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. 2015 Jun;27(6):1579-94.
doi: 10.1105/tpc.114.132795. Epub 2015 Jun 2.

Uncovering DELLA-Independent Gibberellin Responses by Characterizing New Tomato procera Mutants

Sivan Livne  1 , Vai S Lor  2 , Ido Nir  1 , Natanella Eliaz  1 , Asaph Aharoni  3 , Neil E Olszewski  2 , Yuval Eshed  3 , David Weiss  4
Affiliations

Uncovering DELLA-Independent Gibberellin Responses by Characterizing New Tomato procera Mutants

Sivan Livne et al. Plant Cell. 2015 Jun.

Abstract

Gibberellin (GA) regulates plant development primarily by triggering the degradation/deactivation of the DELLA proteins. However, it remains unclear whether all GA responses are regulated by DELLAs. Tomato (Solanum lycopersicum) has a single DELLA gene named PROCERA (PRO), and its recessive pro allele exhibits constitutive GA activity but retains responsiveness to external GA. In the loss-of-function mutant pro(ΔGRAS), all examined GA developmental responses were considerably enhanced relative to pro and a defect in seed desiccation tolerance was uncovered. As pro, but not pro(ΔGRAS), elongation was promoted by GA treatment, pro may retain residual DELLA activity. In agreement with homeostatic feedback regulation of the GA biosynthetic pathway, we found that GA20oxidase1 expression was suppressed in pro(ΔGRAS) and was not affected by exogenous GA3. In contrast, expression of GA2oxidase4 was not affected by the elevated GA signaling in pro(ΔGRAS) but was strongly induced by exogenous GA3. Since a similar response was found in Arabidopsis thaliana plants with impaired activity of all five DELLA genes, we suggest that homeostatic GA responses are regulated by both DELLA-dependent and -independent pathways. Transcriptome analysis of GA-treated pro(ΔGRAS) leaves suggests that 5% of all GA-regulated genes in tomato are DELLA independent.

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Figures

Figure 1.
Figure 1.
The pro∆GRAS Mutant. (A) Schematic presentation of the PRO protein structure. The arrow indicates the position of the mutation in pro∆GRAS converting the amino acid Glu to a stop codon. (B) Five-week-old M82, pro, and pro∆GRAS plants. (C) The mean length (n = 12) ± se of the main stem of 4-week-old M82, pro, and pro∆GRAS plants.
Figure 2.
Figure 2.
Phenotypic Characterization of pro∆GRAS . (A) First leaflet of the fifth leaf in 5-week-old plants. (B) Mean number of leaves to first inflorescence (n = 11 plants) ± se. Letters indicate significant differences, as determined by t test P < 0.05. (C) M82, pro, and pro∆GRAS flowers before anthesis. (D) M82, pro, and pro∆GRAS fruits.
Figure 3.
Figure 3.
The Effect of pro∆GRAS and pro on Anther Development, Pollen Production, and Pollen Tube Elongation. (A) Scanning electron microscopy images of M82, pro, and pro∆GRAS anther cones and single anthers. Flowers were detached prior to anthesis and cut widthwise. Bars in the upper panels = 500 μm; bars in the lower panels = 250 μm. (B) Real-time observation of in vitro germination of M82, pro, and pro∆GRAS pollen. Flowers were detached at anthesis and pollen was incubated in germination solution. Germination and tube elongation were monitored for 6 h using a light microscope. Bar = 50 μm.
Figure 4.
Figure 4.
pro∆GRAS Seeds Are Sensitive to Desiccation. (A) Seeds were harvested from heterozygous pro∆GRAS fruits (after self-pollination) and sown immediately thereafter, or after different periods of dry storage. Values represent the percentages of germinating seedlings with M82 (M82 and heterozygous pro∆GRAS seedlings) or pro∆GRAS (homozygous pro∆GRAS seedlings) phenotypes from total number of seeds. Values are the average of three replicates; each contains 50 seeds ± se. (B) qRT-PCR analyses of ABI3, FUS3-like, LE25, and GOLS expression in M82 and pro∆GRAS seeds. RNA was extracted from fresh M82 and pro∆GRAS homozygous seeds. Values are the average of three biological replicas ± se.
Figure 5.
Figure 5.
pro∆GRAS Is Insensitive to PAC and GA. (A) Six-week-old M82, pro, and pro∆GRAS plants were treated with 10 mg/L PAC three times a week, for 2 weeks (starting at two true leaves), followed by 2 weeks of GA3 application (100 μM, three times a week). (B) Mean length ± se of the main stems of the plants treated as in (A) (n = 8 to 11 plants). (C) Mean chlorophyll content ± se in the first leaflet of the forth leaf taken from 6-week-old plant treated as in (A) (n = 8).
Figure 6.
Figure 6.
Regulation of GA20ox and GA2ox Expression by GA in pro∆GRAS. qRT-PCR analysis of GA20ox1 (A) and GA2ox4 (B) expression. Seedlings were treated with 10 mg/L PAC for 3 d, followed by one application of GA3 (1 or 100 μM). RNA was extracted from young leaves and analyzed. Values (gene-to-TUBULIN ratios) are means of three biological replicates ± se.
Figure 7.
Figure 7.
Regulation of Growth and Gene (GA20ox and GA2ox) Expression by GA in the Transgenic Tomato Overexpressing the Arabidopsis RGA∆17 Mutant Gene. (A) qRT-PCR analysis of RGA∆17 expression in M82 and transgenic tomato plants. RNA was extracted from young leaves of the T2 generation. Values (gene-to-TUBULIN ratios) are means of three biological replicates ± se. (B) M82 and transgenic RGA∆17 plants treated with 100 μM GA3 three times a week for 2 weeks. (C) and (D) qRT-PCR analyses of GA20ox1 (C) and GA2ox4 (D) expression in tomato leaves treated with 10 mg/L PAC for 3 d or PAC for 3 d followed by one application of 100 μM GA3. Values (gene-to-TUBULIN ratios) are means of three biological replicates ± se.
Figure 8.
Figure 8.
Regulation of Arabidopsis GA20ox2, GA2ox4, and GA2ox1 Expression by GA in Arabidopsis. (A) and (B) Seedlings of wild-type Col-0 and dellaP mutant Arabidopsis plants were treated with PAC (5 mg/L) once a day for 3 d followed by a single GA3 application (10 μM). Three hours after the GA treatment, RNA was extracted from the seedlings and analyzed by qRT-PCR for At-GA20ox2 (A) and At-GA2ox4 (B) expression. (C) to (E) Plants (wild-type Col-0 and dellaP) were treated with PAC (5 mg/L) twice a week until flowering and then treated once with 10 μM GA3. Three hours after the GA treatment, RNA was extracted from the flowers and analyzed by qRT-PCR for At-GA20ox2 (C), At-GA2ox4 (D), and At-GA2ox1 (E). (F) Wild type (Col-0) and gid1ac seedlings were treated with PAC (5 mg/L) once a day for 3 d followed by a single GA3 application (10 μM). Three hours after the GA treatment, RNA was extracted and analyzed (qRT-PCR) for At-GA2ox4 expression. Values (gene-to-TUBULIN ratios) in (A) to (F) are means of three biological replicates ± se.
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