The tomato EAR-motif repressor, SlERF36, accelerates growth transitions and reduces plant life cycle by regulating GA levels and responses

Abstract

Faster vegetative growth and early maturity/harvest reduce plant life cycle time and are important agricultural traits facilitating early crop rotation. GA is a key hormone governing developmental transitions that determine growth speed in plants. An EAR-motif repressor, SlERF36 that regulates various growth transitions, partly through regulation of the GA pathway and GA levels, was identified in tomato. Suppression of SlERF36 delayed germination, slowed down organ growth and delayed the onset of flowering time, fruit harvest and whole-plant senescence by 10-15 days. Its over-expression promoted faster growth by accelerating all these transitions besides increasing organ expansion and plant height substantially. The plant life cycle and fruit harvest were completed 20-30 days earlier than control without affecting yield, in glasshouse as well as net-house conditions, across seasons and generations. These changes in life cycle were associated with reciprocal changes in expression of GA pathway genes and basal GA levels between suppression and over-expression lines. SlERF36 interacted with the promoters of two GA2 oxidase genes, SlGA2ox3 and SlGA2ox4, and the DELLA gene, SlDELLA, reducing their transcription and causing a 3-5-fold increase in basal GA₃/GA₄ levels. Its suppression increased SlGA2ox3/4 transcript levels and reduced GA₃/GA₄ levels by 30%-50%. SlERF36 is conserved across families making it an important candidate in agricultural and horticultural crops for manipulation of plant growth and developmental transitions to reduce life cycles for faster harvest.

Keywords: AP2‐ERF domain; DELLA; GA2 oxidase; early maturity; flowering time; gibberellins.

Figure 1

Manipulation of SlERF36 levels alters plant growth and the timing of developmental transitions in tomato. (a) Germination percentage of seeds of control and SlERF36 over‐expression and suppression lines on ½ MS medium. Germination was monitored based on radical emergence. The number of days excludes the two‐day stratification (n = 30). (b) Graphical representation of height of control and transgenic SlERF36 over‐expression and suppression lines during the course of their growth. Measurements were made in nethouse‐grown plants on days 15, 30, 45, 60, 75, 90 120 and 150. Values are averages (± SD) of ten plants per line. The inset shows height of 5‐month‐old plants of control and SlERF36 transgenic lines. (c) Graphical representation of changes in leaf area of control and SlERF36 transgenic lines. Measurements were made in nethouse‐grown plants on days 15, 30, 45, 60, 75, 90 120 and 150 using the 4th leaf from bottom. The values represent the mean (± SD) of leaves of five plants. The inset shows leaves of 5‐month‐old plants of control and SlERF36 transgenic lines. (d) Dry weight of the entire aerial part (inclusive of stem and leaves) and roots of 30‐day‐old plants. The values represent average ± SD of five replicates. (e) Graphical analysis of differences in the leaf number in control and transgenic SlERF36 over‐expression and suppression plants during the course of their development under nethouse conditions. The values are average ± SD of twenty plants per line.

Figure 2

Manipulation of SlERF36 levels alters the timing of flowering, senescence and fruit harvest in tomato. (a) Graphical analysis of differences in the flowering time (time of appearance of the first bud on the plant) in control and transgenic SlERF36 over‐expression and suppression plants grown in the nethouse. The values are average ± SD of twenty plants per line. (b) Representative picture showing progression of whole‐plant senescence in nethouse‐grown plants of control and transgenic SlERF36 lines in the 6th month. (c) Graphical analysis of yield (total fruit weight per plant) in control and transgenic SlERF36 OEx and suppression line plants. The error bars represent SE, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 (Student's t‐test).

Figure 3

Sensitivity of control and transgenic SlERF36 altered lines to exogenous GA₃ and paclobutrazol. Graphical representation of plant height (a) and leaf area (b) in control and transgenic SlERF36 plants upon exogenous application of GA₃ and paclobutrazol. Control and transgenic SlERF36 plants were sprayed with either water (untreated) or with 100 μM GA₃ or 100 μM paclobutrazol in three independent sets at three‐day intervals for 3 months and measurements for height and leaf area taken at 14, 28, 42, 56, 70 and 84 days post‐germination. Bars represent value (± SD) averaged over 10–15 measurements per line. The error bars represent SE, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 (Student's t‐test).

Figure 4

Manipulation of SlERF36 affects the expression of GA pathway genes in different tissues. (a) qRT‐PCR analysis showing relative changes in transcript levels of GA biosynthesis genes (SlGA20ox1, SlGA20ox3 and SlGA3ox1), GA catabolism genes (SlGA2ox3 and SlGA2ox4) and SlDELLA in control and transgenic SlERF36‐altered lines in germinating seeds (3DPI) (upper panel) and in 2‐month‐old leaves (lower panel). The values were normalized against SlCAC and represent means of three biological replicates. The error bars represent SE, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 (Student's t‐test). (b) Real‐time PCR analysis of expression of SlERF36 in leaves of the procera mutant.

Figure 5

SlERF36 manipulation alters GA levels in stem and leaves. Box plots of liquid chromatography‐mass spectrometry estimation of GA₃ and GA₄ in (a) leaves and stems of 60‐day‐old plants of control and transgenic SlERF36 over‐expression and suppression lines and (b) leaves of 120‐day‐old plants and stem of 75‐day‐old plants. The values are average (± SD) of three replicates. The error bars represent SE, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 (Student's t‐test).

Figure 6

SlERF36 functions as a transcriptional repressor and physically interacts with the SlGA2ox3/SlGA2ox4/SlDELLA promoters. (a) SlERF36 functions as a repressor of GAL4 in protoplast transfection assays. The effectors GAL4DBD and GAL4DBD‐SlERF36 under the CaMV35 double promoter and the GAL4(4X)::GUS reporter constructs were co‐transfected into protoplasts extracted from 3 to 4 week‐old Arabidopsis Col‐0 plants. GUS activity was studied after 16 h of transfection in dark. Graph represents the mean ± standard error of three replicates. (b) Yeast one‐hybrid studies showing interactions between SlERF36 and the promoters of the GA catabolism genes P _SlGA2ox3 (−1931 bp, second row), P _SlGA2ox4 (−2498 bp, third row) and the GA signalling inhibitor P _SlDELLA (−1983 bp, fourth row) in different serial dilutions. The positive and negative controls are present in the first and last rows, respectively. The minimal inhibitory concentration of the aureobasidin‐A (AbA) antibiotic for selection of positive bait‐prey interactions was optimized to 100 ng/mL and selection performed on SD/AbA/‐Ura agar plates. (c) Luciferase assays depicting suppression of the above promoters of P _SlGA2ox3, P _SlGA2ox4 and P _SlDELLA by SlERF36 (driven by the CaMV35S promoter) in tobacco leaves. The scheme showing the agro‐injection of constructs in different regions of the leaves (empty vectors at top; promoter‐LUC constructs without SlERF36 on the right; and promoter‐LUC constructs with SlERF36 on the left) is provided in the extreme right panel. The fluorescence in the right part of the leaf indicates basal promoter activity in absence of SlERF36. (d) Electrophoretic mobility shift assays showing binding of SlERF36 to the biotin‐labelled primers containing the GCC box cis element at −1343 nt (upstream of the initiation codon) in the promoter of SlGA2ox3. Biotin‐labelled primer probes (double‐stranded) were electrophoresed with SlERF36 protein on a 6% native polyacrylamide gel as described in methods. Interactions were checked with biotinylated mutated probe and by competition with excess cold probe. The arrow shows the band of shifted probe bound to SlERF36.

Figure 7

Model showing the function of SlERF36 in regulating developmental transitions through its action on the GA pathway and through putative GA‐independent regulators. SlERF36, through co‐repressors and HDACs, suppresses the GA2 oxidases, SlGA2ox3 and SlGA2ox4 and the GA action inhibitor, SlDELLA, to increase the GA pathway output which in turn activates intermediate components (some known, some yet to be identified) that activate various transitions. It also regulates these through suppression of GA‐independent regulators that suppress the transitions through other intermediaries.