Expression of a GALACTINOL SYNTHASE gene in tomato seeds is up-regulated before maturation desiccation and again after imbibition whenever radicle protrusion is prevented

Abstract

Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance.

Figure 1

The gene and cDNA structure of LeGOLS-1 and recombinant protein activity. A, The gene and full-length LeGOLS-1 cDNA are depicted. Untranslated regions are lines, exons (E) are bracketed, and the hatched boxes represent introns (I). The gene-specific primers (GSP) used to retrieve the 5′ portion of the cDNA are indicated. A scale bar of 100 bp is beside the cDNA. The 1,352-nucleotide promoter region and 1,051 3′ nucleotides are not drawn to scale. B, Recombinant LeGOLS-1 protein synthesized a compound identical to that of galactinol. Upper spectrum was obtained from the product of UDP-Gal and myo-inositol incubated with recombinant LeGOLS-1. The lower spectrum is from authentic galactinol obtained from maize seeds. The quality of the match between the two spectra was 95%.

Figure 2

Southern blot of tomato genomic DNA probed with the full coding region of LeGOLS-1. Only single hybridizing bands were detected, even when washed at low stringency (see “Materials and Methods”).

Figure 3

Northern blot of LeGOLS-1 transcript abundance during wild-type tomato seed maturation and germination. A, LeGOLS-1 expression was not detectable before 35 DAA, then increased and remained present throughout seed maturation. B, LeGOLS-1 expression during and after germination on water. After 48 h, seeds were separated into those that had or had not completed germination. G46 detects a constitutively expressed ribosomal protein mRNA used as a loading control (Cooley et al., 1999).

Figure 4

Tissue-specific location of LeGOLS-1 expression in tomato seeds of various genotypes. A, Wild-type seeds were imbibed on water, 100 μm GA₄₊₇, or 100 μm ABA for 0, 24, and 48 h; separated into those that had or had not completed germination when feasible (48 h only); and dissected into endosperm caps (Caps), radicle tips (Tips), and the rest of the seed (ROS). Total RNA was extracted from these seed parts and analyzed for LeGOLS-1 mRNA. G46 is a constitutively expressed loading control. B, A representative tissue print of a mature tomato seed imbibed on water for 3 h at 4°C. LeGOLS-1 mRNA is present throughout the embryo but is noticeably more abundant in the radicle tip. Some LeGOLS-1 mRNA is present in the cells at the periphery of the lateral and micropylar endosperm (endosperm cap). C, Antisense- and sense-probed tissue prints of wild-type, sitiens, and gib-1 mutant tomato seeds imbibed on water for 4 h at 25°C.

Figure 5

The effect of ABA and GA on the maintenance of LeGOLS-1 mRNA abundance in imbibed tomato seeds. A, Wild-type tomato cv Moneymaker seeds were imbibed on either water or 100 μm ABA. LeGOLS-1 transcript abundance decreased substantially after 4 h (water-imbibed) or 8 h (ABA-imbibed). ABA did not maintain LeGOLS-1 transcript abundance, but message was again present after 6 d on ABA, possibly due to the induction of secondary dormancy. B, GA-deficient gib-1 mutant seeds, which require supplemental GA to complete germination, were imbibed on either 100 μm GA₄₊₇ or water. Total RNA extracted after different times of imbibition was hybridized to LeGOLS-1 antisense riboprobe. LeGOLS-1 mRNA abundance decreased substantially within 12 h in GA-imbibed seeds but remained higher when imbibed on water. C, Seeds of the ABA-deficient sitiens mutant were imbibed on water for 0, 24, and 30 h, when they were separated into those seeds that had or had not completed germination. Despite having very low amounts of ABA (Groot and Karssen, 1992), sitiens seeds accumulated LeGOLS-1 transcript during development and maturation desiccation, accounting for its presence in dry seeds. Upon imbibition, transcript amounts declined as they did in wild-type seeds. When sitiens seeds were imbibed on 100 μm ABA, LeGOLS-1 mRNA amounts remained high for at least 30 h, although amounts were declining (compare 24 and 30 h). G46 is a constitutively expressed loading control.

Figure 6

Water stress induces LeGOLS-1 transcription. A, Wild-type tomato cv Moneymaker seeds were imbibed on water for 36 h and then either dried or transferred to −1.3 or −2.0 MPa PEG solutions. Seeds were alternatively imbibed directly on −1.3 MPa PEG. Total RNA was extracted and analyzed for LeGOLS-1 abundance. After a decline in mRNA amounts upon imbibition for 36 h on water, LeGOLS-1 transcript accumulated again in response to desiccation or osmotic stress. Seeds initially imbibed at low water potential also maintained or accumulated the transcript. B, Wild-type tomato cv Moneymaker seeds were imbibed for 24 or 40 h in the dark or under continuous far-red illumination, which prevents completion of germination. Far-red illumination maintained LeGOLS-1 transcript abundance in wild-type tomato cv Moneymaker seeds. C, LeGOLS-1 transcript abundance was also maintained or accumulated in seeds that were in primary dormancy (ungerminated after 14 d on water) or secondary dormancy (imbibed on −1.3 MPa PEG for 6 d, then transferred to water 3 d, radicle not protruded). GA effectively stimulated germination of seeds exhibiting both types of dormancy and decreased LeGOLS-1 mRNA abundance in these seeds.

Figure 7

GOLS enzyme activity from tomato seeds after various treatments. Desiccated seeds of both gib-1 and sit^W mutants had less GOLS activity than did desiccated wild-type seeds. Despite a decline in mRNA amounts after imbibition for 36 h, GOLS enzyme activity did not decrease relative to that present in desiccated seeds. Treatments that prevented the completion of germination maintained GOLS enzyme activity.

Figure 8

LeGOLS-1 is not up-regulated by chilling in seeds. A, Wild-type tomato seeds were imbibed at 25°C for 12 h and then either harvested for RNA extraction or transferred to 20°C, 15°C, 10°C, or 5°C for an additional 12 or 24 h before harvest. Seeds were also imbibed on water at 15°C, 10°C, or 5°C and sampled at 12, 24, and 36 h. LeGOLS-1 transcript was present in all seeds regardless of treatment type or duration. The colder temperatures delayed the decrease in message amounts as imbibition time increased. Temperatures as low as 5°C were not sufficient to increase message abundance beyond that present in mature, desiccated seeds over the brief time course. B, Tissue prints of wild-type tomato seeds imbibed at either 25°C or 4°C for up to 2 d that had not completed germination confirmed that cold temperature was not sufficient to increase LeGOLS-1 mRNA abundance in imbibing seeds but served only to delay its decrease.

Figure 9

LeGOLS-1 transcript accumulates in dehydration-stressed leaves and chilling-stressed seedlings. Mature leaves were detached from 18-d-old tomato plants and dehydrated to different moisture contents. The excised leaves were placed immediately in a plastic bag (0 min, 100% of initial fresh weight) or were allowed to lose water on the bench top for up to 1 h (20 min = 92%, 40 min = 88%, and 60 min = 88% of initial fresh weight) and then were sealed in plastic bags for 6 h before freezing and extraction of RNA. Plants were also sprayed once to run-off with 100 μm ABA and leaves harvested at 0, 6, 12, and 24 h after ABA application. Some plants were subjected to 4°C for 12, 24, and 48 h, and the leaves from some plants harvested, whereas other plants were moved to 25°C for an additional 48 h before the leaves were harvested. Some plants were placed at 37°C for 4, 8, and 12 h and leaves harvested at each time.