Cellular Differentiation Regulated by Gibberellin in the Arabidopsis thaliana pickle Mutant

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Science  04 Jul 1997:
Vol. 277, Issue 5322, pp. 91-94
DOI: 10.1126/science.277.5322.91


The plant growth regulator gibberellin (GA) has a profound effect on shoot development and promotes developmental transitions such as flowering. Little is known about any analogous effect GA might have on root development. In a screen for mutants, Arabidopsisplants carrying a mutation designated pickle(pkl) were isolated in which the primary root meristem retained characteristics of embryonic tissue. Expression of this aberrant differentiation state was suppressed by GA. Root tissue from plants carrying the pkl mutation spontaneously regenerated new embryos and plants.

Gibberellin is required for seed germination and plays a variety of roles during growth and development after germination. GA-deficient plants exhibit defects in germination, time lag to flowering, stem elongation, apical dominance, maintenance of floral meristem identity, and trichome distribution (1). All of these phenotypes primarily affect the shoot. Gibberellin also affects root development by promoting cell elongation (2), but there is essentially no information on the effect of GA on fate determination in roots. We report here a mutant ofArabidopsis that is defective in a GA signaling pathway that promotes the transition of the primary root from an embryonic to an adult differentiation state.

During a screen for Arabidopsis mutants exhibiting abnormal root development, we identified a class of mutants in which the primary root, after a period of apparently normal growth, would thicken and become opaque and green (Fig. 1, A and B). Lateral and adventitious roots did not express this phenotype. Because of the visual appearance of the altered primary roots, we refer to this root phenotype as “pickle.” Genetic analysis revealed that the mutant phenotype was due to a mutation at a single recessive locus located near position 48.4 on chromosome 2, which we namedPICKLE (PKL) (3). Eight independent mutant alleles of the PKL locus have been identified by screening ∼20,000 M2 plants from mutagenized populations.

Figure 1

Phenotypes of Columbia wild-type and pklplants. All comparisons of wild-type and pickle plants are presented at the same magnification unless otherwise noted. Wild-type (A) and pickle (B) primary roots from 10-day-old seedlings. (C) Structures resembling somatic embryos initiated from pickle callus on basal MS media (4). (D) A 10-day-old pkl seedling stained with Fat Red 7B (7). Transmission electron micrograph (17) of wild-type (E) and pickle (F) primary root cells illustrating the presence of oil bodies and starch granules in pickle root cortical cells. Scale bars, 0.5 μm in (E), and 2 μm in (F). M, mitochondrion; S, starch granule; V, vacuole; and O, oil body. (G) Forty-six-day-old wild-type (right) and pkl (left) plants grown for 16 hours under illumination (130 μE m−2 s−1, where E is the energy of 1 mol of photons).

Unusual cell proliferation was observed when roots were removed from mutant plants and placed on synthetic mineral medium without plant hormones (4). The excised pickle roots, at a frequency of 10 to 30%, produced callus-like growths and generated globular- and torpedo-stage embryo-like structures (Fig. 1C). Under identical conditions, excised wild-type roots exhibited neither callus growth nor any event resembling somatic embryogenesis (5). In the absence of any experimental manipulation, pickle root callus produced phenotypically normal pkl plants at a frequency of about 1%.

Because of the ability of excised pickle roots to generate structures resembling somatic embryos, we investigated the possibility that pickle roots express embryonic characteristics before removal from the plant.Arabidopsis embryos accumulate large amounts of triacylglycerols as storage reserves to support early seedling growth (6). When infiltrated with a dye that specifically stains neutral lipids (7), the portion of the pickle roots that had differentiated abnormally were intensely stained red (Fig. 1D), indicating the presence of large quantities of triacylglycerols (8). Analysis of the fatty acid composition of extracted pickle root triacylglycerols by gas chromatography revealed that the fatty acid composition differed from that found normally in roots but was indistinguishable from that of seeds (9). Transmission electron microscopy of sections of pickle root tips revealed the presence of densely packed oil bodies reminiscent of seed oil bodies (Fig. 1, E and F) as well as large starch granules normally not present in either roots or seeds. In addition, transcripts for the oleosin (10) and 2S1 storage protein (11) genes, which are normally expressed only in seeds or pollen, accumulated in pickle roots (Fig. 2). On the basis of the unique differentiation characteristics of pickle roots, we infer that the primary roots of pkl plants either retain or resume some degree of their embryonic differentiation status after germination.

Figure 2

RNA blot analysis. About 15 μg of total RNA (18) was isolated from wild-type leaves (lane 1), wild-type siliques (lane 2), wild-type roots (lane 3), and pickle roots (lane 4). The top panel shows transcripts detected by an oleosin (Ole.) Complementary DNA probe, the middle panel shows transcripts detected by a 2S1 seed storage protein cDNA probe, and the bottom panel shows transcripts detected by an Eif-4A (19) cDNA probe as a loading control.

An important clue in determining how the pkl mutation might result in aberrant primary root differentiation was provided by the observation that expression of the pickle phenotype is suppressed by GA. When seeds were germinated and grown in continuous light on synthetic media plates (4), expression of the pickle phenotype by homozygous pkl plants exhibited low penetrance, typically 1 to 10% depending on the batch of seed. Addition of 10 nM uniconazole-P (12), a GA biosynthetic inhibitor, increased penetrance of the pickle phenotype to greater than 80% (Table1). Addition of 10 μM GA4completely suppressed the ability of 100 nM uniconazole to increase penetrance of the pickle phenotype, indicating that uniconazole is acting by inhibiting GA biosynthesis. Gibberellin also suppressed the pickle root phenotype in pkl1-1 ga1-3 plants (Table2), in which the amount of GA in the plants was determined by the amount of GA exogenously supplied because of the inability of plants carrying the ga1-3 mutation to synthesize GA (13, 14).

Table 1

Effect of uniconazole-P (Un-P) and GA on penetrance of the pickle phenotype. For each treatment, 144 seeds were incubated at 22°C in continuous light (60 μE m−2s−1) on synthetic media (4) containing uniconazole-P or GA4 at the indicated concentrations. Germination and the pickle phenotype were scored at 10 days. Expression of the pickle phenotype was calculated as a percentage of total seeds and as a percentage of seeds that germinated.

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Table 2

Effect of GA on penetrance of the pickle phenotype in pkl-1 ga1-3 seedlings. For each treatment, 72 seeds were incubated at 22°C in continuous light (60 μE m−2 s−1) on synthetic media (4) containing GA4 at the indicated concentrations. Germination and the pickle phenotype were scored at 10 days. Expression of the pickle phenotype was calculated as a percentage of total seeds and as a percentage of seeds that germinated.

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To determine the developmental stage at which the differentiation state of the root was most responsive to uniconazole treatment,pkl seedlings were transferred to or from uniconazole-containing media at different times during the first 96 hours after imbibition (Fig. 3). Thirty-six hours of either regimen was largely sufficient to determine the fate of the primary root meristem; germinating pkl seeds on uniconazole plates for only 24 hours and then transferring them to plates without uniconazole was sufficient to substantially induce the pickle phenotype. Conversely, shifting pkl seeds to uniconazole plates after 36 hours on plates without uniconazole was largely ineffective in inducing expression of the pickle phenotype. At 24 hours after imbibition, the pkl seed coat has split, but the radicle has yet to emerge. Thus, at 24 hours germination is not complete, but the GA-dependent fate of the pkl root meristem may already have been determined. Germination is complete by 36 hours, by which time the fate of the root has been determined. Consequently, GA acts concurrently to promote establishment of adult root fate and germination of pkl seedlings.

Figure 3

Effect of time of application of uniconazole-P on penetrance of the pickle phenotype. pkl seeds were imbibed in water and immediately plated on media (4) containing 10−8 M uniconazole and then shifted at different times to media without uniconazole-P (open circles), or the seeds were plated on media not containing uniconazole-P and then shifted at the indicated times to media containing 10−8 M uniconazole-P (closed circles). The x axis indicates the times at which seeds were shifted. Eleven days after imbibition, the seedlings were scored for the pickle phenotype. The y axis indicates the percentage of seedlings expressing the pickle phenotype for each data point. Forty-eight seedlings were scored at each point on the graph. The experiment was carried out at 22°C in continuous light (60 μE m−2 s−1).

The pkl plants exhibit several shoot phenotypes that are reminiscent of other Arabidopsis mutants deficient in GA biosynthesis (ga1 through ga5) or GA signaling (gai) (1, 15). pkl plants have dark green leaves with short petioles, exhibit delayed bolting and reduced apical dominance, and are reduced in stature (Fig. 1G). All eight defective alleles of the PKL locus result in expression of these phenotypes with 100% penetrance.

Analysis of a pkl-1 ga1-3 double mutant indicated that it is unlikely that PKL codes for a GA biosynthetic enzyme. Because it inactivates the first step of the GA biosynthetic pathway (13, 14), the ga1-3 mutation is expected to be epistatic to any other GA biosynthetic mutant. However,pkl-1 ga1-3 plants exhibited more exacerbated GA-deficient shoot phenotypes than either pkl or ga1 plants. In particular, the pkl-1 ga1-3 plants had rosettes that were 30% smaller than ga1-3 plants and were at least 50% smaller in stature.

To address the possibility that PKL affects GA responsiveness, we examined the effect of the pkl mutation in a gai line. A pkl-1 gai plant exhibited severe GA-deficient phenotypes that were much more extreme than those exhibited by pkl or gai plants. A particularly dramatic effect was seen on flowering time: in continuous light,pkl plants and gai plants flowered 1 day and 3 days, respectively, later than wild-type plants, whereas pkl gai plants flowered 33 days later than wild-type plants on average. The synergistic effect of combining the pkl andgai mutations suggests that PKL, likeGAI, may play a role in GA signal transduction.

At present, the simplest hypothesis to explain our findings is that pkl plants are defective in a GA signaling pathway. This PKL-dependent GA signaling pathway is ubiquitously used by the plant, resulting in pkl plants that express shoot phenotypes that are reminiscent of other GA-deficient plants. In addition, this PKL-dependent GA signaling pathway promotes the transition of root cells from an embryonic to an adult state during germination. The observation that the pickle phenotype is of low penetrance and is suppressed by GA indicates that there is yet another GA signaling pathway in addition to the PKL-dependent pathway governing root differentiation during germination. This additional pathway is unlikely to be GAI-dependent, becausepkl-1 gai plants do not exhibit increased penetrance of the pickle phenotype compared with pkl-1 GAI plants.

The properties of the pkl mutant suggest that GA may play a greater role in determination of root fate inArabidopsis than previously appreciated. In addition, the properties of the pkl mutant indicate that germination and differentiation during germination are genetically separable, GA-regulated events. This separation implies that there is more than one GA response pathway governing the transformation of a dormant seed into an actively growing seedling.

The pkl mutation in Arabidopsis indicates a distinction of the primary root from the secondary roots. We speculate that regulation of PKL expression may be necessary for generation of the specialized primary roots present in other members of the Brassicaceae, such as turnips and radish. In addition, it is notable that the plant permits expression of embryonic characteristics, specifically oil, in a portion of its root system. Very few plant species are known that accumulate significant amounts of oil in roots (16). Characterization of PKL and related gene products may eventually lead to the ability to produce commercially useful amounts of oil in root crops.

  • * To whom correspondence should be addressed. E-mail: jogas{at}


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