Down-regulation of DELLA genes is not essential for germination of tomato, soybean, and Arabidopsis seeds

Abstract

The relationship between expression of a negative regulator of GA signal transduction (RGL2) belonging to the DELLA gene family and repression of Arabidopsis seed germination has been studied (Lee S, Cheng H, King KE, Wang W, He Y, Hussain A, Lo J, Harberd NP, Peng J [2002] Genes and Development 16: 646-658). There is one DELLA gene (LeGAI) present in tomato (Lycopersicon esculentum Mill.), which is expressed in both vegetative and reproductive tissues. During germination of wild-type tomato seed, there was no decline in the expression of LeGAI in either the embryo or the endosperm. Rather, LeGAI transcripts increased in these tissues following imbibition and remained high during and following germination. A similar increase in LeGAI transcripts occurred in the endosperm and embryo of GA-treated gib-1 mutant seed during and following germination. Likewise in soybean (Glycine max) seed, there was no decline in the expression of two DELLA genes in the radicle before or after germination. Upon reexamination of RGL2 in Arabidopsis seeds, a decline in its expression was noted but only after radicle emergence, i.e. after germination had been completed. Taken together, these data are consistent with GA-induced down-regulation of DELLA genes not being a prerequisite for germination of tomato, soybean, and Arabidopsis seeds.

Figure 1.

LeGAI encodes a putative DELLA protein. A, Alignment of the deduced amino acid sequences of various DELLA proteins including LeGAI (tomato, GenBank accession no. AY269087), RGL2 (Arabidopsis, NM_111216), GAI (Arabidopsis, NM_101361), SLN1 (barley, AF460219), and SLR1 (rice, AB030956). Identical residues at each position are marked below the alignment with an asterisk (*), similar residues are marked with a dot (·), and the N-terminal-most amino acid shown for each protein is indicated to the left of the sequence. The conserved -DELLA- domain and semi-conserved -TVH[K]NP- domain are shaded. B, DNA gel-blot analysis at low stringency indicates the presence of a single DELLA gene in tomato. Restriction enzymes used to digest tomato DNA are indicated above each lane and DNA molecular mass markers in kb to the right of the autoradiogram. C, RNA gel-blot analysis of LeGAI transcripts showing expression of this gene in various tissues of tomato. Fluorescence attributed to ethidium-bromide-stained 28S and 18S ribosomal RNA is shown below (C) to illustrate there was equal loading of total RNA samples.

Figure 2.

LeGAI expression in tomato seeds, embryos dissected from intact seeds, and isolated embryos. A, RNA gel-blot analysis of LeGAI expression in whole wild-type tomato seeds cv Trust, either dry or imbibed for 24 h. B, LeGAI expression in tomato embryos of intact wild-type seeds at various times during (24–72 HAI) and following (72–96 HAI) germination. Nongerminated and germinated seeds were separated at 72 HAI (the time at which approximately 50% had completed germination), and their RNA was analyzed. C, LeGAI expression in embryos of intact seeds of the gib-1 mutant cv Moneymaker during and following germination. GA-imbibed seeds (100 μm GA₃) completed germination between 24 and 72 HAI; water-imbibed seeds did not complete germination. D, LeGAI expression during and following germination in embryos isolated from intact gib-1 seeds. Intact seeds were first imbibed on water for 24 h and then embryos excised and placed on water. Isolated embryos completed germination at 33 HAD. Fluorescence attributed to ethidium-bromide-stained 28S and 18S ribosomal RNA is shown below each autoradiogram in A to D to illustrate there was equal loading of total RNA samples. HAI (A–C) or HAD (D) are indicated above each lane.

Figure 3.

LeGAI expression in tomato seed endosperms. A, RNA gel-blot analysis of LeGAI expression in the micropylar and lateral endosperm regions of intact wild-type tomato seeds imbibed in water. Germination was completed between 48 and 96 HAI. Degradation of the micropylar endosperm occurred by 60 HAI, and of the lateral endosperm by 120 HAI. B, LeGAI expression in the micropylar and lateral endosperms of intact tomato seeds of the gib-1 mutant imbibed either in water or in GA (100 μm GA₃). GA-imbibed seeds completed germination between 24 and 72 HAI. Degradation of the micropylar endosperm occurred after 24 HAI, and the lateral endosperm by 96 HAI. Fluorescence attributed to ethidium-bromide-stained 28S and 18S ribosomal RNA is shown below each autoradiogram in A and B to illustrate there was equal loading of total RNA samples. HAI are indicated above each lane in A and B.

Figure 4.

DELLA gene expression and radicle growth in soybean seeds. A, RNA gel-blot analysis with gene-specific probes of GmGAI1 (GenBank accession no. AY269088) and GmGAI2 (AY269089). Germinating (0–12 HAI) and germinated (24–72 HAI) soybean radicles were removed from intact seeds for RNA extraction at the times indicated. Intact dry soybean seeds are represented by 0 HAI. B, Effect of GA (100 μm GA₃) on radicle elongation immediately following the completion of germination (24 HAI) and during soybean seedling growth. Error bars represent the se of two replicates of 15 samples each. C, RNA gel-blot analysis of GmGAI gene expression in germinated radicles of soybean seeds removed at the times indicated following imbibition in either water or GA (100 μm GA₃). Fluorescence attributed to ethidium-bromide-stained 28S and 18S ribosomal RNA is shown below each autoradiogram in A and C to illustrate there was equal loading of total RNA samples. HAI are indicated above each lane in A and C.

Figure 5.

RGL2 expression during and following the completion of germination in Arabidopsis. RGL2 expression in A, wild-type Arabidopsis seeds at various times HAS, as noted above each lane, along with the percentage of seeds that had completed germination; B, ga1-3 Arabidopsis seeds imbibed in 100 μm GA₃ at various times following stratification (HAS). Percentage of the population that had completed germination is shown above the HAS for each time point; C, Arabidopsis seeds separated based on their stage of development: intact ungerminated, testa ruptured signifying incipient radicle emergence, and radicles just emerged from the testa. Fluorescence attributed to ethidium-bromide-stained 28S and 18S ribosomal RNA is shown below each autoradiogram in A to C to illustrate there was equal loading of total RNA samples. D, Germination profiles of wild-type and ga1-3 Arabidopsis seeds. Seeds were imbibed and scored (out of 100) for the completion of germination based upon emergence of their radicles.