Expression of a polygalacturonase associated with tomato seed germination

Abstract

Radicle protrusion from tomato (Lycopersicon esculentum Mill.) seeds to complete germination requires weakening of the endosperm tissue opposite the radicle tip. In common with other cell wall disassembly processes in plants, polygalacturonases (PGs) may be involved. Only calcium-dependent exo-PG activity was detected in tomato seed protein extracts. Chromatographic profiles of a partially acid-hydrolyzed fraction of polygalacturonic acid further digested with seed extract were consistent with the presence of only calcium-dependent exo-PG activity. In addition, a transcript encoding a previously unknown PG was detected prior to the completion of germination. The mRNA, produced from a gene (LeXPG1) estimated by Southern analysis to be represented once in the genome, was also present in flowers (anthers) and in lower amounts in roots and stems. LeXPG1 mRNA abundance was low during seed development, increased during imbibition, and was even greater in seeds that had completed germination. Expression of LeXPG1 during germination predominates in the endosperm cap and radicle tip, and in the radicle appears as a distinct band possibly associated with vascular tissue differentiation. We suggest that PG is involved in cell wall loosening of the endosperm necessary for radicle protrusion from tomato seeds and in subsequent embryo and seedling growth.

Figure 1

PG activity accumulates in conjunction with germination. A, The activity of PG from desiccated developing and mature seeds and mature seeds during germination and subsequent radicle protrusion as assessed using a reducing sugar assay in the presence of 5 mm CaCl₂. From 48 h onward seeds were separated into those that had and had not completed germination. IG, Immature green; MG, mature green; B, breaker; M, mature. B, The effect of 5 mm CaCl₂ on the activity of PG from whole cv Moneymaker seeds allowed to imbibe for 48 h from which the radicle had protruded. Activity is reported as picomoles of GalUA produced per hour per seed.

Figure 2

Profiles of a 1% (w/v) solution of partially hydrolyzed polygalacturonic acid exposed to tomato seed protein extract indicate the activity of an exo-PG. High-pressure liquid chromatography with pulsed amperometric detection (HPLC-PAD) resulted in the fractionation of a 1% (w/v) solution of partially acid hydrolyzed polygalacturonic acid. The solution is comprised of oligomers with a degree of polymerization from 6 to 19 centered on 10 to 12 (G12 fraction; Campbell and Labavitch, 1991). A, Untreated G12; B, G12 spiked with a commercial source of mono- (1) and di-GalUA (2) (made 2.5 nm with respect to both); C, G12 after exposure to tomato seed extract; D, G12 exposed to boiled tomato seed extract; E, G12 exposed to tomato seed extract containing 50 mm EDTA; F, G12 exposed to a commercial source of endo-PG from A. niger. Extracts in chromatograms C to E contained 5 mm CaCl₂. Response bars to the left of each chromatogram represent a 100-mV deflection.

Figure 3

Southern-blot analysis of tomato genomic DNA probed with 1,504 bp of the poly(A)-truncated PG washed at low (5× SSC and 0.1% SDS at 65°C; A) and high (0.2× SSC and 0.1% SDS, 65°C; B) stringency suggest that there is little homology among the PG transcript and other members of this large gene family in tomato. The number of bands visible on the blot corresponds to the number predicted based on the restriction map of the cDNA (accession no. AF138858) and genomic clone (not shown).

Figure 4

Expression of LeXPG1 mRNA. A, mRNA (5 μg lane⁻¹) from developing (IG, immature green; MG, mature green; BR, breaker) or mature wild-type cv Moneymaker tomato seeds allowed to imbibe from 0 to 60 h was hybridized with the LeXPG1 cDNA probe. Upon radicle protrusion, mRNA abundance increased as much as 10-fold over that present in mature, desiccated seeds. G46 is a constitutively expressed cDNA coding for a ribosomal protein that was used as an RNA loading control. Germ., Germinated. B, Northern-blot analysis of 5 μg of total RNA from wild-type cv Moneymaker tomato seed parts revealed that LeXPG1 mRNA is present in the endosperm cap and radicle tip by 24 h after imbibition. The mRNA accumulated in the rest of the wild-type seed 48 h after imbibition only if the seed had completed radicle emergence. UG, Ungerminated; G, germinated. C, Expression of LeXPG1 mRNA is not restricted to tomato seeds but also occurs in the stems, roots, and flowers (anthers) of tomato plants. Each lane was loaded with 5 μg of poly(A) RNA.

Figure 5

PG mRNA accumulates in the embryo and the endosperm cap. A, In situ analysis detected expression of LeXPG1 mRNA in tomato embryos in a distinct band behind the radicle tip where it first emerged from the surrounding endosperm. Some expression was also detected in the remaining pieces of the endosperm cap. B, Section of the same seed as in A but probed with the sense strand RNA. C, At later stages of radicle protrusion, expression of LeXPG1 occurred throughout the embryo, but was most prominent in the vascular trace around the point of egress from the endosperm. Expression remained high in the endosperm cap. D, Section of the same seed as in C but probed with the sense strand RNA. Message was not observed in situ prior to the completion of germination (data not shown). Bars = 1 mm.

Figure 6

Homology of LeXPG1 to other reported PGs. Based on alignment of the deduced amino acid sequences from 31 PGs reported in the literature, the tomato seed PG (LeXPG1) is most homologous to a PG from avocado, sharing 40% similarity at the amino acid level. LeXPG1 does not segregate with PGs associated with pollen but is rather distantly related phylogenetically to PGs associated with abscission zones and fruit ripening that are predicted to contain a prosequence (Hadfield et al., 1998). The numbers above the lines on the tree represent theoretical divergence among PGs proportional to the number of amino acid substitutions.