (NA); AC, Ailsa Craig; MM, Moneymaker; IG, immature green; MG, mature green; En, endosperm; R, radicle; C, cotyledons; T, testa. Bar in all the micrographs = 1 cm unless stated otherwise.

Figure 2.

Sections of the testa and endosperm of WT and mutant tomato seeds. All micrographs are 10-μm-thick sections of: A and B, WT Ailsa Craig; C and D, bs1; E and F, bs2; G and H, WT Moneymaker; I and J, bs4; K and L, WT MT; and M and N, bks1-1. The sections of bks1-2 seeds (not shown) were similar in appearance to those of bks1-1. The first column of micrographs is without stain, whereas the second column has been stained with 1% (w/v) vanillin in 4.5 n HCl for 10 min. Bar in the micrographs = 0.5 mm.

Figure 3.

The bks mutant negatively impacts the completion of germination on water or GA_{4 + 7} but not by impeding imbibition. A, Rate with which the bks mutant commenced radicle protrusion when imbibed on water was retarded relative to that of the WT MT controls. Neither did the bks mutant seeds attain the same final percentage germination as WT MT seeds. Imbibition on GA_{4 + 7} did not affect the germination rate or the final percentage germination of bks mutant seeds. B, Imbibition rate and moisture content attained by the bks mutant seeds was similar to that of WT MT seeds.

Figure 4.

The rate and final germination percentage of tomato seeds is influenced by how they are dried. The physical toughness of the testa (mean and se of the puncture force [Newtons] given in each legend by the appropriate treatment symbol) was influenced by if and how the seeds were dehydrated. Fresh or nitrogen-dried seeds consistently completed germination to the greatest percentage and had weak testae. Fresh, Not dried before germination on water; Air, N₂, and O₂: Dried under an atmosphere of ambient air, pure nitrogen, or oxygen, respectively, in desiccators over activated alumina for 4 d before germination on water. Lowercase letters after the average puncture force values denote significant differences among treatments within a genotype. Uppercase, bold letters denote significant differences among genotypes within a treatment.

Figure 5.

ROS-scavenging enzymes are up-regulated in the bks mutant. Upper: A, PRX; B, CAT; and C, SOD activities present in WT MT and bks mutant tomato seeds. Within an enzyme assay, different uppercase letters within the bars of the histogram represent statistically significant differences at alpha = 0.05 according to Tukey's mean separation test. Lower: A, Native PAGE (12% [w/v], 20 μg of buffer-soluble protein lane^-1) stained for PRX activity from WT MT and bks mutant seeds and a commercial preparation (PRX). B, Native PAGE (9% [w/v], 10 μg of buffer-soluble protein lane^-1) stained for CAT activity from WT MT and bks mutant seeds and a commercial preparation. C, SDS-PAGE (12% [w/v]) of crude protein extracts (20 μg lane^-1) from WT MT and mutant seeds and a commercial preparation (SOD) dissolved in SDS-loading buffer but not boiled and stained for SOD activity. Numbers to the left of the SDS-PAGE represent the molecular mass of the standards in kilodaltons.

Figure 6.

Release of ROS from dry seeds upon imbibition. There were no significant differences in ROS release among dry or 24-h-imbibed (data not shown) bks and WT MT.

Figure 7.

The bks mutant is epistatic to the anthocyaninless mutants: Seeds of double mutants between bks and 12 anthocyaninless lines retain a dark testa color. In these mutants, the anthocyaninless testa phenotype could not be scored. a, anthocyaninless; aa, anthocyanin absent; ae, entirely anthocyaninless; af, anthocyanin-free; ag, anthocyanin gainer; ag-2, anthocyanin gainer 2; ah, Hoffmann's anthocyaninless; ai, incomplete anthocyanin; al, anthocyanin loser; are, anthocyanin reduced; aw, without anthocyanin; bls, baby lea syndrome. Bar in the baby lea syndrome square = 1 cm.

Figure 8.

Physicochemical evidence indicates the presence of un-paired electrons in the bks mutant testa. EPR detected the presence of a single unpaired electron in testae of niger (not shown) and black sunflower (A), both known to contain melanin. A white testa mutant of sunflower (B) did not produce an appreciable signal. Although the bks1-1 mutant testa (C) possessed unpaired electrons, the testa of WT MT did not (D). Black sunflower signal was acquired with one scan, whereas bks1-1, WT MT, and white sunflower were acquired with 100 scans. EPR instrumental parameters are given in “Materials and Methods.”