SlCESTA Is a Brassinosteroid-Regulated bHLH Transcription Factor of Tomato That Promotes Chilling Tolerance and Fruit Growth When Over-Expressed

Abstract

Brassinosteroids (BRs) are required for various aspects of plant growth and development, but also participate in stress responses. The hormones convey their activity through transcriptional regulation and posttranslational modification of transcription factors and one class are basic helix-loop-helix (bHLH) proteins of the BR Enhanced Expression (BEE) subfamily, which in Arabidopsis thaliana include BEE1-3 and CESTA (CES). CES and the BEEs promote the expression of different BR-responsive genes, including genes encoding gibberellin (GA) biosynthetic and catabolizing enzymes, as well as cold-responsive genes. Interestingly, in terms of an application, CES could promote both fruit growth and cold stress tolerance when over-expressed in A. thaliana and here it was investigated, if this function is conserved in the fruit crop Solanum lycopersicum (cultivated tomato). Based on amino acid sequence similarity and the presence of regulatory motifs, a CES orthologue of S. lycopersicum, SlCES, was identified and the effects of its over-expression were analysed in tomato. This showed that SlCES, like AtCES, was re-localized to nuclear bodies in response to BR signaling activation and that it effected GA homeostasis, with related phenotypes, when over-expressed. In addition, over-expression lines showed an increased chilling tolerance and had altered fruit characteristics. The possibilities and potential limitations of a gain of SlCES function as a breeding strategy for tomato are discussed.

Keywords: BEEs; SUMOylation; Solanum; brassinosteroids; gibberellins; hormones.

FIGURE 1

Solyc12g036470 encodes a CES orthologue, which enriches in subnuclear domains in response to BR. (A) Amino acid sequence alignment of CES of A. thaliana (AtCES) and CES of S. lycopersicum (SlCES). Motifs with functional relevance in AtCES are highlighted. (B) SlCES-YFP wild-type and SUMO mutant versions were expressed under control of the 35S promoter in tomato mesophyll protoplasts, which were either left untreated (0) or treated with 1 μM of the BR 24-epibrassinolide (+ BL) or 1 μM of bikinin (Bk). The images show CES-YFP localization in representative protoplasts, analyzed and imaged with fluorescence microscopy. White scale bars: filter = 10 μm, magnified = 6 μm.

FIGURE 2

SlCES over-expression alters GA homeostasis. (A) Expression of SlCES-YFP in two independent over-expression lines (#9 and #44) as compared to wild-type Heinz 1706 (wt). YFP was detected by RT-qPCR in vegetative tissues of the lines. Data show the mean ± SD. n = 4 biological repeats, each measured in 3 technical replicates, normalized to SIACT. (B) Phenotype (left) and quantification of hypocotyl and epicotyl length (right) of 14-day-old, soil-grown plants of the two SlCESoe lines with or without GA treatment. Plants were grown in the incubator in standard growth conditions for 7 days and then treated with DMSO as a control (0), or with 50 μM GA₄₊₇ (GA), once a day for another 7 days, before hypocotyl and epicotyl length was measured. The results are shown as means ± SD (n > 12). The ratio of treated versus untreated is shown. White bar = 5 cm. (C) Illustration of the GA biosynthetic pathway. Different enzyme classes are shown in different colors. (D) GC-MS measurements of different GAs in aerial parts of 14-day-old plants grown in the incubator in the same conditions as in A. The values are given in ng/g fresh weight. The results of four biologically replicates and mean and standard deviations are shown. In all charts: asterisks indicate significant differences determined by Student’s t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

FIGURE 3

Over-expression of SlCES promotes lateral branch growth and fruit set in low ambient temperatures. (A–D) SlCES over-expressing plants and wild-type at normal ambient temperatures of 25 ± 3°C. The plants were grown in soil, in long-day conditions in the greenhouse. Different growth parameters were assessed from 15 plants of each genotype, including epicotyl length after 5 weeks (A), and the length of the first three internodes (B) and the number of leaves (C) after 8 weeks. Photos of representative plants at 7 weeks post germination are shown (D). (E–H) SlCES over-expressing plants and wild-type at low ambient temperatures of 18 ± 3°C. The plants were grown at lower temperatures, but with otherwise comparable conditions as in panel (A–D) and the following growth parameters were assessed: leaves per plant (E), the length of the first three internodes (F) and the length of the first 4 lateral branches (G), all after 8 weeks of development. In addition, the total number of fruits that had developed at least to the green stage was counted at different stages of development from 15 plants per line (H). The results in all bar charts are shown as means ± SD (n = 15). (*P < 0.05; **P < 0.01; Student’s t-test).

FIGURE 4

SlCES over-expression alters fruit development. (A) Photos of representative fruits of SlCESoe plants and wild-type at different developmental stages. For red fruits transverse and longitudinal sections are shown. (B) Average fruit weight of fully ripe fruits of SlCESoe plants. 10–15 fully ripe fruits were harvested and weighed to obtain a total weight, which was then divided by the fruit number, to obtain a mean. Data show the mean ± SD of n = 6 (***P < 0.001; Student’s t-test). (C) Quantification of 100-seed-weight. Seeds were collected from ripe fruits, air-dried and 100 seeds were weighted. Data show the mean ± SD of n = 3 (***P < 0.001; Student’s t-test).